BioSunBlock:Meeting Notes

Call-in info : https://zoom.us/j/820128495 or go to https://zoom.us/join and enter meeting ID: 820 128 495 Meeting 11/14/15 BioCurious: Maria, Jay, Sairah, David H. Zoom: Advait CCL: Patrik, Rikke,  Indiegogo - emailed about tee shirt sizes, will order shirts next week to ship or hand out. Patrik - get Jay a check Rikke - going to check incubator at CCL, see what needs to be done to get us up and going again Ava 358 to 356 in liquid culture which might be viable if it were streaked Rikke will come in during the week (wed?) to try restreak from liquid cultures in incubator Directed evolution piece - what do we need to get to Eric to get it done? need to do a plasmid prep    Meeting 10/31/15 BioCurious: Maria, Sairah Zoom: Advait, Audrey, Rikke CCL: Patrik, DJ  Indiegogo  iGEM wiki edits can be made until Nov 20 Wiki: ="https://www.google.com/url?q=http://2015.igem.org/Team:SF_Bay_Area_DIYBio&amp;sa=D&amp;usg=AFQjCNEqCgKVNFcInnw5XqBBOxIGiiLS6A"&gt;http://2015.igem.org/Team:SF_Bay_Area_DIYBio  iGEM presentation: ="https://www.google.com/url?q=https://drive.google.com/open?id%3D1FvV12vXe0GxkEbP3Ved9sdExBqf4ekycNbJg9QtXxsc&amp;sa=D&amp;usg=AFQjCNH-aGoFUhw3L258PvWMAZ7yzwl_-A"&gt;https://drive.google.com/open?id=1FvV12vXe0GxkEbP3Ved9sdExBqf4ekycNbJg9QtXxsc iGEM poster: ="https://www.google.com/url?q=https://drive.google.com/open?id%3D0Bx0m7X6dHC0mOHdSNENTZURzdEk&amp;sa=D&amp;usg=AFQjCNHqJOnz-hHKxHUEOQPFgWOuc6xqfw"&gt;https://drive.google.com/open?id=0Bx0m7X6dHC0mOHdSNENTZURzdEk   Experiments BioC Jay, Sairah and Meenakshi worked on lab work this week on Thursday and Friday night They cultured the Ava genes and the positive control pglo and negative, of “no plasmid.” Did 2 batches of ligation product: one at 16C-30 minutes/80C-20 minutes on 10/29/15 thurs, transformed right after. Batch #2: 4C/Overnight, transformed after 21 hours on 10/30/2015 friday. <ul class="c14 lst-kix_2dfsughgra6h-0 start"> Neither batch has colonies</li></ul><ul class="c14 lst-kix_k6dzuqwnnwnj-0 start"> find or order more NEB bio brick kit</li></ul> CCL State of the lab at CCL is holding back doing more experiments. Rikke will be doing more organization. Advertise that we will be doing a cleanup on Tuesday if folks can help out. Part of the lab should be useable by Wednesday Need to make glycerine stock. Do a simple class on how to make basic 101 lab techniques like making glycerine stock - Wayne can teach (but needs a week notice) -="https://www.google.com/url?q=https://www.addgene.org/plasmid-protocols/create-glycerol-stock/&amp;sa=D&amp;usg=AFQjCNHTwx0Of2iX3KkZf4V4CVyzY3oP0Q</a>"&gt;https://www.addgene.org/plasmid-protocols/create-glycerol-stock/</a> <ul class="c14 lst-kix_wlqfjaew8rqj-0 start"> need to revive the stock</li> need to do the RCA (talk to eric about it)</li> get plates and do a mini prep (if something grows can put on glycerine)</li></ul>   Email Wayne for a date to teach a short session at CCL on how to make glycerine stocks  (other classes needed, making media, pouring plates, making liquid cultures, basic bacterial culturing) Rikee: liquid media, plate pouring, streaking  ="https://www.google.com/url?q=https://www.washingtonpost.com/news/energy-environment/wp/2015/10/20/after-sunscreen-protects-humans-it-massacres-coral-reefs/&amp;sa=D&amp;usg=AFQjCNEbELnY-iGdgLZuH0LoTUkFJcCdcA</a>"&gt;https://www.washingtonpost.com/news/energy-environment/wp/2015/10/20/after-sunscreen-protects-humans-it-massacres-coral-reefs/</a>   Meeting 10/17/15 BioCurious: Maria, David H. Zoom: Meenakshi CCL: Patrik, Rachel, Maureen Indiegogo Put account information and money should have arrived. Patrik will check bank account $1564 total raise with 19 backers % going to indiegogo Need a list of everyone that needs paying back and expenses thus far Reimbursement will come through CCL via Patrik Need to reach out to funders to get tee shirt sizes etc. Need to send out final email thanking folks and what next steps are etc. Stickers: 2x3”? Experiments BioCurious Tried transformation, nothing came out, no colonies on plate. Making new competent cells re-trying on Thursday evening going to start with re-ligating then trying the transformation again Upcoming Experiments Continue trying to retransform IDT parts at BioCurious Try doing a Directed Evolution round at CCL? Need to check with Rikke. Wiki Need to update iGEM wiki with the figures from poster and presentation OnGoing Will reevaluate how many people are coming and how long to keep the project going on a bi-weekly basis  Meeting 10/3/15  BioCurious: Maria, Jay H., David H., Sairah Zoom: Advait, Adarsh, Audrey, Victoria, Kye, Milo,Meenakshi CCL: Patrik iGEM recap <ul class="c14 lst-kix_jdkhzwjzs9b9-0 start"> We did well, poster looked good, got a bronze medal, missed things on the wiki (they formally check the boxes), gene design wasn’t on there, no page with colabs, weakest link was wiki)</li></ul><ul class="c14 lst-kix_t2jfmev4hfo-0 start"> Judges liked it, didnt have a lot of questions, </li> Easier for HS teams to put together funding and collab with a DIY lab</li> Patrik wants to do an article for BioCoder about DIY labs experience with iGEM</li> two meetings on Sat, discussion session about new tracks at iGEM (hardware, art, etc); large discussion on costs that not only will costs NOT go down might go down and might go to weed out weaker teams, don’t have a grant writer on staff, no discount to community labs), no formal process for grants for 3rd world countries etc.</li> Alternative, join the Labs Program: program for labs to make biobricks and get parts $500/year - http://igem.org/Labs_Program</a></li> DIY Bio labs should set up something for themselves aimed for DIY BioLab open to anyone HS or smaller colleges, lower costs. If a hackathon make it a week long to get something accomplished. Many different possibilities.</li> South American groups are forming their own iGEM alternative - Maria is contact point</li> For community involvement: ask Citizen Schools, which offers a lot of established tech/STEM after school programs for lower-income/minority students, to maybe start an after school DIYBio/Introduction to Synthetic Biology- that way, involving students who may never otherwise experience this exposures</li> Marc Juul is working with new BioBricks Foundation push, a node system for automated freezer to let labs network who has parts, opportunity for a challenge in lab automation</li> Putting together mailing list for people interested in developing educational outreach programs with local schools. Willam&amp;Mary team at iGEM had beautiful 80-page curriculum of simple classes. See also http://www.citizenschools.org/</a></li> WIll hand out certificate for igem participation at CCL and BIoC</li></ul> Reschedule meetings? Maria will send out a google poll ="https://www.google.com/url?q=http://goo.gl/forms/dr3Gacd862&amp;sa=D&amp;usg=AFQjCNGqEJqznWu5bYb8zGFjiUTh_XuxsA</a>"&gt;Link for poll: http://goo.gl/forms/dr3Gacd862</a> Option 1: Stay on Saturday mornings Maybe drop down to meeting every other week? Option 2: Move to Mondays, every other week Alternating with with Real Vegan Cheese Option 3: Merge with Real Vegan Cheese meetings? We have a good amount of overlap with the RVC team anyway. Option 4: any other time slots? DNA synthesis <ul class="c14 lst-kix_u8bmv7cmjt8-0 start"> We may still be able to convince IDT to give us the remaining $1400 in DNA synthesis, but someone will need to negotiate with them - any volunteers?</li> We have the Addgene plasmids, and the initial set of IDT genes - all native cyanobacterial sequence. That good enough? If anything, it should provide lots of room for improvement by Directed Evolution...</li></ul> Website We haven't done a very good job at showing to the general public what this project is about. The iGEM wiki we threw together at the very last minute is missing some major pieces of information. The iGEM wikis do attract a lot of traffic, so now that the wikis are unfrozen we should either clean it up or link out to whatever else we'd like to be the "front page" for this project (="https://www.google.com/url?q=http://2014.igem.org/Team:SF_Bay_Area_DIYbio&amp;sa=D&amp;usg=AFQjCNF9dZ5Bhy9lCi-cbvelX6MQaJA1Gg</a>"&gt;like we did for Vegan Cheese last year). We made some great diagrams for the iGEM poster and presentation that we can easily reuse for a kickass website… Team - Advait, Sairah, Maria, Patrik, Rikke, Milo Indiegogo ="https://www.google.com/url?q=https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen&amp;sa=D&amp;usg=AFQjCNGgybYNEBcAGwpn1Dd6fmlt5eZmPg</a>"&gt;https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen</a> Extend deadline? Add materials from poster, presentation. 6 days for more funding What should be our end goal for this project? <ol class="c14 lst-kix_4hyy1265xhoi-0 start" start="1"> A statistically significant improvement in UV absorption? In E.coli UV resistance?</li> A publishable result?</li></ol> Experiments What is next? Are we ready to do a round of Directed Evolution? Who/When/Where? Next experimental work: At BioCurious; transformation of IDT genes Regular work night at CCL on Wednesdays - Mention on Lab Night meetup description Potentially doing lab work at BioC on Thursday nights led by Jay Need to do RCA nicking, check in with Eric H.  Meeting 9/26/15 AT IGEM JAMBOREE  BioCurious: Maria, Pryianka, Sairah Zoom: Victoria, Shreya, Kye CCL: Tom iGEM: Patrik, Advait, David, Milo  Questions from the Jamboree team: <ul class="c14 lst-kix_jncr633nraik-0 start"> get protocol that was used for UV absorption spectrum: cells grown 20ml overnight in TSB, centrifuged, washed in saline 2x times, how much cell pellet vs 2ml methanol, pellet 10000rpm, collect supernatant</li> how does UV irradiation level compare with sunlight? Need NUMBERS</li></ul><ul class="c14 lst-kix_jncr633nraik-1 start"> 1.5-2 mW/cm2 vs &gt;15 mW/cm2 UVA+B light meter</li></ul><ul class="c14 lst-kix_jncr633nraik-0"> UV spectrum of other light sources (e.g. LED array)?</li> Can we claim Maker Faire? Did we have anything on our project there? - We can claim we recruited members and educated about iGEM</li> was RCA workshop open / on meetup? - it was for iGEM team members only not on Meetup</li> Any more headshots? Yes - Maria will email them, some folks switched out what they have, I will send updated slide or I can send the emails with the pics</li> Can we get a picture of people at BioCurious connecting over zoom, - we can try</li></ul> iGEM labs ~$500 a year and get DNA distribution and can submit parts year round  TOm - at ccl  will send him info on project -  Indiegogo - add new reward of color changing beads, get more people to donate! We will need experiment schedule. Next week regular meeting time - then we will decide about going bi-weekly for meetings. Meeting 9/19/15 ONE WEEK LEFT TO THE IGEM JAMBOREE GOOGLE SLIDES: https://docs.google.com/presentation/d/1t0tGrPprF2e3KcYOzP06BX6zoozQeELeKH73cEZau24/edit#slide=id.gbbca6c085_1_0</a>  Attendees: BioCurious: Maria, Priyanka, Adarsh, David H., Sairah, Shree, Shreya CCL: Patrik, Kye ZOOM: Advait, Victoria, Rikke, Meenakshi  Parts and wiki submitted on time! Yay! Our team presents NEXT SUNDAY in Boston The presentation is probably the most important factor in judging, so we can still get any last-minute results in, over the next week! Judging Forms (REOPENED!) -Required for any special awards (such as outreach, entrepreneurship, etc) -="https://www.google.com/url?q=http://igem.org/2015_Judging_Form?id%3D1839&amp;sa=D&amp;usg=AFQjCNGIGuiCGPZ9evRIX6YUTDhM7e1emw</a>"&gt;http://igem.org/2015_Judging_Form?id=1839</a> Experiments for the week (fluid depending on step before) first round of directed evolution, taking Addgene plasmid (do we have any at BioCurious prepped by Jay? if not then Rikke will be doing a mini prep on liquid cultures) hand off to Eric who will do first step of error prone RCA (to give us mutated plasmids), another round of transformations with the plasmid, take those expose to UV light and then another round of plasmid prep, what ever survives will give us another round and run a gel, and then another round of RCA. IDT Gene Design ask Eric, Wayne (who else?) about issue regarding repeated promoters Poster Milo started on poster layout will go with ppt  ATTENDING IGEM IN BOSTON AND PRESENTING (Please list when you will be arriving, and where you will be staying) Patrik: Likely arriving morning of 25th; haven’t booked yet Advait - Morning of 24th, Staying in Sheraton David H. Milo (Arriving Thursday morning, getting into Logan at 10am. Staying in Cambridge)  Presentation David will put Advait’s into Google all members of the team will work on it  ="https://www.google.com/url?q=https://docs.google.com/presentation/d/1t0tGrPprF2e3KcYOzP06BX6zoozQeELeKH73cEZau24/edit%23slide%3Did.gbbca6c085_1_0&amp;sa=D&amp;usg=AFQjCNEae0cIcAQL_3VZ2480drzySaKGpQ</a>"&gt;https://docs.google.com/presentation/d/1t0tGrPprF2e3KcYOzP06BX6zoozQeELeKH73cEZau24/edit#slide=id.gbbca6c085_1_0</a>  Fundraising ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen&amp;sa=D&amp;usg=AFQjCNGgybYNEBcAGwpn1Dd6fmlt5eZmPg">https://www.google.com/url?q=https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen&amp;sa=D&amp;usg=AFQjCNGgybYNEBcAGwpn1Dd6fmlt5eZmPg</a>"&gt;<a rel="nofollow" class="external free" href="https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen">https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen</a> Minimum everyone donates $1, we need more folks contributing, spread the word Survey To edit Survey: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://docs.google.com/forms/d/19URbYIXXwWi9Hv4B7BM6-iEntHuiCIOcK3TGyA39sjI/edit&amp;sa=D&amp;usg=AFQjCNG_R6XenlMb62g5CmuOy0LYwVDvEw">https://www.google.com/url?q=https://docs.google.com/forms/d/19URbYIXXwWi9Hv4B7BM6-iEntHuiCIOcK3TGyA39sjI/edit&amp;sa=D&amp;usg=AFQjCNG_R6XenlMb62g5CmuOy0LYwVDvEw</a>"&gt;<a rel="nofollow" class="external free" href="https://docs.google.com/forms/d/19URbYIXXwWi9Hv4B7BM6-iEntHuiCIOcK3TGyA39sjI/edit">https://docs.google.com/forms/d/19URbYIXXwWi9Hv4B7BM6-iEntHuiCIOcK3TGyA39sjI/edit</a>  SHARE THIS LINK FOR SURVEY: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://goo.gl/forms/pyqLgoP97Y&amp;sa=D&amp;usg=AFQjCNENwZlcQbiNPIR-vocwlVhybSfltQ">https://www.google.com/url?q=http://goo.gl/forms/pyqLgoP97Y&amp;sa=D&amp;usg=AFQjCNENwZlcQbiNPIR-vocwlVhybSfltQ</a>"&gt;<a rel="nofollow" class="external free" href="http://goo.gl/forms/pyqLgoP97Y">http://goo.gl/forms/pyqLgoP97Y</a> Lessons Learned  ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://docs.google.com/document/d/1TvJ6YEQvp0s7oNeDIaJOSC0VLT_aC3IFNAEo4yH8sqc/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNFkY2ZG_bsxUKlbX_njPz2QhqMiZA">https://www.google.com/url?q=https://docs.google.com/document/d/1TvJ6YEQvp0s7oNeDIaJOSC0VLT_aC3IFNAEo4yH8sqc/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNFkY2ZG_bsxUKlbX_njPz2QhqMiZA</a>"&gt;<a rel="nofollow" class="external free" href="https://docs.google.com/document/d/1TvJ6YEQvp0s7oNeDIaJOSC0VLT_aC3IFNAEo4yH8sqc/edit?usp=sharing">https://docs.google.com/document/d/1TvJ6YEQvp0s7oNeDIaJOSC0VLT_aC3IFNAEo4yH8sqc/edit?usp=sharing</a>  We will meet next week for a shorter experiment update at 10:30am  Meeting 9/12/15 1 WEEK LEFT!!!! Attendees: BioCurious: Sairah, Maria C., Audrey, Priyanka, David Hou CCL: Patrik, Jackie, Kye, Sam, Geoff, Sam ZOOM: Adarsh, Victoria, Sean, Rikke, Meenakshi PARTS SUBMISSION! We need to mail out at least one BioBrick part by Friday, Sept 18! Also need to enter that part into the database beforehand WIKI EDITING! All hands on deck for editing the wiki: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://2015.igem.org/Team:SF_Bay_Area_DIYBio&amp;sa=D&amp;usg=AFQjCNEqCgKVNFcInnw5XqBBOxIGiiLS6A">https://www.google.com/url?q=http://2015.igem.org/Team:SF_Bay_Area_DIYBio&amp;sa=D&amp;usg=AFQjCNEqCgKVNFcInnw5XqBBOxIGiiLS6A</a>"&gt;<a rel="nofollow" class="external free" href="http://2015.igem.org/Team:SF_Bay_Area_DIYBio">http://2015.igem.org/Team:SF_Bay_Area_DIYBio</a> The wiki gets “frozen” Friday, Sept 18, so we have to upload EVERYTHING there first. Lorent has uploaded our logo FUNDRAISING! All hands on deck for getting us finding. Indiegogo is up please donate and spread the word! ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen/x/6697587%23/story&amp;sa=D&amp;usg=AFQjCNHA15p0oVQpFvDJEK0Hk2-wJCNGjQ">https://www.google.com/url?q=https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen/x/6697587%23/story&amp;sa=D&amp;usg=AFQjCNHA15p0oVQpFvDJEK0Hk2-wJCNGjQ</a>"&gt;<a rel="nofollow" class="external free" href="https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen/x/6697587#/story">https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen/x/6697587#/story</a> Experiments: <ol class="c14 lst-kix_ccwntum7i3e2-0 start" start="1"><li class="c3 c4 c17 c11"><h3 style="display:inline"> Mycosporine transformation into HB101 </li></ol> THIS AFTERNOON AT BIOCURIOUS, starting 1pm <ol class="c14 lst-kix_ccwntum7i3e2-0" start="2"><li class="c3 c4 c17 c11"><h3 style="display:inline"> UV spectrum of crude mycosporine methanol extract </li></ol> Here's two protocols for extraction of MAAs from cyanobacteria for spectroscopic analysis. As you can see the first one is very simple: extract in 100% methanol in the fridge overnight, centrifuge, and take the supernatant: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://www.researchgate.net/publication/263709935_Biochemical_characterization_of_sunscreening_mycosporine-like_amino_acids_from_two_Nostoc_species_inhabiting_diverse_habitats&amp;sa=D&amp;usg=AFQjCNH0LOa_dGrRH3oGi2yEr6_IS2W7dg">https://www.google.com/url?q=https://www.researchgate.net/publication/263709935_Biochemical_characterization_of_sunscreening_mycosporine-like_amino_acids_from_two_Nostoc_species_inhabiting_diverse_habitats&amp;sa=D&amp;usg=AFQjCNH0LOa_dGrRH3oGi2yEr6_IS2W7dg</a>"&gt;<a rel="nofollow" class="external free" href="https://www.researchgate.net/publication/263709935_Biochemical_characterization_of_sunscreening_mycosporine-like_amino_acids_from_two_Nostoc_species_inhabiting_diverse_habitats">https://www.researchgate.net/publication/263709935_Biochemical_characterization_of_sunscreening_mycosporine-like_amino_acids_from_two_Nostoc_species_inhabiting_diverse_habitats</a> Cyanobacterial cells were harvested by centrifugation, and MAAs were extracted in 2 mL of 100% high-performance liquid chromatography (HPLC) grade methanol overnight at 4 °C. The methanol extracts were centrifuged at 10,000 rpm for 10 min, and the supernatant was subjected to spectroscopic analysis between 250- and 700-nm wavelengths, using a double-beam spectrophotometer (UV-Vis 2900, Hitachi, Japan). ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://maxwellsci.com/print/crjbs/v3-165-171.pdf&amp;sa=D&amp;usg=AFQjCNHJC6LwvnwyfVAUe8eusuc7bprHRg">https://www.google.com/url?q=http://maxwellsci.com/print/crjbs/v3-165-171.pdf&amp;sa=D&amp;usg=AFQjCNHJC6LwvnwyfVAUe8eusuc7bprHRg</a>"&gt;<a rel="nofollow" class="external free" href="http://maxwellsci.com/print/crjbs/v3-165-171.pdf">http://maxwellsci.com/print/crjbs/v3-165-171.pdf</a> Extraction of 10 mg of dried Aulosira fertilissima in 2 mL 20% Methanol(gradient grade) at 45ºC for 2h. Centrifugation if necessary (10 min 10000 U). 1.5 mL of the supernatant was lyophilised. 2 mL 100 % Methanol was redissolved in the residue further vortexing followed by centrifugation was done. 1.5 mL of the supernatant was evaporated at 45ºC. 1.5 mL H2O was redissolving in the residue, again vortexing and centrifugation was done. Spectroscopic analysis of the supernatant was taken from 200 to 750 nm. Mycosporine like amino acid (MAAs) (Garcia pichelz et al., 1993) Step 1. Took 10 mg fresh biomass and were suspended in 20 ml of methanol             (20%)  and homogenized. Step2. Covered all the test tubes with aluminium foil and were kept at 45℃ in           water bath for 2hrs. Step3. Centrifuged and filtered the supernatant through whatman filter paper           (No.1). Step4. The absorbance of filtrate were measured spectrophotometrically at many          wavelengths (310, 320, 330, 332, 334 and 260). E. coli cells may be even easier to extract, because cyanobacteria tend to have a stronger cell wall&lt;img alt="" src="images/image00.gif" style="width: 20.00px; height: 8.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""&gt; <ol class="c14 lst-kix_ccwntum7i3e2-0" start="3"><li class="c3 c4 c17 c11"><h3 style="display:inline"> Directed Evolution Round 1? </li></ol> Patrik will start writing up a protocol Everybody who has done research on Directed Evolution should help out digging up published protocols. 4. Domestication of gene clusters into IGEM plasmid restriction digest and ligation reaction (Tuesday or Wednesday) We need Pstl; did we get that in the biobrick cloning kit from NEB? Total Participation in iGEM: 102: on google group igem registered: 14 (other), 16(student), 7 (instructors) - total 37  Meeting 9/5/15 2 WEEKS LEFT!!!! Attendees:       BioCurious: Maria, Jay, James, Phil, Prag (biomedical research at Stanford), Sairah, Lawrence (CS), April, Audrey, David H.       CCL: Kye, Megan, Patrik, Patrick, Tom, Maureen        ZOOM: Advait, Adarsh, Shreya, Rikke, David, Victoria  UV Hardware: Drops in the 340nm and 390nm range according to spectra Experiments: Rikke starting another kill curve experiment at 1pm at CCL Jay: transformation in mutagenic strain successful; nothing grew in HB101 (including pGLO positive control). Need to start doing experiments collecting UV absorption spectrum: - Measure UV absorption spectrum of TSB (tryptic soy broth). As you heard from Rikke, there seems to be an issue with fluorescence and/or UVabsorption in the medium we've been using for the kill curve experiments. - Measure E. coli UV absorption spectrum, with and without the pGlo plasmid. (a) in whole cell suspension (b) in cell lysate, (c) in crude protein extract. If we hope to be able to detect a change in UV absorption spectrum in GFP, how should we best measure the spectrum? - Once we have a mycosporine producing strain, we'll need some way to measure its spectrum as well. Eventually, we'd like to separate the MAAs by HPLC, but just like with the GFP, we could generate a spectrum for a crude extract as well. Here's a few protocols for extracting the MAA-containing fraction with methanol: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://www.researchgate.net/publication/263709935_Biochemical_characterization_of_sunscreening_mycosporine-like_amino_acids_from_two_Nostoc_species_inhabiting_diverse_habitats&amp;sa=D&amp;usg=AFQjCNH0LOa_dGrRH3oGi2yEr6_IS2W7dg">https://www.google.com/url?q=https://www.researchgate.net/publication/263709935_Biochemical_characterization_of_sunscreening_mycosporine-like_amino_acids_from_two_Nostoc_species_inhabiting_diverse_habitats&amp;sa=D&amp;usg=AFQjCNH0LOa_dGrRH3oGi2yEr6_IS2W7dg</a>"&gt;<a rel="nofollow" class="external free" href="https://www.researchgate.net/publication/263709935_Biochemical_characterization_of_sunscreening_mycosporine-like_amino_acids_from_two_Nostoc_species_inhabiting_diverse_habitats">https://www.researchgate.net/publication/263709935_Biochemical_characterization_of_sunscreening_mycosporine-like_amino_acids_from_two_Nostoc_species_inhabiting_diverse_habitats</a> Cyanobacterial cells were harvested by centrifugation, and MAAs were extracted in 2 mL of 100% high-performance liquid chromatography (HPLC) grade methanol overnight at 4 °C. The methanol extracts were centrifuged at 10,000 rpm for 10 min, and the supernatant was subjected to spectroscopic analysis between 250- and 700-nm wavelengths, using a double-beam spectrophotometer (UV-Vis 2900, Hitachi, Japan). IDT Order: Need to finalize gene order for Anabaena gene cluster - see ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://docs.google.com/document/d/1AVc2TPD71psi-JXjWkUTxp79HHXIEM2vP6k7G7RxbWE/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNEi_5UvMshIHfAGojmwaO-l2Latzw">https://www.google.com/url?q=https://docs.google.com/document/d/1AVc2TPD71psi-JXjWkUTxp79HHXIEM2vP6k7G7RxbWE/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNEi_5UvMshIHfAGojmwaO-l2Latzw</a>"&gt;<a rel="nofollow" class="external free" href="https://docs.google.com/document/d/1AVc2TPD71psi-JXjWkUTxp79HHXIEM2vP6k7G7RxbWE/edit?usp=sharing">https://docs.google.com/document/d/1AVc2TPD71psi-JXjWkUTxp79HHXIEM2vP6k7G7RxbWE/edit?usp=sharing</a> Jay and Patrik will coordinate on finalizing sequence; can also run by Eric Harness and Wayne Survey: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://docs.google.com/forms/d/19URbYIXXwWi9Hv4B7BM6-iEntHuiCIOcK3TGyA39sjI/viewform?usp%3Dsend_form&amp;sa=D&amp;usg=AFQjCNH5kM_Woa_q-4YrXKTFbiTw_YB81w">https://www.google.com/url?q=https://docs.google.com/forms/d/19URbYIXXwWi9Hv4B7BM6-iEntHuiCIOcK3TGyA39sjI/viewform?usp%3Dsend_form&amp;sa=D&amp;usg=AFQjCNH5kM_Woa_q-4YrXKTFbiTw_YB81w</a>"&gt;<a rel="nofollow" class="external free" href="https://docs.google.com/forms/d/19URbYIXXwWi9Hv4B7BM6-iEntHuiCIOcK3TGyA39sjI/viewform?usp=send_form">https://docs.google.com/forms/d/19URbYIXXwWi9Hv4B7BM6-iEntHuiCIOcK3TGyA39sjI/viewform?usp=send_form</a> <ul class="c14 lst-kix_l6bqxui6mzgc-0 start"> are you concerned about effect of sunscreens on your own health</li> on the environment?</li> difference between UVA, UVB</li> environmental issues are mainly on coral reefs, not coastal environment. and mostly due to other compounds than PABA, TiO2</li> would you wear sunscreen more if there were a more natural alternative?</li></ul> Indiegogo Campaign: Let’s put the fundraiser under CCL Anyone who wants to work on video can do so. Poster: After wiki we will concentrate Wiki: Due on September 18th - everybody should be editing the wiki! Presentation: -Advait presented a talk at the S4 conference, we can use that powerpoint as a template  Meeting 8/29/15 3 Weeks left!! Attendees: BioCurious: Maria, Jay, Audrey, April, Priyanka, Sairah, David H. CCL: Rikke, Kye, Sean       Zoom: David, Adarsh, Victoria, David M.  UV Hardware  Leave as is but UV exposure is not killing bacteria even at 1 hour of exposure, going to try moving closer to lights, right now we are at 8” Problem with kill curve experiment: Some plates with high UV exposure have more growth than plate exposed to lower amounts of UV. In addition, GFP expression across plates are inconsistent. potential hypotheses: <ol class="c14 lst-kix_pyk2nevz22ri-0 start" start="1"> UV light is not close enough to kill majority of bacteria. </li> Bacteria is under stress due to UV irradiation and does not use pGLO plasmid as it doesn’t seem to provide benefit. Note that we are irradiating bacteria with a wide UV spectrum while GFP only absorbs a fraction of it. </li> The TSB media partially absorbs UV light, sheltering bacteria. </li></ol> Need to get anabana transformed into ecoli onto plate and to CCL Jay will send out operan again form addgene  At BioC today will miniprep out 5 of the plasmids from addgene today, wont do transformation today (need to get competent cells ready) And then bring to CCL for kill curve checks  Assume we will do transformations at BioC on Tuesday @ approx 5:30pm to start will take about 3 hours  Patrik start cultures on monday and wednesday and experiments on tuesday and thrusday need to schedule someone to do cell counting within 24 not 48 hours afterwards at CCL EELSI  Survey on sunscreens -  April, Audrey, David, Kye, Priyanka, Sunscreen research -  Wiki - needs more work! Fundraising - stalled for some issues. Poster - After wiki Presentation - Advait first pass   Meeting 8/22/15 - 4 WEEKS LEFT!!!! Attendees: BioCurious: Maria I. C., Priyanka, Audrey, Adarsh, David Hou, Jonathan R.        CCL: Patrik, Kye, Sean Zoom: James, Advait, Lorantto, Rikke, Wayne Deadlines:        -Safety form (final version) due august 28        -="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://2015.igem.org/Main_Page&amp;sa=D&amp;usg=AFQjCNEJKIUothkaPdDnY-VhC6C73CFClw">https://www.google.com/url?q=http://2015.igem.org/Main_Page&amp;sa=D&amp;usg=AFQjCNEJKIUothkaPdDnY-VhC6C73CFClw</a>"&gt;<a rel="nofollow" class="external free" href="http://2015.igem.org/Main_Page">http://2015.igem.org/Main_Page</a>                -Maria, Eric,        -Team banners due September 1st        -Final team rosters due September 11th        -Parts due September 18th (="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://parts.igem.org/DNA_Submission&amp;sa=D&amp;usg=AFQjCNH0lk_pPtAXMbDPyHXeDisctODqHw">https://www.google.com/url?q=http://parts.igem.org/DNA_Submission&amp;sa=D&amp;usg=AFQjCNH0lk_pPtAXMbDPyHXeDisctODqHw</a>"&gt;<a rel="nofollow" class="external free" href="http://parts.igem.org/DNA_Submission">http://parts.igem.org/DNA_Submission</a>) UV Hardware Made a frame to hold UV lamps Spectroradiometer is working Reptile lamp covers UVB really well Supposedly 380nm LED array is really ~410nm - mostly blue visible light! One of the nail curing lamps has nice broad peaks around 365nm and 400nm Reptile lamps + nail curing lamp has reasonable match to solar UV spectrum Kill Curve Experiment Coiled UV lamp in nail curing lamp may be burned out? Only designed to be on 1min at a time. Had to do without. Need to do better job at normalizing initial cell density. pGlo overnight culture had far fewer cells to start with. experiment notes: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://docs.google.com/document/d/1Fio3o6M9dsEPUZZ7Ig5h4nN_9p9mL4UdoYhWkxI7lF0/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNH14yVYTSIn5HD58qzgLmxDPdKcIA">https://www.google.com/url?q=https://docs.google.com/document/d/1Fio3o6M9dsEPUZZ7Ig5h4nN_9p9mL4UdoYhWkxI7lF0/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNH14yVYTSIn5HD58qzgLmxDPdKcIA</a>"&gt;<a rel="nofollow" class="external free" href="https://docs.google.com/document/d/1Fio3o6M9dsEPUZZ7Ig5h4nN_9p9mL4UdoYhWkxI7lF0/edit?usp=sharing">https://docs.google.com/document/d/1Fio3o6M9dsEPUZZ7Ig5h4nN_9p9mL4UdoYhWkxI7lF0/edit?usp=sharing</a> Need to pour another stack of quartered plates (8 left, pour 20 more with LB agar) At CCL this week approx 8pm to 11pm? Monday - figure out UV setup and start overnight batch of liquid culture (Patrik, Sean) Tuesday - Kill curve part 2 (any changes from the first run?) Wednesday - someone make more culture Thursday - Another run of Kill Curve At BioCurious - need to follow up with Jay (Maria emailing)  Need to get our HPLC running - check with Jacob, Eric, Josiah <a rel="nofollow" class="external free" href="https://docs.google.com/document/d/11vkbB8V8bB9dVdSySJ5CqLpqHBNjUqMy10cNifBWoeg/edit">https://docs.google.com/document/d/11vkbB8V8bB9dVdSySJ5CqLpqHBNjUqMy10cNifBWoeg/edit</a> Genes AddGene plasmids arrived; Jay plated them, and has copies for CCL Still need to submit IDT order  description of Anabaena gene cluster: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://docs.google.com/document/d/1AVc2TPD71psi-JXjWkUTxp79HHXIEM2vP6k7G7RxbWE/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNEi_5UvMshIHfAGojmwaO-l2Latzw">https://www.google.com/url?q=https://docs.google.com/document/d/1AVc2TPD71psi-JXjWkUTxp79HHXIEM2vP6k7G7RxbWE/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNEi_5UvMshIHfAGojmwaO-l2Latzw</a>"&gt;<a rel="nofollow" class="external free" href="https://docs.google.com/document/d/1AVc2TPD71psi-JXjWkUTxp79HHXIEM2vP6k7G7RxbWE/edit?usp=sharing">https://docs.google.com/document/d/1AVc2TPD71psi-JXjWkUTxp79HHXIEM2vP6k7G7RxbWE/edit?usp=sharing</a> <ul class="c14 lst-kix_kwe5ipj1odqg-0 start"> exclude restriction sites (used for standard biobricks assembly)</li> add any flanking sequences? Any restriction sites for Gibson? BioBrick prefix/suffix?</li></ul> EELSI <ul class="c14 lst-kix_mxakccboybxi-0 start"> Consolidated all documentation into one google docs: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://docs.google.com/document/d/1jt-huSIabQ6ZlggpiePb7-Lb4-eXLemOtNq-_lZqovM/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNEbDax4xH0mbt3RnBb9YkBgeNVJ6g">https://www.google.com/url?q=https://docs.google.com/document/d/1jt-huSIabQ6ZlggpiePb7-Lb4-eXLemOtNq-_lZqovM/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNEbDax4xH0mbt3RnBb9YkBgeNVJ6g</a>"&gt;<a rel="nofollow" class="external free" href="https://docs.google.com/document/d/1jt-huSIabQ6ZlggpiePb7-Lb4-eXLemOtNq-_lZqovM/edit?usp=sharing">https://docs.google.com/document/d/1jt-huSIabQ6ZlggpiePb7-Lb4-eXLemOtNq-_lZqovM/edit?usp=sharing</a></li> Safety of working with UV light, safety precautions we have taken; include photographs of setup</li> ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen/x/6697587%23/story&amp;sa=D&amp;usg=AFQjCNHA15p0oVQpFvDJEK0Hk2-wJCNGjQ">https://www.google.com/url?q=https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen/x/6697587%23/story&amp;sa=D&amp;usg=AFQjCNHA15p0oVQpFvDJEK0Hk2-wJCNGjQ</a>"&gt;<a rel="nofollow" class="external free" href="https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen/x/6697587#/story">https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen/x/6697587#/story</a></li> </li></ul> WIKI <ul class="c14 lst-kix_o8kdizh6lvgl-0 start"> Moving info over</li> template design for iGEM using logo etc.</li></ul>  Fundraising <ul class="c14 lst-kix_9bd76dha8s1w-0 start"> change tagline on logo to Evolved Sunscreen on indiegogo</li> get BioC EIN</li> figure out which account to send funds to</li></ul>  Poster <ul class="c14 lst-kix_2luqwnv6qrb1-0 start"> layout and elements</li></ul> Presentation        -Advait will also be presenting at S4 on Saturday (link?)   Meeting 8/15/15 - 5 WEEKS LEFT! Attendees:       BioCurious: Maria, Adarash, Jay, Phil, James, Alberto (postdoc at Jbei), Lucan (masters at jbei), Daniel (phd starting at UC Berkeley), Deborah (biologist from Sao Paulo), Audrey (HS), Manakshi, priyanka       CCL: Sean, Kye        Zoom: David, Shreya, Victoria, Wayne, Manali, Advait UV hardware Sean: update on the spectroradiometer? Sean has purchased one off EBay and will see if he can direct pick up from seller in Vacaville. Will let us set up and monitor the kill curve experiments. Victoria &amp; Co: is the light box / stand ready to use? <ul class="c14 lst-kix_c9xl5vyres3i-0 start"> email from Rikke with some issues we are having with the experiments</li> with UV lights will the heat be an issue - add a fan which will add in more contaminants</li> Rig modifications will be with Victoria at CCL today at 1pm</li></ul> Kill Curve experiment Do we have all the cultures, plates, reagents we need? <ul class="c14 lst-kix_2zs5zuyi76wb-0 start"> Have agar plates and bacterial cultures </li> bottle neck is - fan and specifically which wavelengths are hitting the bacteria</li></ul> When do we start the experiment, and how long will it take? <ul class="c14 lst-kix_d8vy7zgoss4k-0 start"> will work on this afternoon and be ready to go this afternoon perhaps?</li> Email to get a protocol and set the date for the experiment (ask Rikke etc)</li></ul> ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://drive.google.com/open?id%3D0B2fL4PwIccWZfmRYc0lDdS1jWV9UeFdwME1EYmZxOG9EakFUZWNzS3pJczJCbU5QNElZSU0&amp;sa=D&amp;usg=AFQjCNEbZ42CVM7MlGtSQ-Evh39ms6yMNQ">https://www.google.com/url?q=https://drive.google.com/open?id%3D0B2fL4PwIccWZfmRYc0lDdS1jWV9UeFdwME1EYmZxOG9EakFUZWNzS3pJczJCbU5QNElZSU0&amp;sa=D&amp;usg=AFQjCNEbZ42CVM7MlGtSQ-Evh39ms6yMNQ</a>"&gt;<a rel="nofollow" class="external free" href="https://drive.google.com/open?id=0B2fL4PwIccWZfmRYc0lDdS1jWV9UeFdwME1EYmZxOG9EakFUZWNzS3pJczJCbU5QNElZSU0">https://drive.google.com/open?id=0B2fL4PwIccWZfmRYc0lDdS1jWV9UeFdwME1EYmZxOG9EakFUZWNzS3pJczJCbU5QNElZSU0</a> Genes 8/21/15 Update from Jay (sorry can’t make today’s meeting) The seven AddGene plasmids arrived. I streaked two sets of plates, one for CCL and one for BioCurious. FYI: Addgene used the Top10 E. coli cell line for delivering these plasmids. They are all Kanamycin resistant for selection. I will grow up some liquid cultures Saturday night and will be doing minipreps of the plasmids on Sunday afternoon starting around 3PM. Anyone interested in joining this miniprep festival is welcome!! I’m in progress designing a GFP-UV gene with His-tag and flanking sequences in the Bio Brick format. As soon as the design is complete and checked we can order from IDT. Anyone interested in helping out on this is certainly welcome. Email me. <ul class="c14 lst-kix_76um8t6vxszq-0 start"> Link to Genome Compiler: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://igem.genomecompiler.com/igem_app&amp;sa=D&amp;usg=AFQjCNGLSfg1bIzs8lLDYJRxy3lFYP4mdA">https://www.google.com/url?q=https://igem.genomecompiler.com/igem_app&amp;sa=D&amp;usg=AFQjCNGLSfg1bIzs8lLDYJRxy3lFYP4mdA</a>"&gt;<a rel="nofollow" class="external free" href="https://igem.genomecompiler.com/igem_app">https://igem.genomecompiler.com/igem_app</a></li> </li></ul> All: what else are we ordering from IDT, and when? <ul class="c14 lst-kix_ujcxgoaonvbc-0 start"> Has anyone looked at Jay’s gene cluster construct? Have someone else review </li> Need to order GFP with histags - Jay will design today in genome compiler folder</li></ul> Fundraising <ul class="c14 lst-kix_qnd92yw2oxph-0 start"> Indiegogo is ready we can launch today</li> Lets launch today!</li></ul> Environmental/Ethical/Legal/Societal Issues (EELSI) Elizabeth, James, Phil, Vikram: any updates? <ul class="c14 lst-kix_dq9dtbe6k4yw-0 start"> needs cleanup but lots of research and info there</li> Community Engagement combined with fundraising (Reddit AMA on project) - to push fundraising - AMA in two weeks Saturday at noon led by James and Phil</li> Vikram (market survey how they are using the product) - Will have sample survey questions by next week</li></ul> Wiki Need to start populating the wiki with our final writeup on some of these topics (e.g. notes on UV spectra, etc.) ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://bayareaigem.herokuapp.com/&amp;sa=D&amp;usg=AFQjCNGlkmaG_PzoltN8fGQo6uQoUdvP9Q">https://www.google.com/url?q=http://bayareaigem.herokuapp.com/&amp;sa=D&amp;usg=AFQjCNGlkmaG_PzoltN8fGQo6uQoUdvP9Q</a>"&gt;<a rel="nofollow" class="external free" href="http://bayareaigem.herokuapp.com/">http://bayareaigem.herokuapp.com/</a> - All experiments need to be documented every single day! ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://bayareaigem.herokuapp.com/index.php?title%3DNotebook&amp;sa=D&amp;usg=AFQjCNHiHRGUEf0j1nBttHMCpF_piEzwNg">https://www.google.com/url?q=http://bayareaigem.herokuapp.com/index.php?title%3DNotebook&amp;sa=D&amp;usg=AFQjCNHiHRGUEf0j1nBttHMCpF_piEzwNg</a>"&gt;<a rel="nofollow" class="external free" href="http://bayareaigem.herokuapp.com/index.php?title=Notebook">http://bayareaigem.herokuapp.com/index.php?title=Notebook</a> Everybody should submit a team bio: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://bayareaigem.herokuapp.com/index.php?title%3DTeam&amp;sa=D&amp;usg=AFQjCNEftsv1aAD_xCBYxK28lg5O-h_06A">https://www.google.com/url?q=http://bayareaigem.herokuapp.com/index.php?title%3DTeam&amp;sa=D&amp;usg=AFQjCNEftsv1aAD_xCBYxK28lg5O-h_06A</a>"&gt;<a rel="nofollow" class="external free" href="http://bayareaigem.herokuapp.com/index.php?title=Team">http://bayareaigem.herokuapp.com/index.php?title=Team</a> <ul class="c14 lst-kix_m4fhd5bv2o4r-0 start"> Maria will updates with submitted bios.</li></ul>   Meeting 8/8/15 Attendees:        BioCurious: Maria, Eric H., Daniel C, John McG, Zack, David H. (HS), Vikram, Manali (molecular Biologist), James (MD), Phil (materials scientist), Jonathan R. (yoga instructor)       CCL: Patrik, Tom, Sean, Elizabeth (visiting from NC research triangle iGEM team)        Zoom: Advait, Shreya, Vardaan, Rikke, Wayne, David M, VIctoria, Milo Safety Training! Everybody who wants to work in the wetlab needs to take the online safety quiz! CCL:="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://docs.google.com/forms/d/1n_R0MB1xJCMgdaEv1fklwtv-BRY0WmaPWhVQnNSg9cs/viewform&amp;sa=D&amp;usg=AFQjCNGu6zNWVkwwxyF0Yye096Z18SRfJw">https://www.google.com/url?q=https://docs.google.com/forms/d/1n_R0MB1xJCMgdaEv1fklwtv-BRY0WmaPWhVQnNSg9cs/viewform&amp;sa=D&amp;usg=AFQjCNGu6zNWVkwwxyF0Yye096Z18SRfJw</a>"&gt;<a rel="nofollow" class="external free" href="https://docs.google.com/forms/d/1n_R0MB1xJCMgdaEv1fklwtv-BRY0WmaPWhVQnNSg9cs/viewform">https://docs.google.com/forms/d/1n_R0MB1xJCMgdaEv1fklwtv-BRY0WmaPWhVQnNSg9cs/viewform</a> BioC:="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://docs.google.com/forms/d/16m9xRfmq0obBe_aNBZwtNxUME_vDjVUieloK_jI6DVA/viewform&amp;sa=D&amp;usg=AFQjCNFvvECHoDFUWJNc2As69FAfU3Hpkg">https://www.google.com/url?q=https://docs.google.com/forms/d/16m9xRfmq0obBe_aNBZwtNxUME_vDjVUieloK_jI6DVA/viewform&amp;sa=D&amp;usg=AFQjCNFvvECHoDFUWJNc2As69FAfU3Hpkg</a>"&gt;<a rel="nofollow" class="external free" href="https://docs.google.com/forms/d/16m9xRfmq0obBe_aNBZwtNxUME_vDjVUieloK_jI6DVA/viewform">https://docs.google.com/forms/d/16m9xRfmq0obBe_aNBZwtNxUME_vDjVUieloK_jI6DVA/viewform</a> (You only need to take one - they are essentially identical. Please send us feedback if anything in the quiz is confusing) Anyone working in CCL also needs to be a member CCL will offer anyone on the iGEM team free or discounted membership. ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://docs.google.com/forms/d/16YqJ00D7EYAk5IIXmCLkFLEhzqeMRP5LiCLQGdSikKE/viewform&amp;sa=D&amp;usg=AFQjCNHpnAfJrtWJYQf-HJzvAOEKwYqorA">https://www.google.com/url?q=https://docs.google.com/forms/d/16YqJ00D7EYAk5IIXmCLkFLEhzqeMRP5LiCLQGdSikKE/viewform&amp;sa=D&amp;usg=AFQjCNHpnAfJrtWJYQf-HJzvAOEKwYqorA</a>"&gt;Apply for sponsored membership here. Or ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://counterculturelabs.org/join&amp;sa=D&amp;usg=AFQjCNHd4SBue0MJF-pbo3rW9jXBeFnR-A">https://www.google.com/url?q=https://counterculturelabs.org/join&amp;sa=D&amp;usg=AFQjCNHd4SBue0MJF-pbo3rW9jXBeFnR-A</a>"&gt;become a full paying member of CCL here. Experiments update See ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://bayareaigem.herokuapp.com/index.php?title%3DNotebook&amp;sa=D&amp;usg=AFQjCNHiHRGUEf0j1nBttHMCpF_piEzwNg">https://www.google.com/url?q=http://bayareaigem.herokuapp.com/index.php?title%3DNotebook&amp;sa=D&amp;usg=AFQjCNHiHRGUEf0j1nBttHMCpF_piEzwNg</a>"&gt;<a rel="nofollow" class="external free" href="http://bayareaigem.herokuapp.com/index.php?title=Notebook">http://bayareaigem.herokuapp.com/index.php?title=Notebook</a> <ol class="c14 lst-kix_v7wc2w3tbbfj-0 start" start="1"> pGLO transformation at BioCurious (Jay)</li></ol> The transformation we did on Saturday was successful. We got some transformants on plates 1B, 3B and 2B expressing GFP. I attached images showing all the plates and the GFP glowing under UV (let me know if you need a higher res image). Below is a table of the plate by plate outcomes. We used pGLO plasmids from different sources and some were over a year old so we expected that not all would successfully transform. Thanks to everyone who helped out on Saturday!! I'll streak an HB101/pGLO plate for CCL. I plan to do a miniprep to purify the pGLO plasmid on Thursday night starting around 7pm if anyone wants to participate. Patrik will be at Biocurious on Thursday night and he can pick up a plate with the transformed HB101 and some plasmid to take back to CCL. Plate                       Observation 1                        Lawn LA plate no ARA 1B                       &gt; 20 colonies expressing GFP 2                       No growth 2B                        &gt; 100 colonies expressing GFP 3                       ~ 6 colonies - no GFP (did not glow under UV) 3B                       &gt; 20 colonies expressing GFP 4A                       No growth 4B                        Lawn        LA plate No ARA 5A                        No growth 5B                        No growth 6A                        No growth 6B                        No growth 7                        No growth  <ol class="c14 lst-kix_v7wc2w3tbbfj-0" start="2"> UV sources</li></ol> See ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://docs.google.com/document/d/1frmVyd8b8TmWgwaUV8lN5-0OlswaJP0t5B3H_hwRWGo/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNEB3eJ43UpBA-jO90jCx-53OBTw4g">https://www.google.com/url?q=https://docs.google.com/document/d/1frmVyd8b8TmWgwaUV8lN5-0OlswaJP0t5B3H_hwRWGo/edit?usp%3Dsharing&amp;sa=D&amp;usg=AFQjCNEB3eJ43UpBA-jO90jCx-53OBTw4g</a>"&gt;Notes on UV Spectra - all the hardware is documented there  <ol class="c14 lst-kix_v7wc2w3tbbfj-0" start="3"> UV kill curve experiments at CCL (Rikke)</li></ol> Need to normalize bacterial cultures - need to get spectrophotometer online to calculate cell count based on optical density (OD) Use drop spot plate method for plate counts - saves a lot on plates Use 96 well plates and multipipetters to do dilutions Genes Jay found the Anabaena genes available on plasmids at Addgene - $65 each! ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://www.addgene.org/Christopher_T_Walsh/&amp;sa=D&amp;usg=AFQjCNHf59FCFhdqqg6iZL9iYzc8mmYcwA">https://www.google.com/url?q=http://www.addgene.org/Christopher_T_Walsh/&amp;sa=D&amp;usg=AFQjCNHf59FCFhdqqg6iZL9iYzc8mmYcwA</a>"&gt;<a rel="nofollow" class="external free" href="http://www.addgene.org/Christopher_T_Walsh/">http://www.addgene.org/Christopher_T_Walsh/</a> Let’s order what we already know we want to get from IDT asap. No need to order everything at once. <ul class="c14 lst-kix_n57cj1mvdprg-0 start"> codon optimized Anabaena genes (with separate promoters / in separate biobricks?)</li> Nostoc final enzyme</li> GFPuv with strong promoter, strong RBS, transcriptional terminator</li></ul> Need to decide which exact E.coli strain to use with RCA (especially whether or not it should be a RecA / DNA repair mutant!)         DH5 alpha sequence ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://www.ncbi.nlm.nih.gov/bioproject/205928&amp;sa=D&amp;usg=AFQjCNGnvD4boUwKWSYz9dRDD6czeo4kgg">https://www.google.com/url?q=http://www.ncbi.nlm.nih.gov/bioproject/205928&amp;sa=D&amp;usg=AFQjCNGnvD4boUwKWSYz9dRDD6czeo4kgg</a>"&gt;<a rel="nofollow" class="external free" href="http://www.ncbi.nlm.nih.gov/bioproject/205928">http://www.ncbi.nlm.nih.gov/bioproject/205928</a>  Fundraising <ul class="c14 lst-kix_9i52ajiuliz3-0 start"> Indiegogo editable ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=https://www.indiegogo.com/projects/biosunblock-looking-at-sunscreen/x/6697587%23/story&amp;sa=D&amp;usg=AFQjCNEX6vGRRpYUNe7PuVfCttq4erREBA">https://www.google.com/url?q=https://www.indiegogo.com/projects/biosunblock-looking-at-sunscreen/x/6697587%23/story&amp;sa=D&amp;usg=AFQjCNEX6vGRRpYUNe7PuVfCttq4erREBA</a>"&gt;<a rel="nofollow" class="external free" href="https://www.indiegogo.com/projects/biosunblock-looking-at-sunscreen/x/6697587#/story">https://www.indiegogo.com/projects/biosunblock-looking-at-sunscreen/x/6697587#/story</a></li> Need to finalize </li></ul>  FYI regarding images for the Indiegogo campaign: Instead of buying stock photos, look for images on Google Images that are labeled for reuse: ="<a rel="nofollow" class="external free" href="https://www.google.com/url?q=http://screencast.com/t/bnrtTRvhBX&amp;sa=D&amp;usg=AFQjCNHLn8ezxv0TLdft6lcCiT22Xq5k6w">https://www.google.com/url?q=http://screencast.com/t/bnrtTRvhBX&amp;sa=D&amp;usg=AFQjCNHLn8ezxv0TLdft6lcCiT22Xq5k6w</a>"&gt;<a rel="nofollow" class="external free" href="http://screencast.com/t/bnrtTRvhBX">http://screencast.com/t/bnrtTRvhBX</a> EELSI Would be great to have 1-2 people who are dedicated to this. Join slack channel if interested. Elizabeth, James, Phil, Vikram **email invitations to slack coming shortly  why is paba banned in EU damage to coral reefs nano/Australia AddGene licenses not compatible w biobrick licenses? Meeting 8/1/15 Attendees <ul><li> Biocurious: Maria C., Adarash, Phil, David Hou, Leo, Daniel (age 11), April, Zack (visiting from Atlanta EE), Jay Hanson, James, Johan, Meenakshi, Priyanka </li><li> CCL: Patrik, Andrew, Sean </li><li> Zoom: Rikke, Shreya, Antonio, Vardhaan, Victoria </li></ul> Jamboree Who signed up to go? Patrik, Advait, Eri (may go as media) Fundraising <ul><li>Did we decide on platform? Indiegogo </li><li>Need a logo - April </li><li>Budget: $6000 (minus pledges donated to team) </li><li>Thank you, Stickers, Tee Shirt </li><li>Research color changing beads in UV, tee shirt that changes colors in sunlight </li><li>Quantum dot jewelry research as a stretch goal </li><li>Make a gadget with UV LED as well </li><li>Small GFP kit with plasmid $100 </li></ul> UV spectra <ul><li>mycosporine-glycine + shinorine/porphyra-334 + GFP covers UV spectrum nicely </li><li>UV lamps for pet reptiles may be good choice for cheap broad spectrum UVA/B (add some leds for near UV?) </li></ul> LEDs at 395-420nm are very easy to find at any electronics store (Radio Shack, Fry’s,...), but barely qualify as UV <ul><li>UV LEDs <a rel="nofollow" class="external free" href="http://www.qphotonics.com/UVTOP-LEDs/">http://www.qphotonics.com/UVTOP-LEDs/</a> </li><li>395nm LEDs <a rel="nofollow" class="external free" href="http://www.ebay.com/itm/like/321364837871?lpid=82&amp;chn=ps">http://www.ebay.com/itm/like/321364837871?lpid=82&amp;chn=ps</a> </li><li>may need to hack one of our UV-specs to accept external light </li><li>Start doing kill curve expts asap, on wild type e coli &amp; mutator strain. </li></ul> Start doing experiments at CCL on UV on Wednesday Finalize IDT gene order <ul><li>Patrik can do codon optimization </li><li>Which promoter &amp; RBS to add? Or assemble everything from existing biobricks? </li><li>Which flanking sequences do we need, for which assembly method? </li><li>Do we include biobrick prefix/suffix? </li></ul> Let’s use Genome Compiler - there’s a free iGEM version Contact Minnesota team! Patrik will contact Maria will contact iGEM about DNA distribution kit Order free NEB kits Decide what to order now Assembly methods: <a rel="nofollow" class="external free" href="https://j5.jbei.org/j5manual/pages/1.html">https://j5.jbei.org/j5manual/pages/1.html</a> Gibson and InFusion: <a rel="nofollow" class="external free" href="https://j5.jbei.org/j5manual/pages/22">https://j5.jbei.org/j5manual/pages/22</a>. Golden Gate: <a rel="nofollow" class="external free" href="https://j5.jbei.org/j5manual/pages/23.htmlhtml">https://j5.jbei.org/j5manual/pages/23.htmlhtml</a> Biobrick Assembly: <a rel="nofollow" class="external free" href="https://j5.jbei.org/j5manual/pages/21.html">https://j5.jbei.org/j5manual/pages/21.html</a> GFP (BFP?) transformation today! - GFP or BFP? GFP! - just a warmup, or will we be able to use this construct together with the MAA pathway? Take notes on all experiments in group notebook and person notebooks. Lookinto benchling.com There are several deadlines this month. Be sure that your team meets the following: August 07: Track selection August 07: Title and abstract August 28: Final Safety Form Meeting 7/27/15 Attendees <ul><li> Biocurious: Eric H., Maria T. Chavez, Patrik, Eric A, Adarash, James, Rikke, Victoria, Dmitry, Jay, Erik I, Greg, Shreya, Michael, Samera, David, Meekakshi, Johan </li></ul> General Eric’s talk on RCA/RCR - Rolling circle amplification, Rolling circle replication A way for viruses to replicate their DNA, a compact way to replicate their DNA needing only 1 strand of DNA. Uses enzyme phi 29. A way to produce lots of DNA at a low temperature. Need a circle, need an initiation point (a single stranded circle), 529 will initiate this at the lesion in a double stranded DNA. Phi 29 has a fidelity for doing this that is unreplicated so far from bacillus setellus (???) RCA is not old technology, it was developed to make copies, for Sanger sequencing. To make a lot of copies of something that is a replication. Make a copy of a whole circle, strand replace and start again making a new circle of DNA. Pic of process - <a rel="nofollow" class="external free" href="https://en.wikipedia.org/wiki/Rolling_circle_replication">https://en.wikipedia.org/wiki/Rolling_circle_replication</a>. For our process is to NOT use phi 29 to introduce mutagenesis. DNB is a DNA nanoball. Eric today took M13 and put a nick in it to use it today. MB-BSM1 is an asymmetric cutting enzyme and will make a nick in the DNA either top strand (MB is the bottom strand). Eric will post Protocol used for tonight. Meeting 7/25/15 Attendees <ul><li> Biocurious: David M, Eric H, Jay, Phil, James, lafia, Meenakshi </li><li> CCL: Patrik, Andrew, Ramsel, Catherine (catherinesoneda@sbcglocal.net), Kye, Laurent (lorantto@gmail.com) </li><li> Zoom: Adarsh, David Hou, Shreya T, Rikke, Milo </li></ul> Plasmid.com for preparing 1 mg of plasmid for $99 <a rel="nofollow" class="external free" href="http://www.plasmid.com/">http://www.plasmid.com</a> When are we doing the GFP experiment? Antonio Lamb (Amlamb@ucsc.edu), Jay, David, Rikke, David Hou, Shreya Eric can teach a class on RCA: this Monday or Wednesday? PIPE cloning papers: <a rel="nofollow" class="external free" href="http://technology.sbkb.org/portal/page/334/">http://technology.sbkb.org/portal/page/334/</a> <a rel="nofollow" class="external free" href="http://www.ncbi.nlm.nih.gov/pubmed/18988020">http://www.ncbi.nlm.nih.gov/pubmed/18988020</a> Growing mutator strain without accumulating mutations in chromosome (see section 3.1): <a rel="nofollow" class="external free" href="http://www.chemengr.ucsb.edu/~ceweb/faculty/daugherty/images/9-nguyen.pdf">http://www.chemengr.ucsb.edu/~ceweb/faculty/daugherty/images/9-nguyen.pdf</a> Fundraising Andrew, Catherine, Laurent, David Hou Eric contacted NEB, will follow up with their iGEM coordinator and other contacts at NEB adarsh: contact Agilent for directed evolution reagents. Agilent does not offer discounts for iGEM Eric: just need cloning kit &amp; buffers for RCA Gene designs Anyone to contact Minnesota 2012 team (Jay) Link to Minnesota 2012 iGEM Team UV-Protective Compounds : <a rel="nofollow" class="external free" href="http://2012.igem.org/Team:Minnesota/Project/UV_Absorption">http://2012.igem.org/Team:Minnesota/Project/UV_Absorption</a> Need to finalize gene designs, including any flanking sequences Milo, Jay, Patrik Look into possible DNA assembly methods: <ul><li><ul><li>Gibson </li><li>Golden Gate </li><li>Golden Braid </li><li>Infusion Cloning - Clonetech </li></ul> </li></ul> Other Genes: <dl><dd>-UV sensitive promoters? (see also SulAp) </dd><dd>-reporter genes? </dd></dl> UV spectra and hardware mycosporines absorb in UVA (334nm) GFP absorbs in UVA (395nm) we have various UV illuminators (320nm, 365nm) - copy out some model numbers, and look up spectrum <ul><li><ul><li>Stratagene crosslinker </li></ul> </li></ul> More research on killing E. coli with UVA, UVB, sunlight - what doses at what wavelengths <dl><dd>-Rikke, Patrik </dd></dl> UV Sources: <ul><li>At Biocurious: “Strategene crosslinker” - has replaceable / swappable UV bulbs, so will need to know what type of bulb is in there to determine radiation specs </li><li> Actual crosslinking is usually done with a very short-wave, very narrow-band UVC bulb (254nm), which would not be suitable for our purposes </li><li> Manual says it also has 302nm Midrange (UVB) bulbs (# 34-0042-01) and 365 nm Longwave (UVA) bulbs (# 34-0006-01), </li></ul> UVP Cross linker Midrange 302nm UV:40 watts <a rel="nofollow" class="external free" href="http://uvp.com/crosslinker.html">http://uvp.com/crosslinker.html</a> Midrange 302nm UV:40 watts <ul><li> <a rel="nofollow" class="external free" href="http://www.uvp.com/pdf/302wf.pdf">http://www.uvp.com/pdf/302wf.pdf</a> </li><li> <a rel="nofollow" class="external free" href="http://www.uvp.com/pdf/302.pdf">http://www.uvp.com/pdf/302.pdf</a> </li></ul> Meeting 7/18/15 Attendees <ul><li> Biocurious: Priyanka,jon,Eric Aker,Phillip, Audrey, April, Jay, David M, Adarsh, Johan </li><li> CCL: Patrik, Vishnu, Lorent, Andrew, Kye, Sean, Nathan </li><li> Zoom: Antonio, Rikke, Victoria, Advait </li></ul> Fundraising (experiment.com) Maria, Patrik Initial Experiment Antonio Lamb (Amlamb@ucsc.edu), Jay, David, Rikke, David Hou Eric can teach a class on RCA New People Email(CCL): <ul><li>Andrew.g.elkins@gmail.com </li><li>lorantto@gmail.com </li><li>eic@mycopathologia.net </li><li>nathan.eisenberg@att.net </li></ul> Meeting 7/11/15 Attendees <ul><li> Biocurious: Priyanka,jon,Eric Aker,Phillip, Audrey, April, Jay, David M, Adarsh, Johan </li><li> CCL: Patrik, Ramsel, Jay, Kye </li><li> Zoom: Maria, David Hou, Milo Toor, Rikke, Elsa,Victoria </li></ul> Please fill out the biography form - <a rel="nofollow" class="external free" href="https://docs.google.com/forms/d/1D_fcWZVLaq6O3WoKQCFjSdUaI6MODaEFjqO_hauagxU/viewform?usp=send_form">https://docs.google.com/forms/d/1D_fcWZVLaq6O3WoKQCFjSdUaI6MODaEFjqO_hauagxU/viewform?usp=send_form</a> Fundraising <ul><li> iGEM registration fee $4000, bio bricks kit, and IDT discount for ordering DNA </li><li> NEB has gotten back to us and is NOT offering community labs the discount for iGEM ** We should follow up and talk to them about this </li><li> Options for fundrasing - Crowdsource funding through Indiegogo, Experiment.com, gofundme, sponsorships, self funding </li><li> Need to build a budget, each subgrup to let us know a very general cost, and what materials we need ordered (so we can look into getting supplies donated </li></ul> General  Which genes do we need to order? <a rel="nofollow" class="external free" href="http://2015.igem.org/Sponsors/IDT*">http://2015.igem.org/Sponsors/IDT*</a> *Teams will receive value equivalent to twenty 1000 bp gBlocks Gene Fragments in their local currency. Promotion expires September 24, 2015.  Page with intro to gBlocks video: <a rel="nofollow" class="external free" href="http://www.idtdna.com/pages/landing/igem-2015">http://www.idtdna.com/pages/landing/igem-2015</a> <ul><li> Shinorine pathway: <ul><li> mycosporine-glycine pathway from Minnesota 2012 team is NOT available; need to reorder! Anabaena variabilis Ava_3855 to 3858 </li><li> We could also order Nostoc punctiforme NpF5597 to 5600 as an alternative, and codon optimized versions of both </li><li> The Minnesota team could not get Ava_3855 to work, but we've identified 9 other enzymes that can do the final step in the pathway </li></ul> </li><li> pABA biosynthesis <ul><li> BBa_K137055 from Caltech 2008 (promoter + RBS + PabA; IN 2015 IGEM KIT!) </li><li> BBa_K909014 from Zurich 2012 (P + RBS + PabB + RBS + PabA + term; IN IGEM 2015!) </li><li> express antisense RNA for pABA consuming enzyme? </li></ul> </li><li> GFP <ul><li> What is the most favorite GFP biobrick? </li></ul> </li><li> UV sensitive promoters <ul><li> BBa_J22106 RecA SOS promoter (in stock, not on distribution) </li><li> What else? </li></ul> </li><li> Any reporter genes (other than GFP!? </li></ul> Need someone to look into UV light spectrums - could look into what UV lights we should be using (LED's that are mostly UVA, braod spectrum UV light) What spectrum, what hardware, what level of elimination to kill e-coli, what has been used by other labs - [Rikke] Directed Evolution Update -   Ethical, Legal, Social Issues  April and Audrey will dig more into the issues around PABA  To-Do <ul><li> directed evolution look up $ estimates for experiments w mutator strain vs RCA </li><li> come up with concrete list of genes to order from IDT </li><li> transfer all notes to Wiki: <a rel="nofollow" class="external free" href="http://bayareaigem.herokuapp.com/">http://bayareaigem.herokuapp.com/</a> </li></ul>  Meeting 7/4/15   Attendees <ul><li> Biocurious: Maria, David M., Priyanka, Pam, April, Jeff, </li><li> CCL: </li><li> Zoom: Advait, Victoria, </li></ul> 96 well plates price link: <a rel="nofollow" class="external free" href="https://www.sigmaaldrich.com/catalog/product/sigma/p2487?lang=en&amp;region=US">https://www.sigmaaldrich.com/catalog/product/sigma/p2487?lang=en&amp;region=US</a> RCA kit price link: <a rel="nofollow" class="external free" href="http://www.sigmaaldrich.com/catalog/product/sigma/ge25640010?lang=en&amp;region=US">http://www.sigmaaldrich.com/catalog/product/sigma/ge25640010?lang=en&amp;region=US</a> Reminder for using Slack - check in on threads folks are supposed to be working on Updates iGEM Stuff/Deadlines: <ul><li> <a rel="nofollow" class="external free" href="http://2015.igem.org/Main_Page">http://2015.igem.org/Main_Page</a> </li><li> <a rel="nofollow" class="external free" href="http://2015.igem.org/Calendar_of_Events">http://2015.igem.org/Calendar_of_Events</a> </li><li> Judging info: <a rel="nofollow" class="external free" href="http://2015.igem.org/Judging">http://2015.igem.org/Judging</a> </li><li> Register to attend jamboree by July 31 </li><li> Project description: <a rel="nofollow" class="external free" href="http://2015.igem.org/Safety/About_Our_Project">http://2015.igem.org/Safety/About_Our_Project</a> </li><li> Future dates: <ul><li> Title and abstract (Aug 7) </li><li> Final Safety form (Aug 28) </li><li> Team rosters finalized (September 11) </li><li> Judging form, Part Submission, Wiki freeze (September 18) </li><li> Jamboree (September 24) </li></ul> </li></ul> UV Compounds: <ul><li> Ran organism specific BLAST for protein homologs; see google drive folder/slack for results (shinorine &amp; porphyra-334 enzymes) </li><li> <a rel="nofollow" class="external free" href="https://drive.google.com/folderview?id=0Bx0m7X6dHC0mflJOeDR3R3U4Wldya0ZJRzZ3N01IUXlnVXZwOHQwYW5ud1p5dEpnRlJvWkk&amp;usp=sharing">https://drive.google.com/folderview?id=0Bx0m7X6dHC0mflJOeDR3R3U4Wldya0ZJRzZ3N01IUXlnVXZwOHQwYW5ud1p5dEpnRlJvWkk&amp;usp=sharing</a> </li></ul> Directed Evolution - RCA (rolling circle amplification) requires the least work, with the nutator strain we will have to transform the bacteria two times. It will take alot more time. <ul><li> next step is to develop an experiment to verify that RCA is the way to go, using GFP and ecoli, after class on biobricks, best result can be used for initial experiments </li><li> thread needs to set up on Slack about a bio bricks class - </li></ul> Previous BioBricks parts <ul><li> need more members researching. Will email list. Victoria, and April will help </li><li> <a rel="nofollow" class="external free" href="https://pad.riseup.net/p/pastigemsunscreen">https://pad.riseup.net/p/pastigemsunscreen</a> </li></ul> <ul><li> iGEM - maria will work on making Advait an instructor, and confirm we have finished About Our Team </li><li> Survey of Members - Maria will send out to get member bios </li><li> Confirm who has taken safety training </li><li> Registration for Jamboree ends July 30th </li><li> For next week set our timelines based on iGEM dates. </li><li> Find out how to get NEB pricing (Adarsh sent them email this past week) to price out costs for directed evolution. </li><li> Next week priority will be budget </li></ul> Meeting 6/27/15 Call-in info : <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> or go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and and enter meeting ID: 820 128 495 Attendees <ul><li> Biocurious:Maria, David H., Eric H., Johan, Audrey, Adrash, Brian, Ryan </li><li> CCL: Patrik, Eric A., Solna, Victoria, Erik I. (einnocent@gmail.com), April (HS student), Jeff, Pam, Goldie, Dana </li><li> Zoom: Anja, Nathan, David, Advait, Vicky, David M., Vardhaan,Shreya, Maria Theresa, Rikke </li></ul> Membership code (to join team): bf9bfd General Forms which may be due? Will check on igem registration - Need to add other instructors to Igem team list - Maria, Eric, Advait Need to send out iGEM team member background and bio survey - Maria UV protective compound homework: <ul><li> found analogs for two genes - </li><li> Patrik is planning to do a quick practical bioinformatics intro soon, focusing on BLAST </li></ul> Directed Evolution group <ul><li> We have a list of projected maerials and costs on google doc </li><li> two to three days to have initial varations and diversity, and how many generations can we fit in before iGEM </li><li> approach a company who makes a kit for donations or support - Needs follow up and more info </li><li> to do - fix how many high thruput plates? 96 well plates? </li><li> Literature search of mutators strains, RC strain - </li><li> Have we choosen the pathway? GFP (some UV protection) </li></ul> <ul><li> should do an intro to working with biobricks class, insert gfp into plasmid, do a Gibson assembly (Advait, Maria, Johan, Shreya, Vardhaan,Audrey, Adarsh, Nathan, David M) </li><li> Safety Training quiz to all who havent already taken and passed - Maria will send out. </li><li> Drive by Safety Training for all at this meeting! </li><li> Wednesday 6pm official/makeup cant take too much safety </li></ul> iGEM parts - <ul><li> <a rel="nofollow" class="external free" href="https://pad.riseup.net/p/pastigemsunscreen">https://pad.riseup.net/p/pastigemsunscreen</a> </li></ul> Ethical, Legal, Social Issues <ul><li> More PABA research to writue up (Maria) </li><li> Another question about UV radiation as a sterilization technique is there an issue with creating UV/ sterilization resistant strain that could pass on issues to pathogenic strains? </li><li> some sunblock compounds damage corals (big issue with snorkelers); natural compounds like mycosporine that come from algae or coral should be much better for coral ecosystems <ul><li> Other environmental effects? </li><li> Human toxicity/disease effects? </li></ul> </li></ul> Marketing - Logo Design <ul><li> Will send out and vote (who?) </li></ul> Drive by Safety Training - Eric Harness (Formal on Wednesday at 6pm) <ul><li> BSL1 labs (all three labs) </li><li> We wear gloves in the lab for protection, product and experiment protection, protects your experiment from you, if you work with acids or bases </li><li> no loose fitting clothing, should be longer than knee length </li><li> no sandles, must be closed toed, no heels, not evne a 1" heel, dont want folks to slip and fall </li><li> Tie back long hair! </li><li> Eating and drinking - NO EATING AND DRINKING IN THE LAB! This includes chewing gum </li><li> Most dangerous equip in lab - Centerfuge (must be balanced, if not then they wobble and it can cause accidents), Autoclave (requires special traning, be careful handling hot items, things are hot and under pressure), Hood (Careful of open flames in the hood, remember its to protect your experiment from you not you from your experiment) </li><li> Communicate with others in the lab, ask if you dont know. </li><li> BioHazardous waste - red bin at Bioc (any waste that has touched bacteria, tips, media, agar) it gets picked up, put something in to absorb liquids before putting it in the bag, paper towels etc.), when bleacking it needs to be 10% bleach and minimum contact time 10 mins, spore bacteria then autoclave can autoclave in bleach solution, should always be freshly made or it will lose strength </li><li> Broken Glass - BioC has a container if free of biohazard, if biohazaard but in cardboard container then add to red bag, pick up with tweezers and then steralize them with bleach </li><li> Chemical safety, if you are working with chemicals let the safety committee know what you are bringing in and how to work with them, might need PPE </li><li> Last - if we use agar, please dont dump it down the drain that makes a huge mess </li></ul> BioInformatics <a rel="nofollow" class="external free" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3194895/figure/F3/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3194895/figure/F3/</a> Nostoc punctiforme ATCC 29133 NpF5597* * ATP-grasp; D-ala D-ala ligase homolog* ACC83902 Anabaena variabilis ATCC 29413 Ava_3855* * NRPS-like*   ABA23460 <a rel="nofollow" class="external free" href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> Meeting 6/20/15 Attendees <ul><li> Biocurious - Priyanka, David, Adarsh, Audrey, Eric, Clara, Jason, April, Reena, Phil, James(jwantuck@gmail.com), Charlie, Jerry </li><li> CCL - Patrik, Milo, Sean, Anthony(rushanthony@gmail.com), Sasha </li><li> Zoom- Advait, Shreya, Vardhaan, Maria </li></ul> REIGSTER FOR iGEM 2015: <a rel="nofollow" class="external free" href="http://igem.org/IGEM_2015_Registration">http://igem.org/IGEM_2015_Registration</a> Slack address: <a rel="nofollow" class="external free" href="https://bayareaigem.slack.com/">https://bayareaigem.slack.com</a> To-Do UV protective compound homework: <ul><li> absorbtion spectra for UV-absorbing compounds (what frequencies, how wide is the spectrum for each): mycosporine-glycine, shinorine, porphyra-334, DHQ, 4-DG, PABA </li><li> shinorine / porphyra-334 - identify homolog genes in the shinorine/porphyra-334 orgs using BLAST <a rel="nofollow" class="external free" href="http://blast.ncbi.nlm.nih.gov/">http://blast.ncbi.nlm.nih.gov/</a> </li><li> PABA - look into previous iGEM projects </li></ul> Directed evolution homework: <ul><li> research and report various directed evolution methods that may be applicable to our needs </li><li> for promising methods, report protocols including expenses, materials, time needed </li></ul> iGEM parts <ul><li> which ones are actually available? (Reena)  - see latest update at <a rel="nofollow" class="external free" href="https://pad.riseup.net/p/pastigemsunscreen">https://pad.riseup.net/p/pastigemsunscreen</a> - results of search - several biobricks are labelled as "planning", majority are labelled "unavailable", only one marked as "available". Refer to link for detailed notes. </li></ul> Ethical, Legal, Social Issues <ol><li> Why was PABA banned (at least from sunscreens) in the EU? - (James, Maria, Phil) </li><li> some sunblock compounds damage corals (big issue with snorkelers); natural compounds like mycosporine that come from algae or coral should be much better for </li><li> coral ecosystems </li><li> Other environmental effects? </li><li> Human toxicity/disease effects? </li></ol> Logistics team members join iGEM team officially safety training gibson assembly class? !!Please collect emails of new people and put them here so I can invite to slack.com and the Google group, etc!! (jwantuck@gmail.com) Reena Nagpal - RNagpal@me.com &lt;-- both invited Updates on research <ul><li> UV protective compounds (David) <ul><li>Consider doing GFP as well! (WT excitation max at 395nm) </li><li>Plan of action: <ul><li> DHQ (dehydroquinate) —&gt; 4-DG (4-deoxygadusol) —&gt; mycosporine-glycine —&gt; shinorine / porphyra-334k </li></ul> </li><li>4-DG and mycosporine-glycine already have UV absorption! </li><li>Previous team was able to make mycosporine-glycine, but not shinorine or porphyra-334 </li><li>Definite producers of shinorine / porphyra-334: <ul><li> Chondrus yendoi (shinorine) </li><li> Mytilus galloprovincialis (both) </li><li> Porphyra tenera (porphyra-334) </li></ul> </li></ul> </li><li> Directed evolution (Adarsh) <ul><li>Did anyone talk with Glowing Plant group yet? Who will commit to doing that? </li><li>Tasks: <ul><li> Find recent review article on directed evolution to learn newest techniques </li><li> What is a typical protocol for directed evolution? What does it require from the team? </li></ul> </li><li>Excellent review: Random Mutagenesis Methods for In Vitro Directed Enzyme Evolution Required reading! </li><li>Rolling Circle Error-Prone PCR seems like a promising technique, since it introduces mutations specifically into the plasmid. </li><li>See also One-step random mutagenesis by error-prone rolling circle amplification </li></ul> </li><li> Previous iGEM teams &amp; available biobricks (Advait) <ul><li><a rel="nofollow" class="external free" href="https://pad.riseup.net/p/pastigemsunscreen">https://pad.riseup.net/p/pastigemsunscreen</a> </li><li>Need more people working on this </li><li>Future steps: go through all of the biobricks </li><li>Find if they work and are available in the distribution kit, can be ordered, or do not work </li></ul> </li></ul> iGEM logistics All iGEM teams must complete the About our Lab and About our Project questionnaires by June 26! Tell us about your laboratory and your primary project idea. <a rel="nofollow" class="external free" href="http://2015.igem.org/Safety/About_Our_Lab">http://2015.igem.org/Safety/About_Our_Lab</a> <ul><li> Maria wrote and submitted </li></ul> <a rel="nofollow" class="external free" href="http://2015.igem.org/Safety/About_Our_Project">http://2015.igem.org/Safety/About_Our_Project</a> Need to schedule a hands-on workshop on Gibson assembly and biobricks soon (Anthony can hgelp - rushanthony@gmail.com) Meeting 6/13/15 Call-in info : <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> or go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and and enter meeting ID: 820 128 495 Attendees <ul><li> Biocurious- Priyanka, David M,Eric, Charlie, Adarsh, Verdan, Audrey, John, Eva, Phil, James, April, Greg </li><li> CCL- Patrik </li><li> Zoom- Advait, Milo, David, Jeff, Shreya, Maria Teresa, Johan </li></ul> Updates on research <ul><li> mycosporine and other UV protective compounds (David leads; Thomas, Eric Aker, David M, Nathan Zhang) <ul><li> This team tried something similar to our project: <a rel="nofollow" class="external free" href="http://2012.igem.org/Team:ETH_Zurich/Project_overview">http://2012.igem.org/Team:ETH_Zurich/Project_overview</a> </li></ul> </li><li> Directed Evolution (Adarsh leads; Advait, David M,Thomas, Shreya, Eric H., priyanka, Charlie, Johan, Milo, Nathan, Greg) <ul><li>Oxford iGEM 2014 used directed evolution in their project </li><li> <a rel="nofollow" class="external free" href="http://2014.igem.org/Team:Oxford/directed_evolution">http://2014.igem.org/Team:Oxford/directed_evolution</a> </li><li> Adarsh - look at previous directed evolution teams </li><li> Eric took people on a visit at DNA2.0 <ul><li> combinatorial assembly to mix and match pieces </li><li> mutagenic PCR to introduce more diversity </li><li> oxford team 2014, UC Davis 2012, UChicago 2014, used this (find more teams through iGEM team seeker </li><li> use fluorescent or chromogenic proteins as reporter: <a rel="nofollow" class="external free" href="https://www.dna20.com/products/protein-paintbox">https://www.dna20.com/products/protein-paintbox</a> </li></ul> </li></ul> </li><li> iGEM teams and biobricks (Advait leads; Adarsh, David Hou, Charlie Huang, David M., Patrik, Victoria, Vardhaan) <ul><li> <a rel="nofollow" class="external free" href="http://igem-qsf.github.io/iGEM-Team-Seeker/dist/">http://igem-qsf.github.io/iGEM-Team-Seeker/dist/</a> </li><li> Adarsh - look at previous directed evolution teams </li><li> Minnesota has a large collection of relevant biobricks - <a rel="nofollow" class="external free" href="http://2012.igem.org/Team:Minnesota/Project/UV_Absorption">http://2012.igem.org/Team:Minnesota/Project/UV_Absorption</a> </li></ul> </li></ul> WIKI: <ul><li> <a rel="nofollow" class="external free" href="http://bayareaigem.herokuapp.com/">http://bayareaigem.herokuapp.com/</a> </li></ul> Meeting 6/6/15 Call-in info: <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> Or, go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and enter meeting ID: 820 128 495 Attendees <ul><li> BioCurious -Priyanka,jerry,charlie,David, Eric A, Eric H (no please send zoom id - 820 128 495, Joh </li><li> CCL - Sean, Kai (HS senior at College prep) </li><li> Zoom - Maria, Adarsh, Ilya, Thomas, David,NATHAN, Shreya, </li></ul> Agenda  And the winner is.......  Let's form a couple of sub-teams to work on different aspects of the project. <ol><li> more mycrosporine research on bio chemistry (Thomas, Eric Aker, David M,Nathan Zhang) </li><li> a separate team researching how to do the evolution experiments - that's something that could be done entirely separate from the synthetic biology work. Maybe talk with the folks at Glowing Plant - they've been using directed evolution to optimize their bioluminescence output! (David M,Thomas, Shreya, Eric H., priyanka, Charlie). Look into other iGEM teams who have used directed evolution as well! </li><li> Get list of iGEM teams who have worked on relevant projects, including mycosporine/shinorine, PABA, UV sensors, etc. For each of those teams, check their wiki to see how far they managed to take their project (may require digging into their experiment notebooks!) (Adarsh, David Hou, Charlie Huang, David M.) <ol><li> Check what biobricks they've entered in the repository, and what the status of those biobricks is (not available, available from iGEM, or included in 2015 DNA distribution?) <ol><li> <a rel="nofollow" class="external free" href="http://parts.igem.org/Main_Page">http://parts.igem.org/Main_Page</a> </li><li> <a rel="nofollow" class="external free" href="http://parts.igem.org/Catalog">http://parts.igem.org/Catalog</a> </li><li> <a rel="nofollow" class="external free" href="http://parts.igem.org/Catalog#Browse_parts_and_devices_by_function">http://parts.igem.org/Catalog#Browse_parts_and_devices_by_function</a> </li></ol> </li></ol> </li><li> Begin building the team wiki which site to use (media wiki - sean, google sites, medium, synbiota) (Maria, Eric Aker, Shreya) </li><li> signup everyone on the igem site (Maria, Patrik, Advait) </li><li> Rough outline of goals with due dates with iGEM timeliens and milestones (Maria, </li><li> build a questionaire for member bios to put on the wiki (Maria) </li></ol> <a rel="nofollow" class="external free" href="https://docs.google.com/document/d/1K8jbQY9_NuRrR_gaVLxLtee262zZTU9Aipf6nB_zqas/edit#headinge=h.mco3u3oj2jsp">https://docs.google.com/document/d/1K8jbQY9_NuRrR_gaVLxLtee262zZTU9Aipf6nB_zqas/edit#headinge=h.mco3u3oj2jsp</a> To-Do  - Focus on budget for project - Begin planning funding (crowd source or otherwise) - Marketing ideas for a catchy name to make logos - Safety training (july 11th for eric to run find someone else otherwise)  Meeting 5/30/15   Attendees <ul><li> BioCurious - David, Priyanka, Johan, John, Nathan, </li><li> CCL - Patrik, Maria, Kye </li><li> Zoom - Adarsh, Laura, Shreya, Rikke,shashank, Milo, Vardhaan </li><li> phone - Maria </li></ul>  Agenda  new google drive folder with the 3 one-slide presentations: <ul><li> <a rel="nofollow" class="external free" href="https://drive.google.com/folderview?id=0Bx0m7X6dHC0mfkdZNEV1SU5KUUhQeGlOT3RnUFQxcHdjWnhSX3BpcTFqenduU1NESVo3X2M&amp;usp=sharing">https://drive.google.com/folderview?id=0Bx0m7X6dHC0mfkdZNEV1SU5KUUhQeGlOT3RnUFQxcHdjWnhSX3BpcTFqenduU1NESVo3X2M&amp;usp=sharing</a> </li></ul> voting system - how to record votes We could use this form <a rel="nofollow" class="external free" href="http://goo.gl/forms/G2SEOifv7a">http://goo.gl/forms/G2SEOifv7a</a> <ul><li> can one vote for more than one project </li><li> anonymous or visible voting </li><li> 72 hour voting window ok? </li></ul> Nnext steps <ul><li> put together a skills list for each member a survey of who, background, availability, skills </li><li> begin a simple charter of purpose for group </li><li> schedule a safety training session for iGEM team members (Eric H. Or others to teach) </li></ul> Meeting 5/23/15 Attendees <ul><li> BioCurious - Maria, Charlie (13yo, 8th grade Miller Middle School, Simon (software eng), Nathan (8th grade, Miller), </li><li> CCL - Patrik, Don (Arcturus Biocloud, iGEMx2), Alex, Kye, Tom (BricoBio) </li><li> Zoom - Jeff, Vardhan, Advait, Johan, Adarsh, Laura, Johan, Milo, Shreya </li></ul> Agenda <ul><li> Review of Project </li><li> Overview of iGEM competition </li><li> Proposed to do a 1 page with a template from Patrik </li></ul>  Projects <ul><li> A Alergen detection with antibody attached to receptor </li><li> TEAM - Milo, Adarsh, Patrik, Nathan, Kye </li><li> B Alergen detection of with fluorescent aptamers </li><li> TEAM - Ilya, Johan, Vardhaan, Shreya, Charlie </li><li> C - UV Expression of Mycrosporine </li><li> TEAM - Shahsank, Patrik, David, Don, Jeff, Tom, Advait </li></ul>  Meeting 5/9/15 <ul><li>Call-in info: <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> Or, go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and enter meeting ID: 820 128 495 </li></ul> Attendees <ul><li> BioCurious - Maria, Priyanka, Adarsh(bioengineering at Santa Clara), Anja </li><li> CCL - Patrik, Rikke, Sean </li><li> Zoom - Advait, Ilya (BioEngineering), David (Gunn HS), Jeff, Shreya, Japna,milo </li></ul> NO MEETING NEXT WEEK - May 16! Agenda Project road maps: <ul><li> <a rel="nofollow" class="external free" href="https://docs.google.com/document/d/1K8jbQY9_NuRrR_gaVLxLtee262zZTU9Aipf6nB_zqas/edit#heading=h.k4xtoch10wib">https://docs.google.com/document/d/1K8jbQY9_NuRrR_gaVLxLtee262zZTU9Aipf6nB_zqas/edit#heading=h.k4xtoch10wib</a> </li></ul> p-coumaric acid pathway is already well worked out. Our role could be to optimize production, or find an interesteing compound we can make from it. See: <a rel="nofollow" class="external free" href="http://en.wikipedia.org/wiki/Aroma_compound">http://en.wikipedia.org/wiki/Aroma_compound</a> Narrowed down the allergen detection project to two approaches: <ul><li> linking antibody (or aptamer) binding to to a cell signalling response <ul><li> possible approach: blocking an existing receptor (e.g. involved in chemotaxis) by binding to allergen </li></ul> </li><li> making DNA or RNA aptamers <ul><li> How do we make this a synthetic biology project? </li><li> How do we use the aptamer, once we have something that binds the target? </li></ul> </li></ul> Promising target: Casein! Cow's milk protein allergy (could we distinguish cow casein vs goat casein?) Utilize milk allergen Need to organize a meetup after Maker Faire to onboard any new people: <ul><li> intro to synthetic biology </li><li> what is iGEM, what resources are available </li><li> our top two project ideas: detecting allergens; UV resistance </li></ul> Need group to work on onboarding: Maria, Jeff, Sean, Advait UV team: Shashank, Advait, Patrik Allergen team: Ilya, Rikke Google Doc for Maker Faire: <ul><li> <a rel="nofollow" class="external free" href="https://docs.google.com/spreadsheets/d/1vR7Suk8gTvNFb7B6-r_Jsjez8AXTHGQ3RI0RW43DaDU/edit#gid=0">https://docs.google.com/spreadsheets/d/1vR7Suk8gTvNFb7B6-r_Jsjez8AXTHGQ3RI0RW43DaDU/edit#gid=0</a> </li></ul> For next meeting - start at 10am for onboard members, syn bio presentation, presentation of two final ideas, voting, housekeeping Meeting 5/2/15 <ul><li>Call-in info: <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> Or, go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and enter meeting ID: 820 128 495 </li></ul> Attendees <ul><li> BioCurious - Maria, Johan, Priyanka, Jay, John McGowen, David, Advait </li><li> CCL - Patrik, Rikke, Jeff Morris </li><li> Zoom - Ilya, Nikola, Sean, Adrash, Shreya, David Hou, Emi, Shashank </li></ul> General Need a link to the iGEM medal standards <a rel="nofollow" class="external free" href="http://2015.igem.org/Judging/Medals">http://2015.igem.org/Judging/Medals</a> - medal requirements Make a flyer for MakerFaire explaining about iGEM - Shreya, Advait, Priyanka Specific medal requirements for Community lab track: <ul><li> The medal requirements have changed from last year (interaction with another iGEM team is not required anymore for community track, but still required for normal track). </li><li> Video still required for gold (like the crowdfunding videos we showed at the Jamboree last year) </li></ul> Community Labs Bronze - Your team must convince the judges you have achieved the following 6 goals: <ul><li> Register for iGEM, have a great summer, and attend the Giant Jamboree. </li><li> Complete the Judging form. </li><li> Create a Team Wiki. </li><li> Present a poster and a talk at the iGEM Jamboree. See the 2015 poster guidelines for more information. </li><li> Create a page on your team wiki with clear attribution of each aspect of your project. This page must clearly attribute work done by the students and distinguish it from work done by others, including host labs, advisors, instructors,sponsors, professional website designers, artists, and commercial services. </li><li> Interact with your community by creating an engaging activity that showcases iGEM, synthetic biology and community labs. This activity could involve teaching a class to your community, running a public forum to promote the discussion of synthetic biology, or another activity. </li></ul> Silver - In addition to the Bronze Medal requirements, your team must convince the judges you have achieved the following 3 goals: <ul><li> Help any registered iGEM team from a high-school, different track, another university or institution by, for example, mentoring a new team, characterizing a part, debugging a construct, modeling/simulating their system or helping validate a software/hardware solution to a synbio problem. </li><li> Demonstrate a functional prototype of your project OR a substantial improvement on a previous iGEM project. You can work on a previous project by your team or by another team. Show this system working under real-world conditions (biological materials may not be taken outside the lab). </li><li> iGEM projects involve important questions beyond the bench, for example relating to (but not limited to) ethics, sustainability, social justice, safety, security, and intellectual property rights. Demonstrate how your team has identified, investigated and addressed one or more of these issues in the context of your project. Your activity could center around education, public engagement, public policy issues, public perception or other activities (See the human practices hub for more information and examples of previous teams exemplary work). </li></ul> Gold - In addition to the Bronze and Silver Medal requirements, your team must convince the judges you have achieved at least two of the following goals: <ul><li> iGEM projects involve important questions beyond the bench, for example relating to (but not limited to) ethics, sustainability, social justice, safety, security, and intellectual property rights. Expand on your silver medal activity by demonstrating how you have integrated the investigated issues into the design and/or execution of your project OR demonstrate an innovative human practices activity that relates to your project (this typically involves educational, public engagement, and/or public perception activities; see the human practices hub for information and examples of previous teams comprehensive and innovative activities). </li><li> Document at least one new standard BioBrick Part or Device central to your project and submit this part to the iGEM Registry (submissions must adhere to the iGEM Registry guidelines). You may also document a new application of a BioBrick part from a previous iGEM year, adding that documentation to the part main page. </li><li> Interact with your community by creating an engaging activity that showcases iGEM, synthetic biology and community labs. This activity could involve teaching a class to your community, running a public form to promote the discussion of synthetic biology, or another activity. Take the activity you came up with for your bronze medal and create a video showing how you interacted with your community. </li><li> Experimentally validate that at least one new BioBrick Part or Device of your own design and construction works as expected. Document the characterization of this part in the Main Page section of that Part’s/Device’s Registry entry. This working part must be different to the part documented in gold medal criteria #2. </li></ul> Deadlines (iGEM) <ul><li> No deadline - IDT giving 20kB DNA free: <ul><li> <a rel="nofollow" class="external free" href="http://2015.igem.org/Sponsors/IDT">http://2015.igem.org/Sponsors/IDT</a> </li></ul> </li><li> May 15 SYNERGENE is helping other teams: "Teams working on biorefineries, intellectual property or other related open science issues are especially encouraged to apply!" <ul><li> Proposals due May 15th </li><li> <a rel="nofollow" class="external free" href="http://2015.igem.org/SYNENERGENE_Calls_for_Proposals">http://2015.igem.org/SYNENERGENE_Calls_for_Proposals</a> </li></ul> </li></ul> <ul><li> May 27 SimBiology and MATLAB for Modeling Synthetic Biology Systems <ul><li> MathWorks will host a webinar to provide iGEM teams with an introduction to modeling, simulation and analysis with MATLAB and SimBiology using an example from synthetic biology literature </li></ul> </li></ul> <ul><li> June 26 Safety Forms due </li><li> <a rel="nofollow" class="external free" href="http://2015.igem.org/Safety">http://2015.igem.org/Safety</a> <ul><li> Fulfill the safety requirements for iGEM participation: </li></ul> </li><li> April-August: Review your organisms &amp; parts against the White List / submit any necessary Check-Ins </li><li> June 26: About Our Lab questionnaire due </li><li> June 26: About Our Project questionnaire due (and remember to update when your primary project idea changes!) </li><li> August 28: Final Safety Form due </li><li> September 18 Wiki freeze <ul><li> Cannot edit wiki anymore </li><li> Parts due to registry </li></ul> </li></ul> Cancelling meeting on 5/16 for MakerFaire if you want to volunteer please mail maria@biocurious.org Detection of foods or allergens - Broadly, 4 different approaches: <ul><li> Link antibodies to a signalling pathway, so a genetic circuit can detect binding and produce an intelligent response </li><li> Making fluorescent / colored antibodies (very mainstream approach) (ruled out) </li><li> Making DNA (or RNA) aptamers </li><li> Making peptide aptamers with phage display </li></ul> Need to make 1-page flyer about iGEM. - Shreya? Meeting 4/25/15 <ul><li>Call-in info: <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> Or, go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and enter meeting ID: 820 128 495 </li></ul> Attendees <ul><li> BioCurious - Jay, Anitha, Maria, David, Advait, Priyanka </li><li> CCL - Sean, Patrik </li><li> Zoom - Shashank, Johan, Laura, Adarsh, Sean, Shreya, Emi, Nikola, Japna, Milo </li></ul> Agenda <ul><li> Projects Doc: <ul><li> <a rel="nofollow" class="external free" href="https://docs.google.com/document/d/1K8jbQY9_NuRrR_gaVLxLtee262zZTU9Aipf6nB_zqas/edit#heading=h.k4xtoch10wib">https://docs.google.com/document/d/1K8jbQY9_NuRrR_gaVLxLtee262zZTU9Aipf6nB_zqas/edit#heading=h.k4xtoch10wib</a> </li><li> Discussion of Nikola's idea - Bacteria that form an internal magnet and move along magnetic field lines, to engineer magnetic e-coli - Nikola, Johan, Milo <ul><li> Allergen detection with Aptamers - **, Genestamp, a stealthmode startup in Menlo park doing food allergen detection -- Jay, Ilya, Shreya, Laura, Anitha </li><li> Bacterial batteries using electrogenic bacteria <ul><li> Already a lot of research being done in this field </li></ul> </li></ul> </li></ul> </li><li> Not much to expand (less traction) <ul><li> UV Protective mycosporine under UV promoter <ul><li> Lots of interest Patrik, Advait, Shashank, David, Priyanka </li></ul> </li></ul> </li><li> Resveratrol producing yogurt with p-coumaric acid <ul><li> YoVivo attempted to do this Adarsh, Shreya, Maria </li></ul> </li><li> 3D pattern formation toolkit </li><li> Bacteria that form an internal magnet and move along magnetic field lines, to engineer magnetic e-coli - Nikola, Johan, Milo </li><li> Potential rubric for each topic in order to compare all of them: <ul><li> step by step roadmap of the project with estimated timeline </li><li> Skills, instructions, and techniques needed for the project <ul><li> Bioinformatics, Assay, etc </li></ul> </li><li> pros/cons of the project; What are going to be the hardest parts/roadblocks we need to overcome? What are the risks? </li><li> future applications (possibly beyond iGEM), public interest, commercialization, previous research and traction </li></ul> </li></ul> To-Do <ol><li> Each team should flesh out each project according to the rubric, create a presentation, and present next time. </li><li> Potential to vote on the project in the next 1-2 weeks. </li><li> Address if we should change the meeting date or time. Next week will be 10:30am. </li></ol> Next meeting May 2nd 10:30am, will discuss a permenant meeting time and send out a new doodle poll Meeting 4/18/15 <ul><li>Call-in info: <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> Or, go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and enter meeting ID: 820 128 495 </li></ul> Attendees <ul><li> Biocurious - Maria, Eric, Terron, Anya, Jake, Adrash, Nikola, Emi, Bianca, Jay, Priyanka </li><li> CCL - Bruce, Alex, Sam, Vicky </li><li> Zoom - Laura (bio med eng), David (HS), Japna, Shreya (HS), Johan (web/past iGEM) </li></ul> iGEM ideas <ul><li> <a rel="nofollow" class="external free" href="https://docs.google.com/spreadsheets/d/1GtMmn_8sFxBc2s10JBQySlSQdceOSNHP06JeLVJ3YGk/edit#gid=0">https://docs.google.com/spreadsheets/d/1GtMmn_8sFxBc2s10JBQySlSQdceOSNHP06JeLVJ3YGk/edit#gid=0</a> </li></ul> <ul><li> Road maps: </li><li> <a rel="nofollow" class="external free" href="https://docs.google.com/document/d/1K8jbQY9_NuRrR_gaVLxLtee262zZTU9Aipf6nB_zqas/edit#">https://docs.google.com/document/d/1K8jbQY9_NuRrR_gaVLxLtee262zZTU9Aipf6nB_zqas/edit#</a> </li></ul> <ul><li> Potential timeline of events - <ul><li> Choose the project by the end of the April </li><li> ~2 weeks to start the IndieGogo Campaig </li><li> Market and try to raise money during MakerFaire (May 15-17th) </li><li> Get team members with experience to volunteer and teach sessions on diffferent topics such as intro to genetics and how to do plasimid design, basic wet lab work, safety training etc. We would like to run these at both CCL and BioC at the end of May beginning of June and throughout the project as needed. Please email Maria if you want to teach. </li></ul> </li></ul> Meeting 4/11/15 <ul><li>Call-in info: <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> Or, go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and enter meeting ID: 820 128 495 </li></ul> Attendees <ul><li> Biocurious - Maria, David, Advait, Johan, Shreya </li><li> CCL - Patrik, Bruce </li><li> Zoom - Teja, David H, Shashank, Ilya, Vardhaan, Adrash </li></ul> iGEM ideas <ul><li> <a rel="nofollow" class="external free" href="https://docs.google.com/spreadsheets/d/1GtMmn_8sFxBc2s10JBQySlSQdceOSNHP06JeLVJ3YGk/edit#gid=0">https://docs.google.com/spreadsheets/d/1GtMmn_8sFxBc2s10JBQySlSQdceOSNHP06JeLVJ3YGk/edit#gid=0</a> </li></ul> Make sure to look up (and read!) similar iGEM projects from previous years here: <ul><li> <a rel="nofollow" class="external free" href="http://igem-qsf.github.io/iGEM-Team-Seeker/dist/">http://igem-qsf.github.io/iGEM-Team-Seeker/dist/</a> </li></ul> And look up related BioBrick parts in the registry here: <ul><li> <a rel="nofollow" class="external free" href="http://parts.igem.org/Catalog">http://parts.igem.org/Catalog</a> </li></ul> Our top ideas so far fall into a couple categories: 1) Detecting and/or killing pathogens (Topics L, N, V) Check out the list of previous year's projects pasted into the spreadsheet, or search for "pathogen" on <a rel="nofollow" class="external free" href="http://igem-qsf.github.io/iGEM-Team-Seeker/dist/">http://igem-qsf.github.io/iGEM-Team-Seeker/dist/</a> A number of good attemnpts already, but they're really only scratching the surface so far. We coudl definitely come up with some other pathogen sensors, or combine input from multiple sensors [issue with Livermore - Patrik would not be able to participate in this, for IP reasons] [Quick Vote and this idea is voted as being off the table] 2) Detection of foods or allergens If we could link antibodies to a signaling pathway, that could be HUGE! See: <ul><li> <a rel="nofollow" class="external free" href="http://2008.igem.org/Team:Illinois">http://2008.igem.org/Team:Illinois</a> </li><li> <a rel="nofollow" class="external free" href="http://2010.igem.org/Team:KAIST-Korea">http://2010.igem.org/Team:KAIST-Korea</a> </li><li> Will help if we narrow down to a target allergy or a group of similar foods to detect <ul><li> Needs more research, literature search, antibody research, did any of the faculty advisors publish anything along these lines, doing general publication and patent searches. </li></ul> </li></ul> 3) Bacterial batteries, using electrogenic bacteria <ul><li> Geobacter (<a rel="nofollow" class="external free" href="http://en.wikipedia.org/wiki/Geobacter">http://en.wikipedia.org/wiki/Geobacter</a>) </li><li> Where is there room for us to find a niche - doing something useful with it, link to having a cell receive electrical signals, produce electricity by breaking down compost, take advantage of turning carbon into electricity, and making electricial circuits (linking bio to electricity from a control perspective, turn bacteria into electrial signal or turn it into an input), More groups are working on microbial fuel cell systems is the main area of research </li></ul> 4) Express UV-protective mycosporine under UV sensitive promoter; then grow engineered E. coli under UV to do directed evolution. Sequence the plasmids from strains evolved towards higher UV resistance. <ul><li> Biosynthesis pathway has been found out but no one had done an igem project with mycosporine, reverse engineer the pathways, this would wbe a new biobrick for iGEM, very few if any teams have done directed evolution in igem </li></ul> 5) Engineer plant cells to differentiate under (green?) light sensitive promoter <ul><li> Patrik opinion PITA for iGEM the timeline for working with plant cells to grow and to get to a plant (might be able to cut it down to 2 1/2 months if everything goes perfect and assays will not be easy) </li></ul> [Voted to take this off the table for lack of additional info] 6) Resveratrol producing yogurt. Producing p-coumaric acid is the key here - if we can do that, we can make hundreds of plant flavonoids in bacteria, not just resveratrol! Think caffeine, ginger, vanilla, capsaicin, you name it... <ul><li> <a rel="nofollow" class="external free" href="http://biocyc.org/META/NEW-IMAGE?type=PATHWAY&amp;object=PWY1F-FLAVSYN">http://biocyc.org/META/NEW-IMAGE?type=PATHWAY&amp;object=PWY1F-FLAVSYN</a> </li><li> <a rel="nofollow" class="external free" href="http://biocyc.org/META/NEW-IMAGE?type=PATHWAY&amp;object=PWY-361">http://biocyc.org/META/NEW-IMAGE?type=PATHWAY&amp;object=PWY-361</a> </li><li> <a rel="nofollow" class="external free" href="http://biocyc.org/META/NEW-IMAGE?object=PHENYLPROPANOID-SYN&amp;detail-level=1">http://biocyc.org/META/NEW-IMAGE?object=PHENYLPROPANOID-SYN&amp;detail-level=1</a> </li><li> ALREADY ATTEMPTED (the indiegogo campaign didn't do as well): <a rel="nofollow" class="external free" href="https://www.indiegogo.com/projects/yovivo-yogurt-naturally-inspired-synthetic-biology-meets-global-health">https://www.indiegogo.com/projects/yovivo-yogurt-naturally-inspired-synthetic-biology-meets-global-health</a> </li><li> Has many larger applications and can go to other flavonoids, the impact is in having the first steps of the pathway </li></ul> 7) Light switchable tRNA / nucleotides: awesome idea. How would we achieve this, and then waat would we do with them? <ul><li> Needs more elaboration </li></ul> [Voted to take this off the table for lack of additional info] 8) 3D pattern formation toolkit:: express cellulose or other biopolymers under light induced promoter. Construct solid 3D structures by shining a light pattern on engineered bacteria. <ul><li> A couple of other standalone ideas were mentioned in some of the top voted ones, but it's not clear if anyone voted for them specifically. These should probably have been broken out as separate ideas on the spreadsheet: <ul><li> Suicide vectors </li><li>Use E. coli to remove phosphorus contamination from wastewater and to create precursors for the synthesis of LiMPO4, a promising battery cathode. </li><li> Making biopolymers using light, could eventually turn into 3D printer using bacteria or a photographic tenchique, use enzymes that degrade or deposits material or carves it out </li></ul> </li></ul> Ideas for voting - making a commitment for what you would work on, are you excited and which aspect of the project you want to work on and how much effort are you personally willing to put into it An outline for each project and which steps would we need to take, a project road map By next week lets make a google doc with all the projects and we can add more in depth notes and project road maps and names of people that want to work on them and what they want to work on for each. Meeting 4/4/15 <ul><li>Call-in info: <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> Or, go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and enter meeting ID: 820 128 495 </li></ul> Attendees <ul><li> Biocurious - Maria,Eric, Solna, David, Anya, Jake, Johan, priyanka </li><li> CCL - Patrik, Milo, Rebecca </li><li> Zoom - David Hou, Laura, Fei, Vardhaan </li></ul> General PLEASE VOTE! <a rel="nofollow" class="external free" href="https://docs.google.com/spreadsheets/d/1GtMmn_8sFxBc2s10JBQySlSQdceOSNHP06JeLVJ3YGk/edit#gid=0">https://docs.google.com/spreadsheets/d/1GtMmn_8sFxBc2s10JBQySlSQdceOSNHP06JeLVJ3YGk/edit#gid=0</a> iGEM now giving 20 kb worth of synthesis to teams Send money to Maria or Patrik if you pledged Project ideas <ul><li> Continued discussion of ideas in spreadsheet </li><li> Will reduce number of ideas and email group to let them know what's left </li><li> Target selecting idea within next 2 weeks, would like to launch crowdfunding campaign before MakerFaire </li></ul> Meeting 3/30/15 <ul><li>Call-in info: <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> Or, go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and enter meeting ID: 820 128 495 </li></ul> Attendees <ul><li> Biocurious - Maria </li><li> CCL - </li><li> Zoom - Advait, Jay, </li></ul> Meeting 3/28/15 <ul><li>Call-in info: <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> Or, go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and enter meeting ID: 820 128 495 </li></ul> Attendees <ul><li> BioCurious - Maria, Itai, Shreya, Terron,priyanka </li><li> CCL - Patrik, Sean, Milo </li><li> Zoom - Eddy (BBL startup, Molecules for Life - antiviral lectins), Advait, Laura, Yafei, Vardhaan, David, Jay </li></ul> General Notes AGENDA <ul><li> Funding: <ul><li> Need 8 to 10 folks to pledge/contribute a significant portion towards the $4000 registration fee in the next 2 days ($400 to $500 each) </li><li> Need to decide or at least begin discussion of long term funding plans (crowd source or self funded) </li></ul> </li><li> IF Crowd funding need folks to start a sub group working on this </li><li> <a rel="nofollow" class="external free" href="http://2015.igem.org/Registration">http://2015.igem.org/Registration</a> </li><li> People interested in creating a campaign: Advait, Shreya </li></ul> Project Ideas Google Spreadsheet: <ul><li> <a rel="nofollow" class="external free" href="https://docs.google.com/spreadsheets/d/1GtMmn_8sFxBc2s10JBQySlSQdceOSNHP06JeLVJ3YGk/edit#gid=0">https://docs.google.com/spreadsheets/d/1GtMmn_8sFxBc2s10JBQySlSQdceOSNHP06JeLVJ3YGk/edit#gid=0</a> </li></ul> Continued discussion Everyone should pick one project to research and advocate for Meeting times: Maria to send out doodle poll to vote on regular meeting date/time Meet this Monday to discuss funding/application for iGEM Sean will be getting an OpenTrons and OpenPCR - would love to work on automating some experiments with these machines using Antha - <a rel="nofollow" class="external free" href="https://www.antha-lang.org/">https://www.antha-lang.org/</a> Meeting 3/21/15 <ul><li>Call-in info: <a rel="nofollow" class="external free" href="https://zoom.us/j/820128495">https://zoom.us/j/820128495</a> Or, go to <a rel="nofollow" class="external free" href="https://zoom.us/join">https://zoom.us/join</a> and enter meeting ID: 820 128 495 </li></ul> Follow stream of Alex's SynBio 101 class here: mrk.tv/1FSt3Lz Attendees <ul><li> BioC: Maria, Advait (High School, RVC), John (Physics), Priyanka (Botany), Eric (Physics and Computer Science), Laura (Biomedical Engineering), Teja (biomedical engineer), Hugo (Economics and Law), Japna, David (High School) </li><li> CCL: Patrik, Bruce, Shawn, Steve (bioE) </li><li> Sean Van der Linden sdvdl@hotmail.com </li><li> Bruce Kim bruce_kim@hotmail.com </li><li> Steve Bates steverbates@gmail.com </li><li> eric@ericelias.org Eric Elias </li><li> Berkeley: </li><li> Zoom: Ilya (BioE), Joseph NM (nonpracticing Physicist, 3D printing fan boy) </li></ul> General notes <ul><li> join the mailing list! </li><li> funding: can self-fund, crowd-fund, or some combination </li><li> Patrik recommends either doing a project that will complement RVC project or one that is totally different so same resources are not required </li><li> prefer to use E. coli so that iGEM kits can be used </li><li> efficiency of transformation kits is low </li><li> google doc will be sent out with list of ideas, add ideas and background information. Do not delete anything! </li><li> next meeting is next Saturday 3/28/2015 </li><li> should decided on funding next week </li></ul> iGEM ideas list from last year: <a rel="nofollow" class="external free" href="https://docs.google.com/document/d/1PHt2bsw7AT6FyyXgKLsKKw_MqS4oHIrROuut9_wGl5c/edit?usp=sharing">https://docs.google.com/document/d/1PHt2bsw7AT6FyyXgKLsKKw_MqS4oHIrROuut9_wGl5c/edit?usp=sharing</a> Ideas for this year <ul><li> Food detecting bio marker label turn color </li><li> Glue mollusk, water tolerant (already done) </li><li> Bio inks, with different properties </li><li> Uv activated polymers made cheaper (ex Form 1 printer) </li><li> Structural stuff for 3d printing </li><li> Bacteria that respond to different colors optimize for different wave lengths of light </li><li> Better luminescent proteins </li><li> Refine titanium from ore, (<a rel="nofollow" class="external free" href="https://books.google.com/books?id=J4OkqlEJgl0C&amp;pg=PA169&amp;lpg=PA169&amp;dq=bacteria+that+absorb+gold&amp;source=bl&amp;ots=mtGVvzE2pw&amp;sig=WXtdOIiDfxx-HGrHOZUW-RvUaBU&amp;hl=en&amp;sa=X&amp;ei=nrsNVaCDMJCMoQSH1YLgDw&amp;ved=0CB4Q6AEwAA#v=onepage&amp;q=bacteria%20that%20absorb%20gold&amp;f=false">https://books.google.com/books?id=J4OkqlEJgl0C&amp;pg=PA169&amp;lpg=PA169&amp;dq=bacteria+that+absorb+gold&amp;source=bl&amp;ots=mtGVvzE2pw&amp;sig=WXtdOIiDfxx-HGrHOZUW-RvUaBU&amp;hl=en&amp;sa=X&amp;ei=nrsNVaCDMJCMoQSH1YLgDw&amp;ved=0CB4Q6AEwAA#v=onepage&amp;q=bacteria%20that%20absorb%20gold&amp;f=false</a>) </li><li> Cupriavidus metallidurans takes up metals such as gold </li><li> Vern - plant astragalus manufactures compounds cycle astragal and that is extracted and it's a telemorase activator, look at the plant to find out the gene sections that control that reaction and put it into bacteria or other plant. Huge cost to buy. </li><li> Proteins that form certain color matrices scaffold self assembles to different colors </li><li> <a rel="nofollow" class="external free" href="http://2009.igem.org/Team:Cambridge">http://2009.igem.org/Team:Cambridge</a> </li><li> <a rel="nofollow" class="external free" href="http://2010.igem.org/Team:Cambridge">http://2010.igem.org/Team:Cambridge</a> </li><li> <a rel="nofollow" class="external free" href="http://2011.igem.org/Team:Cambridge">http://2011.igem.org/Team:Cambridge</a> </li><li> Make a clock that changes in a novel way </li><li> Circuits in cells to produce a protein to kill a cell (oscillating genes) </li><li> The Bathtub Mechanism (Cancer Research Project) </li><li> Cancer cells frequently, perhaps always in some cases, have anabnormal number of chromosomes, often about double the normal number. The Bathtub Mechanism is a theoretical mechanism to selectively kill cells with too many (or too few) chromosomes -- or indeed any numerical abnormality. Most healthy people have twenty-three pairs of chromosomes (46 total) in each cell. A small minority have one or two extra or too few chromosomes. Cancers cells often, perhaps always in some cases, havean abnormal number of chromosomes, often about double the normal number -- around eighty to ninety chromosomes. This abnormal number of chromosomes is known as aneuploidy. </li><li> The Bathtub Mechanism works in analogy to an old-fashioned bathtub with two faucets and a single drain with a limited capacity. Turn on one faucet full and the drain will be able to remove the water as it is added. The water level in the bathtub remains low, less than a half-inch or a centimeter. Turn on both faucets full and the drain will not be able to remove the water as it is added, the water level rises and the bathtub overflows. </li><li> In the Bathtub Mechanism, each chromosome is a "faucet" adding a mild toxin to the cell -- ideally a chemical that is not toxic in low concentrations at all. The concentration of the cell killer is analogous to the water level in the bathtub. A drain is added to the cell. In cells with the normal number of chromosomes, the concentration of the cell killer toxin remains very low. In cells with double the normal number of chromosomes, the concentration of the cell killer toxin builds up to lethal levels because the drain cannot remove the toxin rapidly enough. </li><li> Mechanism.Author/Point of Contact: John F. McGowan, Ph.D. (jmcgowan79@gmail.com) </li><li> Articles on the Bathtub Mechanism </li><li> <a rel="nofollow" class="external free" href="http://math-blog.com/2011/07/11/can-mathematics-cure-cancer/">http://math-blog.com/2011/07/11/can-mathematics-cure-cancer/</a> </li><li> <a rel="nofollow" class="external free" href="http://math-blog.com/2011/10/07/tackling-cancer-with-math/">http://math-blog.com/2011/10/07/tackling-cancer-with-math/</a> </li><li> <a rel="nofollow" class="external free" href="http://math-blog.com/2011/10/31/animations-of-a-possible-cure-for-cancer/">http://math-blog.com/2011/10/31/animations-of-a-possible-cure-for-cancer/</a> </li><li> In principle, the concept could be demostrated with yeast which have chromosomes. Different species of yeast have different numbers of chromosomes. The demonstration would be a system of biochemicals that selectively kill yeast with a larger number of chromosomes. Yeast come in two large groups, one with about eight chromosomes and one with about sixteen chromosomes. The cutoff could be set at around twelve chromosomes. </li><li> Pathogen-eating E. coli </li><li> Biological thermometer (heat sensing bacteria) </li><li> Light induced protein expression </li><li> Bacteria that secrete metal oxides in response to varying light levels (light induced sunscreen, complementary to project above) </li><li> E. coli that produces alcohol </li><li> Bacterial batteries, using electrogenic bacteria </li><li> Linking quantum dots to bioluminescence </li><li> Controlling resistance with bacteria - bacterial transistor </li><li> from last year:d </li></ul>

Votes Project