BioSunBlock - SunScreen Evolved
BioSunblock is comprised of organic compounds called Mycosporine-like amino acids (MAAs). MAAs, also known as “microbial sunscreen,” are compounds that exhibit UV-protective qualities. MAAs are naturally produced by organisms that usually live in marine environments with high amounts of UV exposure. The plan for this project is to genetically engineer bacteria to produce these compounds for sunscreen applications.
Our existing chemical sunscreens cause various health and environmental problems. Some are effective at blocking the sunburn inducing UVB rays but not the UVA rays that cause long-term skin damage. PABA was shown to induce DNA damage when applied to human skin cells in vitro and exposed to UV light, despite its UV protection of the skin from incident UV rays. PABA’s use in sunscreens has been banned in the EU since this study. Other sunscreen compounds such as oxybenzone are so toxic to coral reefs that the equivalent of a single drop in half-dozen Olympic-sized swimming pools can lead to coral bleaching - a significant issue in areas popular to beach goers and snorkelers.
Mycosporine-like amino acids (“MAAs”) are not known to be toxic to humans. In fact, many MAAs have simultaneous anti-oxidant properties in addition to their UV absorbing properties and thus may actually be beneficial in certain human conditions and when used for certain applications. They are also naturally produced by algae and corals, and should be far more friendly to the environment.
How does it work?
Our plan is to insert into E. coli a set of genes from cyanobacteria that are known to produce shinorine, one of the most common MAAs, based on prior work by Baskus and Walsh. As a side effect, this should also make the E. coli cells more resistant to UV radiation. We will then use a technique called Directed Evolution to evolve for increased UV resistance, thereby optimizing shinorine production in E. coli or potentially even evolving novel alternative mycosporine-like amino acids. Finally, we will sequence the resulting genes to figure out what changes have occurred that improved our engineered construct.
(From Patrik - 8/4/15 email)
The Ava_3858-1855 gene cluster is described in the 2010 Science paper by Balskus and Walsh:
All the essential details are in the Supplementary Materials:
They don't list the full sequence of the gene cluster, but Table S4 has the PCR primers and restriction sites. If you blast the primer sequences against the Anabaena genome, you get the following 6460bp sequence:
They cloned the gene cluster into pET-29b, which has an IPTG-inducible T7 promoter. If we want to do things more the iGEM way, we could use BBa_J23100 as a strong constitutive promoter, BBa_B0030 as a strong RBS, and end with double terminator BBa_B0015.
Alternatively, we could replicate this 4102bp long sequence with the first three enzymes that the Minnesota succesfully demonstrated to produce a UV absorbing compound:
Experiment 1: Transformation of E. coli with pGLO (BioC, 7/31/2015)
A preliminary transformation experiment was prepared in order to familiarize new team members with lab protocol/safety. A subculture of competent E. coli cells were prepared to be transformed with a pGLO plasmid. The resulting recombinant bacteria could then be used in further experiments to determine whether UV absorption by the expressed fluorescent protein has any effect on the survival of bacteria cultured under UV light.
LB Media 75mM CaCl2 E. coli pGLO Plasmid Ampicillin plates (50ug/ml)
Competent Cell Preparation (7/31/2015)
1. A single colony was selected from a plate and stored at 4 ºC.
2. The colony was innoculated into 3ml of LB in a 15ml culture tube.
3. The cells were incubated at 37ºC overnight for 24 hours.
4. After incubation, 1ml of overnight culture was added to 10 ml of fresh LB in a 50ml Falcon Tube.
5. The cells were incubated at 37ºC with shaking for 3 hours to enter log phase.
6. Cells were then centrifuged at 3,500 RPM for 10 minutes.
7. The cell solution was then decanted carefully as to not disturb the cell pellet and was respuspended in 2ml of ice-cold 75mM CaCl2
8. The sell suspension was again centrifuged at 3,500 RPM for 5 minutes
9. The cell suspension was then decanted and resuspended in 1ml sterile ice cold 75mM CaCl2 and put in a cryogenic freezer at -150ºC
Transformation of Competent Cells (8/1/2015)
11. 50ul of cell solution was added into 6 PCR tubes kept on ice.
12. 3ul of pGLO plasmid was added to the 6 tubes with 50ul of cells and run through a thermal cycler with the following parameters:
i. 30 minutes @ 4ºC
ii. 45 seconds @ 42ºC
iii. 5 minutes @ 4ºC
13. 7 1.5 ml microcentrifuge tubes were then filled with 950ul sterile LB at room temperature (1:10 diluton of transformed cells), and prewarmed in a water bath to 37ºC
i. 6 tubes with plasmid
ii. 1 tube without plasmid (negative control)
14. When the thermocycling process had terminated, 50ul of transformed cells were inserted into pre-warmed 450ul LB to create a total of 500ul in each centrifuge tube.
15. The cells were then incubated for one hour at 37ºC with shaking.
16. The cultured cells were then centrifuged at 12,000 RPM for 30 seconds.
17. The cells were then decanted, leaving 150ml.
18. The pellet was then redissolved to make the cells more concentrated.
19. The transformants were then plated on 13 LB plates
1 plate with no ampicillin (+) 1 plate E. coli w/o plasmid (-) 11 plates with transformants on selectable media
20. Plated cells were then stored and incubated at 37ºC and observed for colonies after 24 hours.
Transformants were plated and stored at 370C at approximately 3:45pm.
Fluorescence observed on plates 1B, 2B, 3B
Plate 1.........................Lawn, LA plate no ARA
Plate 1B.........................> 20 colonies expressing GFP
Plate 2:.........................No growth
Plate 2B:.........................> 100 colonies expressing GFP
Plate 3:.........................~ 6 colonies - no GFP (did not glow under UV)
Plate 3B:.........................> 20 colonies expressing GFP
Plate 4A:.........................No growth
Plate 4B:.........................Lawn LA plate No ARA
Plate 5A:.........................No growth
Plate 5B:.........................No growth
Plate 6A:.........................No growth
Plate 6B:.........................No growth
Plate 7:.........................No growth
Experiment 2: Kill curve of E. coli exposed to UV (CCL, 8/5-8/9/2015)
Our goal for this experiment is to get our UV exposure harware sorted out, and to do a first experiment testing the effect of various of levels of UV exposure on survival of E. coli cells with and without the pGLO plasmid.
- UV sources: we decided to use two 15W Exo Terra Repti-Glo 10.0 fluorescent lamps, for their good match to solar spectrum in the far UVA to UVB range (365nm - 295 nm), supplemented by a 380nm Keedox 20W Fast Curing UV Light LED lamp to cover the near-UVA range (which should also give us the most GFP UV absorbance and fluorescence).
- E. coli strains:
- HB101 transformed with pGLO, from Experiment 1
The UV light sources (two 15W 18" fluorescent tubes, and one 20W LED array) will be mounted in an opaque plastic box to avoid eye damage. The lamps will be suspended from cords to make them height-adjustable, and powered from a timer. We will check the temperature inside the box, and if necessary, install a fan in the lid of the box to extract some of the heat generated by the bulbs (50W total!).
Experiment 3: Transformation of HB101 and CFSC #3600 bacteria with shinorine producing genes (BioCurious, 9/1-9/3/2015)
We transformed 2 different strains of bacteria (HB101 and CGSC #3600) and inserted the pET29b plasmid (with shinorine producing genes).
- HB101 E. Coli strain
- CGSC #3600 E. Coli Strain (mutagenic strain from the Yale Coli Genetic Stock Center http://cgsc.biology.yale.edu)
- LB Media
- 75mM CaCl2
- pET29b plasmid backbone (Ava3858-3855, Ava3858-3856, Ava3858-3857 are genes of interest)
- pGLO plasmid
- kanamycin (50ug/ml)+ampicillan (100ug/ml) plate
We ran transformations with the following plasmids for each E. coli strain.
1. pET29b-Ava3858-3855 (Kan plate)
2. pET29b-Ava3858-3856 (Kan plate
3. pET29b-Ava3858-3857 (Kan plate)
4. pGLO (+control) (Amp plate)
5. no plasmid (-control)(Amp plate)
Competent Cell Preparation:
1. Pick single colony from plate stored at 4 oC.
2. Innoculate colony into 3 ml of LB in a 15 ml culture tube
3. Incubate at 37oC with shaking ~ 16 - 24 hours (overnight)
4. Add 1ml of the overnight culture to 10 ml fresh LB in a 50 ml Falcon tube(provides more aeration than a 15 ml tube but not as much as an Erlenmeyer flask)
5. Incubate @ 37oC with shaking 2.5 – 3.5 hours - The culture will be in log phase and turbid
6. Centrifuge at 3,500 RPM for 10 mins (big floor centrifuge) - balance w/water-filled Falcon tube or use the two tubes as prepared in step 4 to balance each other.
7. Decant supernatant and re-suspend in 2 ml sterile ice cold 75mM CaCl2 (11 g CaCl2•2H2O/100 ml water) CaCl2•2H2O MW = 147.01
8. Centrifuge at 3,500 RPM for 5 mins (big floor centrifuge)
9. Decant supernatant and re-suspend in 1 ml sterile ice cold 75mM CaCl2
10. Put 50 μL of cell solution into PCR tubes ON ICE
11. Add DNA (1-2 μL plasmid DNA or 10-20 μL PCR product) to 50 μL of cells and run in thermal cycler on program TRANS: (30 min 4 ̊C, 45 sec 42 ̊C, 5 min 4 ̊C)
12. While thermocycler is running, fill sterile 1.5 ml microcentrifuge (Eppi) tubes with 450 μL sterile LB (1:10 dilution of transformed cells), and pre warm in water bath to 37
13. After thermocycler is finished, transfer the 50 μL of transformed cells into pre-warmed 450 μL LB Eppi tube to yield a total of 500 μL in each eppi tube
14. Incubate 45 min-1hr at 37oC with shaking
15. Centrifuge grown out cells at 12,000 RPM for 30 sec - 1 min.
16. Decant most of the supernatant--leave about 150-200 μL.
17. Resuspend cells in this solution
18. Transfer cells onto solid growth media
19. Incubate plates at 37 ̊C, look for colonies the next
We got transformants in the CGSC #3600 strain for the following plasmids.
1. pET29b-Ava3858-3855 (Kan plate)
- This is the full Ava Gene cluster to produce shinorine
2. pET29b-Ava3858-3856 (Kan plate)
4. pGLO (+ control) (Amp plate)
The HB101 had no transformants. We suspect that there was a problem with the HB101 competent cells since we got nothing on any of the HB101 plates.