BioSunBlock Evolved Bacterial Sunscreen Team SF Bay DIYbio
The BioSunBlock Team Who are we? We are a collective group of local Biohackers and Bio-Enthusiasts teaming up to compete in iGEM. Who is on our team? A diverse group of newbies and experts; from programmers, chemists, bioinformaticians, to students and even high schoolers! Where are we located? We are based in community labs BioCurious, Counter Culture Labs, and Berkeley BioLabs, all located in the San Francisco Bay Area.
Why BioSunBlock? Our existing chemical sunscreens cause various health and environmental problems: PABA has been banned in the EU; linked to skin damage under UV • Titanium dioxide is linked to peroxide pollution in coastal waters • Other sunscreens are known to bleach corals • Is there a healthier, more environmentally friendly alternative? Other susncreens
Mycosporine-like Amino Acids (MAAs) In wide range of organisms exposed to sunlight • Cyanobacteria, micro- and macroalgae, corals, fungi, ... Microbial sunscreen • MAA present! “Natural!” Induced upon exposure to UV light • Efficient UV absorbers + protect against oxidative damage • Marketed as “Eco - Compatible” sunscreen • But expensive, since it needs to be extracted from algae Other potential applications • UV resistant bacteria for terraforming Mars! • Non-antibiotic selectable marker •
The Plan: Experimental Overview 1. Express Shinorine biosynthesis pathway in E. coli Also explored other UV absorbers, like GFP (!) and PABA pathway Measure MAA production based on UV absorption spectrum 2. Improve UV resistance in E. coli using Directed Evolution! Quantify UV resistance based on kill curve of E. coli under UV exposure 3. Sequence plasmid and figure out what improvements evolution came up with
Anabaena variabilis shinorine gene cluster Baskus and Walsh (2010) identified a gene cluster in A. variabilis that produced shinorine, a well-studied MAA The U. Minnesota 2012 iGEM team successfully transformed the entire gene cluster, but did not observe the production of shinorine, the final metabolite in the pathway. No parts submitted to Registry. Baskus and Walsh (2010)
Gene Design Anabaena variabilis shinorine biosynthesis gene cluster: Ava_3858 Ava_3857 Ava_3856 Ava_3855 6459bp BBa_K1839000 BBa_K1839002 BioBrick prefix overlap for & suffix * * BBa_K1839001 Gibson assembly * IDT gBlocks: removed restriction * site * Additional gBlocks to stitch together the full gene cluster with Gibson Reconstructed gene cluster:
Alternative Mycosporine enzymes • The Minnesota 2012 team was not able to get the final step in the pathway, so we also searched for alternative enzymes in other cyanobacteria • Nostoc punctiforme ATCC 29133: final enzyme produces shinorine and porphyra-334 • Identified 7 other homologous genes from known shinorine producing cyanobacteria
Measuring UV absorbing compounds UVB UVA Vis MAA’s can be extracted with methanol: Ava_3858-3855 (shinorine) Grow 20ml culture overnight in TSB • Ava_3858-3856 Spin down & wash in saline 2x • (mycosporine-glycine) Extract in 2ml methanol at 4C overnight • Pellet at 10,000rpm, collect supernatant • Collect UV absorption spectrum: • E. coli HB101 • normalized to 3 no-plasmid controls HB101+Ava_3858-3855 (full shinorine pathway) • HB101+Ava_3858-3856 (up to mycosporine-glycine only) • Yay - we’re producing UV absorbing compounds! Further analysis will have to wait until we get our HPLC up and running...
Information on UV Spectra UVA: 320- 400nm. “Tanning” wavelengths. Long -term free radical damage • UVB: 280-320nm. Causes sunburn and direct DNA damage • UVC: 100-280nm. Rapid skin and retinal damage (e.g.: germicidal UV in BSC) • We want to mimic solar UV: broad-band UVA+UVB, but not UVC! After testing many UV sources, we settled on: UVB basking lamp for pet reptiles • UVA nail curing lamp • UVB UVA Vis UVB UVA UVB UVA Vis Vis Combined spectrum UVB reptile lamps UVA nail curing lamp
Building the UV Exposure Box
Safety: UV light in the Lab “Aren’t you burning your retinas, working with that much UV light?” Our lamps only produce wavelengths found in natural sunlight - no UVC! • Strong visible light component; acts as a built-in safety warning • Overall intensity is 1/10th lower than walking in bright sunlight • All the UV exposure work is done inside the biosafety cabinet, behind a • layer of glass Turning on the germicidal UV lamp built into the biosafety cabinet, or staring into a gel illuminator is more dangerous than the UVA/UVB lamps we are working with!
UV Kill Curve Experiments We need to establish how much UV to expose the cells to, to provide the right selective pressure for Directed Evolution: kill most cells, not all . Expose to different UV durations, and count colony forming units For each time point, make a 1:10 dilution series; spot on plates to count colonies
Procedure Shaker broken? Make another one! 1. Grow E. coli overnight in TSB
2. Spin down and wash three times in saline. Put cells in saline in shallow plates
3. Expose E.coli to UV light, inside biosafety cabinet
4. Take samples every 15 min, and make 1:10 dilution series down to 10 -5
5. For 10 -2 to 10 -5 , spot 6 drops of 2ul culture onto quadrant of LB agar plate
10 -3 10 -2 10 -3 10 -2 10 -4 10 -5 Countable number of colonies at 10 -5 dilution 0 min 10 min 60 min 6. Count colonies, at whichever dilution yields countable spots
UV Kill Curve Experiments: Results UV kill curve was generated for E.coli HB101, to establish baseline exposure HB101 + pGLO for Directed Evolution We also tested HB101 + pGLO plasmid, to check if GFP has a protective effect. E.coli HB101 4 log decrease in viable cells after 75min for HB101, 30min for pGLO If anything, pGLO has a negative effect on UV resistance!
Future Work: Directed Evolution UV resistance is an ideal target for Directed evolution: Introduce mutations • Select for UV resistance • Repeat! • Error-prone Rolling Circle Amplification Phi29 DNA Polymerase +MnCl 2
Ethical, Environmental, Legal, Societal Issues “Are we creating a superbug that cannot be killed by UV?” • Only solar UV; germicidal UV lamps are much shorter wavelength! • Extensively researched health and environmental impacts of • existing sunscreen compounds Educated members on UV safety - including safety issues with • existing lab equipment Do not use the biosafety cabinet with the UV on!
Sunscreen and UV-Awareness Survey 99 respondents • Asked questions about the public’s knowledge and acceptance of sunscreen: • Are you concerned about the effects Would you wear sunscreen if there Are you concerned about the effects of sunscreen on the environment? were a more natural alternative? of sunscreen on your health? I am un- concerned No this doesn’t affect No I don’t worry, I trust I was unaware of any effects of how much I would sunscreens to protect me Yes I would wear sunscreen on the environment wear sunscreen sunscreen more Yes, I worry about how I am concerned about effects of safe sunscreen is sunscreen on our environment
Accomplishments Researched UV sources and built UV exposure rig to mimic solar UV • Determined exposure level needed for >4 log decrease in CFU • Demonstrated GFP is NOT an effective UV protectant for E.coli • Synthesized and transformed A. variabilis shinorine biosynthesis genes • Submitted one gene (Ava_3856) to Parts Registry • Demonstrated that we produced UV absorbing compounds •
We don’t do community outreach We are the community outreach 36 people signed up on our team on iGEM website • 105 people on our mailing list for this project • Many more joined for 1-2 meetings, or came to one of our classes (biweekly Intro • to Synthetic Biology, RCA workshop, Intro to Bioinformatics, & more…) Weekly team meetings Sat morning, open to the public and posted on our Meetup • lists, >3000 members. Meetings held jointly at BioCurious and CCL, with video link. Yes, we had a meeting yesterday! This is an ongoing project - YOU can join our team! Next BioSunBlock meeting is Oct 3 Next Real Vegan Cheese meeting Oct 5.
Collaborations Participated in Building with Biology demos at Chabot Space & Science • museum Discussed projects and shared ideas with the UCSF, Stanford-Brown, • Santa Clara, and UCSC iGEM teams Presented project to an audience of undergraduates interested in • synthetic biology at the regional iGEM meetup in Reno Thanks to The Tech / Wellesley team for very last-minute emotional and enzyme support!
Special Thanks!
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