BioSunBlock Evolved Bacterial Sunscreen Team SF Bay DIYbio The - - PowerPoint PPT Presentation

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

Other susncreens Is there a healthier, more environmentally friendly alternative?

• In wide range of organisms exposed to sunlight

Cyanobacteria, micro- and macroalgae, corals, fungi, ...

• Microbial sunscreen
• 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

Mycosporine-like Amino Acids (MAAs)

MAA present! “Natural!”

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

Ava_3858 Ava_3857 Ava_3856 Ava_3855 Anabaena variabilis shinorine biosynthesis gene cluster: BBa_K1839000 BBa_K1839001 BBa_K1839002 IDT gBlocks:

• verlap for

Gibson assembly BioBrick prefix & suffix Additional gBlocks to stitch together the full gene cluster with Gibson

* * * * *

removed restriction site Reconstructed gene cluster:

6459bp

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

MAA’s can be extracted with methanol:

• Grow 20ml culture overnight in TSB
• Spin down & wash in saline 2x
• Extract in 2ml methanol at 4C overnight
• Pellet at 10,000rpm, collect supernatant
• Collect UV absorption spectrum:
• E. coli HB101
• 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...

Ava_3858-3855 (shinorine) Ava_3858-3856 (mycosporine-glycine) normalized to 3 no-plasmid controls

UVB UVA Vis

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

UVA UVB Vis UVA UVB Vis UVA UVB Vis

UVB reptile lamps UVA nail curing lamp Combined spectrum

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 0 min 10 min 60 min 10-5 10-4 Countable number of colonies at 10-5 dilution 10-3 10-2

• 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 for Directed Evolution We also tested HB101 + pGLO plasmid, to check if GFP has a protective effect.

HB101 + pGLO 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!

+MnCl2

Error-prone Rolling Circle Amplification

Phi29 DNA Polymerase

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

• f sunscreen on the environment?

I am concerned about effects of sunscreen on our environment I was unaware of any effects of sunscreen on the environment I am un- concerned

Are you concerned about the effects

• f sunscreen on your health?

No I don’t worry, I trust sunscreens to protect me Yes, I worry about how safe sunscreen is

Would you wear sunscreen if there were a more natural alternative?

Yes I would wear sunscreen more No this doesn’t affect how much I would wear sunscreen

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!