Team:SF Bay Area DIYBio/Results

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Accomplishments

Researched UV sources and built UV exposure rig to mimic solar UV

• 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

Determined exposure level needed for >4 log decrease in CFU

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.

Demonstrated GFP is NOT an effective UV protectant for E.coli

4 log decrease in viable cells after 75min for HB101, 30min for pGLO

If anything, pGLO has a negative effect on UV resistance!

Synthesized and transformed A. variabilis shinorine biosynthesis genes

Submitted one gene (Ava_3856) to Parts Registry

Demonstrated that we produced 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 (control)
  • 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...