Difference between revisions of "Team:SF Bay Area DIYBio/Results"

 
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{{SF_Bay_Area_DIYBio}}
 
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[[File:UV Absorption Curve.png]]
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__NOTOC__
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==Accomplishments==
  
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===Researched UV sources and built UV exposure rig to mimic solar UV===
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*<font size=2>UVA: 320-400nm. “Tanning” wavelengths. Long-term free radical damage
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*UVB: 280-320nm. Causes sunburn and direct DNA damage
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*UVC: 100-280nm. Rapid skin and retinal damage (e.g.: germicidal UV in BSC)</font>
  
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[[File:Kill Curve results.png]]
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We want to mimic solar UV: broad-band UVA+UVB, but not UVC! After testing many UV sources, we settled on:
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*<font size=2>UVB basking lamp for pet reptiles
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*UVA nail curing lamp</font>
  
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<html>
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[[File:Spectra.png|950px]]
  
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<h2> Project Results</h2>
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===Determined exposure level needed for >4 log decrease in CFU===
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UV kill curve was generated for E.coli HB101, to establish baseline exposure for Directed Evolution
  
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<p>Here you can describe the results of your project and your future plans. </p>
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We also tested HB101 + pGLO plasmid, to check if GFP has a protective effect.
  
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<h5>What should this page contain?</h5>
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[[File:Kill Curve results.png|500px]]
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<ul>
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project </li>
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<li> Considerations for replicating the experiments </li>
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</ul>
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===Demonstrated GFP is NOT an effective UV protectant for E.coli===
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4 log decrease in viable cells after 75min for HB101, 30min for pGLO
  
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If anything, pGLO has a negative effect on UV resistance!
  
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===Synthesized and transformed A. variabilis shinorine biosynthesis genes===
  
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===Submitted one gene (Ava_3856) to Parts Registry===
  
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<h4> Project Achievements </h4>
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===Demonstrated that we produced UV absorbing compounds===
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MAA’s can be extracted with methanol:
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*<font size=2>Grow 20ml culture overnight in TSB
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*Spin down & wash in saline 2x
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*Extract in 2ml methanol at 4C overnight
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*Pellet at 10,000rpm, collect supernatant
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*Collect UV absorption spectrum:
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**E. coli HB101 (control)
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**HB101+Ava_3858-3855 (full shinorine pathway)
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**HB101+Ava_3858-3856 (up to mycosporine-glycine only)</font>
  
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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[[File:UV Absorption Curve.png|500px]]
  
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<ul>
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Yay - we’re producing UV absorbing compounds! Further analysis will have to wait until we get our HPLC up and running...
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<li>A list of linked bullet points of the successful results during your project</li>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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</ul>
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<h4>Inspiration</h4>
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<p>See how other teams presented their results.</p>
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<ul>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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</ul>
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</div>
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</html>
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Latest revision as of 13:14, 21 November 2015

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

Spectra.png

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.

Kill Curve results.png

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)

UV Absorption Curve.png

Yay - we’re producing UV absorbing compounds! Further analysis will have to wait until we get our HPLC up and running...