Team:Yale/Our Project/Future Work

From 2010.igem.org

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<li>Make our generator inducible!</li>
<li>Make our generator inducible!</li>
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<b>Prepare TSI agar plates with PDMS molds and inoculate with cells</b>
<b>Prepare TSI agar plates with PDMS molds and inoculate with cells</b>
<li>2. Use of TSI plates (see Cu localization page) will initially serve as a proof-of-principle for cell localization.  The TSI agar will make the plates turn black in the pattern of our PDMS mold.  Moving forward, we can use these molds to create ordered arrays/wires with deposited covallite and check these for conductivity (see applications page).</li>
<li>2. Use of TSI plates (see Cu localization page) will initially serve as a proof-of-principle for cell localization.  The TSI agar will make the plates turn black in the pattern of our PDMS mold.  Moving forward, we can use these molds to create ordered arrays/wires with deposited covallite and check these for conductivity (see applications page).</li>
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<li>Limiting copper uptake</li>
<li>Limiting copper uptake</li>
<li>Limiting copper concentration by binding to other proteins: in this strategy, copper would be sequestered using binding proteins to both limit the amount of free copper available to kill our cells and possibly to locally increase the concentration of copper to facilitate covallite formation.  In our search we focused on genes for small proteins that are readily made and exported by E. coli cells.</li>
<li>Limiting copper concentration by binding to other proteins: in this strategy, copper would be sequestered using binding proteins to both limit the amount of free copper available to kill our cells and possibly to locally increase the concentration of copper to facilitate covallite formation.  In our search we focused on genes for small proteins that are readily made and exported by E. coli cells.</li>
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<!------------- FUTURE WORK: NEEDS TO BE EDITED ------------->
<!------------- FUTURE WORK: NEEDS TO BE EDITED ------------->

Revision as of 03:58, 28 October 2010

iGEM Yale

future work

    1. Finish cloning promoter Q04121 to phsABC
  • Make our generator inducible!

  • Prepare TSI agar plates with PDMS molds and inoculate with cells
  • 2. Use of TSI plates (see Cu localization page) will initially serve as a proof-of-principle for cell localization. The TSI agar will make the plates turn black in the pattern of our PDMS mold. Moving forward, we can use these molds to create ordered arrays/wires with deposited covallite and check these for conductivity (see applications page).

  • 3. Other copper/cell localization strategies
  • We have also discussed other strategies to localize copper and/or cells in order to make ordered deposits of covallite. One method is to use a copper binding proteins which could be affinity tagged and then paired with a ligand that has been put on a surface. For example a copper binding protein could be fused to a biotinylation sequence, which could then be paired with a glass surface that has avidin on it. By depositing avidin in a specific arrangement, we could thus control where copper is localized and where covallite formation is most likely. A variety of methods to functionalize glass with reactive compounds or ligands are available.

  • 4. Research and develop assay for covallite formation
  • Simple UV-VIS (max absorbance of 860nm) may not be sensitive enough to verify we are actually forming covellite crystals in solution. There are a variety of more exotic ways to probe the structure of precipitated copper compounds (Cu K-edge X-ray absorption near-edge structure [XANES] or plasma optical emission spectrometry), some of which are available at Yale, that we can turn to after we have advanced our precipitation system sufficiently.

  • 5. Develop more Biobricks to enhance E. coli copper survivability. Currently we’ve thought of three strategies:
  • Copper export proteins: our team is working to acquire a copper export gene
  • Limiting copper uptake
  • Limiting copper concentration by binding to other proteins: in this strategy, copper would be sequestered using binding proteins to both limit the amount of free copper available to kill our cells and possibly to locally increase the concentration of copper to facilitate covallite formation. In our search we focused on genes for small proteins that are readily made and exported by E. coli cells.