IGEM:MIT/2006/System brainstorming/Autotrophic bacteria

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This has been of enough interest to various team members to pursue the Calvin cycle component of the project, the dedicated project page can be found here.

Contents

Description

Construct an autotrophic E.coli. Specifically, "domesticate" a light-driven proton pump and utilize the generated proton gradient in order to drive a mechanism to metabolize carbon (possibly from CO2 via the Calvin cycle).

  • Deliverable: E.coli that grow on minimal media with no carbon source.

Requirements

Design

Light-driven Proton pump (Proteorhodopsin)

This paper describes co-transforming E.coli, with pACCAR16<delta>crtX and a plasmid containing the blh gene and the Proteorhodopsin gene each under inducible control.

  • pACCAR16<delta>crtX - contains crtE,crtB,crtI,andcrtY genes, which are responsible for B-carotene biosynthesis in E.herbicola
    • Originally, specified in this paper written by researchers at Kirin, makers of a very fine product.
  • Blh protein cleaves B-carotene creating retinal

in particular see this figure where they demonstrate inducing both Blh and proteorhodopsin and getting red E.coli (an indicator of proteorhodopsin bound to retinal). The particular Blh-homolog used in this study was found in a BAC library from the same paper.

This project would involve "domesticating" this system by getting the relevant genes either chromosomally integrated or all on a single BioBrick plasmid. We would be able to tell its working at a first pass by just seeing that the cells turn red, and we could follow up by doing pH measurements as specified by Ed Delong's group.

Calvin Cycle

So check this out. In an effort to do directed evolution on RuBisCO, these folks partially reconstructed the Calvin cycle in E.Coli (wt E.coli naturally expresses 8 of 11 of the required calvin cycle enzymes), by adding RuBisCO & phosphoribulokinase from cyanobacteria PCC6301 and PCC7492, respectively. Anyway, these cells needed CO2 to grow (as well as a pentose C-source), so this looks to be something close to the other piece of the puzzle. I have to do my e.coli metabolism HWK, but it seems like it might be possible to fill in the other calvin cycle genes, hook up the light-driven proton pump and then grow on minimal media.

Construction

Testing

Feasibility & Timeline

Rought timeline:

  1. Order strains/plasmids/synthesis
  2. Research the existence proofs for each of the parallel tracks
  3. Research if the metabolic "fluxes" work out -- could we get sufficient energy out of the proton pumps?
  4. build the proton pump / metabolically engineer the calvin cycle in
  5. test each one
  6. integrate

Parallelizability

Seems parallelizable:

  1. Get the proton pump working
  2. Plug the proton gradient into the Calvin cycle

Intermediate goals

  1. Working proton pump (As tested via methods from DeLong group)
  2. Working calvin cycle, by the methods specified here.
  3. Final goal: integrate the two systems.

Significance

  1. Think of all the glucose that could be saved!
  2. Better model organism for studying "photosynthesis-sort-of", rather than slow growing cyanobacteria
  3. Better power supply/chassis?

Safety & ethics

  1. Would these be more fit then native e.coli? could they displace them in the environment?

Summary (pros/cons)

References

pACCAR16 plasmid structure
pACCAR16 plasmid structure
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