IGEM:MIT/2006/Autotrophic E.coli

Progress
6/30/2006 - recieved 4 strains from Ichiro Matsumura (thanks!).

Overall Strategy

 * Reconstitute functional Calvin Cycle in E. coli.
 * making use of Synechococcus RuBisCO and PrkA from Parikh et al. (Protein Engineering, 2006)
 * making use of sedoheptulose-1,7-bisphosphatase (SBPase, EC 3.1.3.37) or a dual-purpose SBPase/fructose-1,6-bisphosphatase (SBPase/FBPase, EC 3.1.3.11)
 * making use of proteorhodopsin from Beja et al. (Science, 2000)


 * General metabolism pathway reference (Sigma)


 * Review of autotrophic CO2-fixation mechanisms in Archaea


 * Jason is poop.

To Do List

 * 1) Biobrick the three Calvin cycle enzymes
 * 2) Find appropriate metbolite for testing the function of the three enzymes
 * 3) Can we run the Calvin cycle and glycolysis in the same cellular compartment?
 * 4) We need to do energy/mass balances on Calvin Cycle, etc

Research Questions

 * 1) Is there an intermediate metabolite in the calvin cycle that would be a decent test for whether all 3 new enzymes were working properly?
 * 2) Detailed work-up of the energy flux that is needed? Would we be able to get enough out of PR in order to drive the Calvin cycle?
 * 3) Will we have trouble getting the Calvin cycle to run in the same compartment as glycolysis? In plants they seem to use the chlorplast to help isolate metabolites.  However, I think cyanobacteria do it all in one compartment.
 * 4) How does reverse e-transport work in detail?
 * 5) *journal issue about Complex I - the proton pump that oxidzes NADH (which needs to operate in reverse in our case.)
 * 6) **E. coli Complex I review - E. coli has two NADH:ubiquinone oxireductases, NDH-1 and NDH-2. Only NDH-1, a 13-subunit membrane-bound complex, couples NADH oxidation (to NAD+) and ubiquinone reduction (to ubiquinol) to transmembrane proton flux.
 * 7) **Ref. 18
 * 8) **Ref. 19
 * 9) **Figure 3 from Ingledew and Poole, "Respiratory Chains of E. Coli." Microbiol Rev, Sept. 1984:

Strains
''Strains are stored in -80C in the Endy lab in Autotrophic E.coli box. AE = Autorphic E.coli, we will use that convention for non-BB strains that are stored for this project.''
 * AE1 -
 * pBR322
 * pLysS
 * K-12, LB-amp-chl
 * AE2 -
 * pBR322
 * arap-6his-WT-prkA-pLysS
 * K-12, LB-amp-chl
 * AE3 -
 * rbcLS-pMAL-p2x
 * pLysS
 * K-12, LB-amp-chl
 * AE4 -
 * rbcLS-pMAL-p2x
 * arap-6his-WT-prkA-pLysS
 * K-12, LB-amp-chl