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?
   • journal issue about Complex I - the proton pump that oxidzes NADH (which needs to operate in reverse in our case.)
     • 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.
     • Ref. 18
     • Ref. 19
     • 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

AUTOTROPHIC E. COLI

1	AE1	AE2	AE3	AE4	..........	..........	..........	..........	..........
10	AE1	AE2	AE3	AE4
19
28
37
46
55
64
73