IGEM:MIT/2006/Autotrophic E.coli
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Timeline
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)
- Jason is poop.
To Do List
- Biobrick the three Calvin cycle enzymes
- Find appropriate metbolite for testing the function of the three enzymes
- Can we run the Calvin cycle and glycolysis in the same cellular compartment?
- We need to do energy/mass balances on Calvin Cycle, etc
Research Questions
- Is there an intermediate metabolite in the calvin cycle that would be a decent test for whether all 3 new enzymes were working properly?
- 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?
- 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.
- 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:
- journal issue about Complex I - the proton pump that oxidzes NADH (which needs to operate in reverse in our case.)
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
1 | AE1 | AE2 | AE3 | AE4 | .......... | .......... | .......... | .......... | .......... |
---|---|---|---|---|---|---|---|---|---|
10 | AE1 | AE2 | AE3 | AE4 | |||||
19 | |||||||||
28 | |||||||||
37 | |||||||||
46 | |||||||||
55 | |||||||||
64 | |||||||||
73 |