Export to Periplasm
- It appears our hydrogenase is exported to periplasm by the Tat pathway. Now R. etli also has this pathway, as it is involved somehow with N2 fixation.
- The Sec pathway appears to mess up protein folding "For example, green ﬂuorescent protein (GFP) can be translocated via both the Sec and Tat pathways but is only ﬂuorescent in the periplasm when exported by the Tat pathway." doi: 10.1146/annurev.micro.60.080805.142212
- Since R.etli has this pathway, as well as the Sec one, we should avoid having a promiscuous signal. "A net charge of +2 or higher resulted in Tat speciﬁcity while an overall charge of −1 or lower resulted in Sec and Tat promiscuity" doi: 10.1146/annurev.micro.60.080805.142212
- It appears that the chaperones encoded in the operon of Tat substrates are responsible for the delay in export to periplasm. We should look into our system and see if the HydA requires a chaperone, if said chaperone is present in R.etli / E.coli, and if not then who folded HydA in the E.coli experiments? Do we require said chaperone to avoid our protein getting shipped prematurely to periplasm? "Cytoplasmic chaperone-like proteins that function in the assembly of a number of cofactor and/or multimeric Tat substrates prevent targeting to the translocase until assembly is complete. The genes encoding these proteins are often found in the same operon as their corresponding Tat substrate proteins. These types of chaperones have recently been termed redox enzyme maturation proteins (REMPs)" doi: 10.1146/annurev.micro.60.080805.142212
- See reference 47 of said paper for more info on "engineering. Recently, a clever technique was developed for the selection of polypeptides that fold into a stable and soluble conformation."
- Signal peptide cleavage site?! Apparently, after translocation, our exported peptide will get cleaved at the signal site. Therefore, we would be required to add the localization AA motif to a terminal that is unrequired...
- If we design two types of chassis, one that fixes N2 and another that un-fixes H2, and mix them in the nodule, what would we expect?
- The nodule as a whole subunit must fix N2 to avoid termination by the plant, therefore N2 fixers are required.
- However, H2 un-fixers are more efficient since they do not have the metabolic load of N2 fixers, so they are favored.
- Is this a snowdrift game? doi:10.1038/nature07921
Adding the PFOR
- If we add the PFOR mentioned in the Silver paper (see previous entry), that would add extra electrons to the hydrogen reaction, tilting the production balance towards H2 production and against H2 fixation. This is an added bonus.