Ralstonia eutropha TF93 Is Blocked in Tat-Mediated Protein Export

Use Phenazine methosulfate to visualise H2ase activity?
What's the Sec pathway in E coli?
broad-host-range vector pGE151

Expression of hydrogenase in Alcaligenes spp. is altered by interspecific plasmid exchange.
Doubling time of Ralstonia eutropha = 230 minutes

Co-translocation of a Periplasmic Enzyme Complex by a Hitchhiker Mechanism through the Bacterial Tat Pathway
Tat signal sequence on small subunit -> small + large getting transported
Sec pathway -> unfolded export

The Photobiological Production of Hydrogen: Potential Efficiency and Effectiveness as a Renewable Fuel
max efficiency with hydrogenase + water splitting = 40.7%

Screening technique patent
holy shit..so fucking clever. Couple hydrogenase expression with h2 uptake sensor and link that with expression of reporter gene!
HupR = regulator of uptake hydrogenase. Splice GUS in (not claimed by patent) and stick some X-gal plus IPTG in the media and there you go! Blue fucking cells! Brilliant!



Escherichia coli hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities
E coli h2ase = oxygen sensitive but maybe a good target for directed evolution>?

Development of Selection and Screening Procedures for Rapid Identification of H₂-Producing Algal Mutants with Increased O₂ Tolerance
Use of a chromagenic film to directly detect H2 production

The bacterial tryptophan reverse mutation assay with Escherichia coli WP2
Another possible screening mechanism? Couple h2ase 1/2 (uptake enzymes) with tryptophan synthesis enzyme lacking in E coli WP2 strain. Cells producing H2 will also produce the trp enzyme and thus be viable.

Protein engineering of hydrogenase 3 to enhance hydrogen production
- ePCR followed by gene shuffling followed by saturation mutagenesis at the change site was very effective in getting enhanced mutants
- They cultured anaerobically. Maybe aerobic culture would promote oxygen insensitive h2ase mutants? Use uptake h2ase from R eutropha to screen?