1 / 9

Reverse genetics: Site-directed mutagenesis in Thiomicrospira crunogena

Reverse genetics: Site-directed mutagenesis in Thiomicrospira crunogena. Terminologies. Forward genetics: Start with a phenotype. Find the genes responsible for a phenotype. Reverse genetics: Start with a/some gene(s) Figure out the traits they confer. Carbonic anhydrase reaction.

Download Presentation

Reverse genetics: Site-directed mutagenesis in Thiomicrospira crunogena

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Reverse genetics: Site-directed mutagenesis in Thiomicrospiracrunogena

  2. Terminologies • Forward genetics: • Start with a phenotype. • Find the genes responsible for a phenotype. • Reverse genetics: • Start with a/some gene(s) • Figure out the traits they confer

  3. Carbonic anhydrase reaction Slow (uncat.) Fast + carbonic anhydrase

  4. One possible vacuum ‘mechanism’ HCO3- HCO3- HCO3- CA CO2 CO2 Ru bisco biomass

  5. 3 carbonic anhydrase genes in T. crunogenome Two b-carbonic anhydrase genes One a-carbonic anhydrase gene

  6. Signal P 3.0 output, a-CA

  7. Elucidating gene function using site-directed mutagenesis 2. Ligate PCR product intoworkhorse plasmid (pRC3.1); TCCE Amplify target genesfrom T. crunogenagDNAvia PCR 3. Subject plasmid to Tn5-mediatedmutagenesis in vitro; TCCE 7. Mate into T. crunogena 4. Screen clones for Tn5-interruptedtarget gene 5. Amplify interrupted target genes via PCR 6. Ligate interrupted genesinto mating plasmid (pLD55); TCCE

  8. Do the CA genes play a role in CO2 vacuuming? HCO3- HCO3- HCO3- CA CO2 CO2 Ru bisco biomass

  9. Steps for testing whether CA is helpful for CO2 vacuuming • Try to grow site-directed mutants of CA genes on: • Hi CO2 plates (positive control) • Low CO2 plates (experimental)

More Related