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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.

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Reverse genetics: Site-directed mutagenesis in Thiomicrospira crunogena

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  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)

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