Reverse genetics site directed mutagenesis in thiomicrospira crunogena
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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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Reverse genetics site directed mutagenesis in thiomicrospira crunogena

Reverse genetics: Site-directed mutagenesis in Thiomicrospiracrunogena


Terminologies

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

Carbonic anhydrase reaction

Slow

(uncat.)

Fast

+

carbonic

anhydrase


One possible vacuum mechanism

One possible vacuum ‘mechanism’

HCO3-

HCO3-

HCO3-

CA

CO2

CO2

Ru

bisco

biomass


3 carbonic anhydrase genes in t cruno genome

3 carbonic anhydrase genes in T. crunogenome

Two b-carbonic anhydrase genes

One a-carbonic anhydrase gene


Signal p 3 0 output a ca

Signal P 3.0 output, a-CA


Elucidating gene function using site directed mutagenesis

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


Do the ca genes play a role in co 2 vacuuming

Do the CA genes play a role in CO2 vacuuming?

HCO3-

HCO3-

HCO3-

CA

CO2

CO2

Ru

bisco

biomass


Steps for testing whether ca is helpful for co 2 vacuuming

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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