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Forward genetics. Finding genes in Thiomicrospira crunogena that are necessary for growth under low CO 2 conditions. Terminologies. Forward genetics: Start with a phenotype. Find the genes responsible for a phenotype. Reverse genetics: Start with a/some gene(s)

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forward genetics

Forward genetics

Finding genes in Thiomicrospiracrunogenathat are necessary for growth

under low CO2 conditions

  • 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
random mutagenesis of thiomicrospira crunogena

Random mutagenesis of Thiomicrospiracrunogena

Finding genes responsible

for ‘CO2 vacuuming’

thiomicrospira crunogena

Hydrothermal vent chemolithoautotroph


Oxidizes sulfur cpds for energy

RAPID growth rate

Erratic environment

Bright and Scott, 1998

the co 2 vacuum
The ‘CO2 vacuum’

Can “trap” high conc’ns of bicarbonate inside their cells

Dobrinski, Longo, and Scott, 2005

J. Bact. 187: 5741-5766.

one possible vacuum mechanism
One possible vacuum ‘mechanism’










co 2 vacuuming genes via knockouts
CO2-vacuuming genes via knockouts

Knockout mutagenesis

Mate w/E. coli

Interrupt genes at random with a transposon

Screen for loss of CO2-vacuuming ability

Larsen, Metcalf et al., 2002

more details on the e coli host and prl27
More details on the E. coli host and pRL27
  • Host E. coli BW20767 genome encodes:
    • transfer functions necessary for pRL 27 transfer
    • Phage genes necessary for oriR6K replication (pir)
  • pRL27 encodes:
    • oriT for transfer
    • oriR6K for theta replication in appropriate host
    • Tnptransposase OUTSIDE of transposon
    • aph = kan resistance

Grow T. crunogenaand E. coli separately

Allow them to mate <3 <3 <3

Cultivate T. crunogenatransconjugants on recovery plates (+ kanamycin)

Larsen, Metcalf et al., 2002

mating e coli and t crunogena
Mating E. coli and T. crunogena
  • Mix suspensions of both types of cell
  • Pipette onto solid growth medium to create biofilm
  • Let mate overnight
  • ‘Mating medium’: keep both E. coli and T. crunogenahappy overnight
    • No antibiotic (T. crunogena)
    • Thiosulfate (T. crunogena)
    • Low salt (E. coli )
    • Yeast extract, tryptone (E. coli )
    • 32-34°C (E. coli, T. crunogena)
selection for t crunogena transconjugants
Selection for T. crunogenatransconjugants
  • Scrape mating biofilms off mating plates
  • Wash cells to remove tryptone and yeast extract
  • Spread cells on selective medium
    • Seawater salt concentrations, thiosulfate
      • T. crunogena , E. coli 
    • Does not contain tryptone and yeast extract
      • T. crunogena , E. coli 
    • Contains kanamycin
      • T. crunogena wild type , T. crunogena mutants 
1 2 wks later isolating kanamycin resistant knockout mutants
1-2 wks later: Isolating kanamycin-resistant knockout mutants
  • Pick colonies from mating plates to isolate them
  • Screen them to make sure they’re T. crunogenaand not E. coli
    • Acid production from thiosulfate
screen t crunogena mutants for co 2 sensitivity
Screen T. crunogenamutants for CO2 sensitivity
  • Have any of them lost their ‘CO2-vacuuming’ ability?

unable to grow under low-CO2 conditions

recap forward genetics to find genes responsible for co 2 vacuuming
Recap: Forward genetics to find genes responsible for CO2 vacuuming
  • Mate transposon into T. crunogena
  • Collect transconjugantT. crunogena
    • Each has one interrupted gene
  • Screen transconjugants for CO2 sensitivity