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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

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
random mutagenesis of thiomicrospira crunogena

Random mutagenesis of Thiomicrospiracrunogena

Finding genes responsible

for ‘CO2 vacuuming’

thiomicrospira crunogena
Thiomicrospiracrunogena

Hydrothermal vent chemolithoautotroph

.g-proteobacterium

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’

HCO3-

HCO3-

HCO3-

CA

CO2

CO2

Ru

bisco

biomass

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
steps
Steps

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