Forward genetics
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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

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Forward genetics

Finding genes in Thiomicrospiracrunogenathat are necessary for growth

under low CO2 conditions


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 Thiomicrospiracrunogena

Finding genes responsible

for ‘CO2 vacuuming’


Thiomicrospiracrunogena

Hydrothermal vent chemolithoautotroph

.g-proteobacterium

Oxidizes sulfur cpds for energy

RAPID growth rate

Erratic environment

Bright and Scott, 1998


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’

HCO3-

HCO3-

HCO3-

CA

CO2

CO2

Ru

bisco

biomass


Which genes encode the components?


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

  • Host E. coli BW20767 genome encodes:

    • transfer functions necessary for pRL 27 transfer

    • Transposase inhibitor

    • 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

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

  • 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. 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

  • 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. 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 CO2 vacuuming

  • Mate transposon into T. crunogena

  • Collect transconjugantT. crunogena

    • Each has one interrupted gene

  • Screen transconjugants for CO2 sensitivity


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