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

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


Which genes encode the components

Which genes encode the components?


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

    • 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

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


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