Upload
justice-hindson
View
214
Download
0
Tags:
Embed Size (px)
Citation preview
Forward genetics
Finding genes in Thiomicrospira crunogena that 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
Thiomicrospira crunogena
–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
0
2
4
6
8
10
12
0 0.05 0.1 0.15 0.2 0.25
Extracellular DIC (mM)
Intr
ac
ellu
lar
DIC
(m
M)
Dobrinski, Longo, and Scott, 2005J. Bact. 187: 5741-5766.
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
– Tnp transposase OUTSIDE of transposon
– aph = kan resistance
Steps•Grow T. crunogena and E. coli separately•Allow them to mate <3 <3 <3•Cultivate T. crunogena transconjugants 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. crunogena happy 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
• 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. crunogena and not E. coli– Acid production from thiosulfate
Screen T. crunogena mutants for CO2 sensitivity
• Have any of them lost their ‘CO2-vacuuming’ ability?unable to grow under low-CO2 conditions