Genetics Chapter 9.1. Genetics Genetics - the study of genes and heredity.
Forward genetics Finding genes in Thiomicrospira crunogena that are necessary for growth under low...
-
Upload
justice-hindson -
Category
Documents
-
view
213 -
download
0
Transcript of Forward genetics Finding genes in Thiomicrospira crunogena that are necessary for growth under low...
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