1. Forward genetics Finding genes in Thiomicrospiracrunogenathat are necessary for growth under low CO2 conditions

2. 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

3. Random mutagenesis of Thiomicrospiracrunogena Finding genes responsible for ‘CO2 vacuuming’

4. Thiomicrospiracrunogena Hydrothermal vent chemolithoautotroph .g-proteobacterium Oxidizes sulfur cpds for energy RAPID growth rate Erratic environment Bright and Scott, 1998

5. The ‘CO2 vacuum’ Can “trap” high conc’ns of bicarbonate inside their cells Dobrinski, Longo, and Scott, 2005 J. Bact. 187: 5741-5766.

6. One possible vacuum ‘mechanism’ HCO3- HCO3- HCO3- CA CO2 CO2 Ru bisco biomass

7. Which genes encode the components?

8. 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

9. 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

10. 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

11. 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)

12. 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 

13. 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

14. Screen T. crunogenamutants for CO2 sensitivity • Have any of them lost their ‘CO2-vacuuming’ ability? unable to grow under low-CO2 conditions

15. 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