1. Reverse genetics on a non-model organism

2. Reverse genetics Gene in hand. What’s its function?

3. Thiomicrospiras JANNASCH (H.W.), WIRSEN (C.O.), NELSON (D.C.) and ROBERTSON (L.A.): Thiomicrospira crunogena sp. nov., a colorless, sulfur-oxidizing bacterium from a deep-sea hydrothermal vent. Int. J. Syst. Bacteriol., 1985, 35, 422-424.

4. Competence? Conjugation? What works on the relatives? Genetically manipulating a nonmodel organism

5. Take a comprehensive EKS approach – Competence Electroporation – Buffers, voltages, growth stage Chemical competence – Buffers, growth stage, heat shock conditions Natural competence – Growth stage Vector – Conjugation Mating with E. coli = success! – pRL27 for random mutagenesis – pLD55 for site-directed mutagenesis

6. Things to tweak for mating Growth stage of recipient cells – Exponential, stationary? Mating medium – FW heterotroph + SW lithoautotroph = TLA? – TASW + LB, 30 o C Mating interval (o/n) Recovery interval (o/n) Strength of initial counterselection – Antibiotic conc’n

7. Functional genomics project Microbial physiology (MCB 4404L) Seniors 50 students (2X25) Bio/Microbio/BMS majors 2 student assistants (volunteers from class)

8. Our system: Thiomicrospira crunogena Chemolithoautotroph *requirements for growth: O 2, thiosulfate, CO 2, ammonia or nitrate, phosphate Motile 14 methyl-accepting chemotaxis protein (MCP) genes *14 ‘noses’ to sense nutrients or toxins *what does each MCP detect????

9. Steps to find what each MCP detects 1.Characterize chemotaxis in wild-type 2.Make 14 mutant strains 3.Screen the phenotype of the mutant strains 4.Write this up in a lab report.

10. 1. Characterize chemotaxis in wild-type T. crunogena We tested for chemotaxis toward: – High O 2 – Low O 2 – Thiosulfate – Phosphate – Nitrate – Ammonia – Bicarbonate

11. Chemotaxis assay Cell suspension Chemotaxis solution

12. 2. Make 14 mutant strains, each with one of its methyl-accepting chemotaxis genes interrupted

13. 2. Making 14 MCP mutant strains using site-directed mutagenesis 1.Amplify target genes from T. crunogena gDNA via PCR 2. Ligate PCR product into workhorse plasmid (pRC3.1); TCCE 3. Subject plasmid to Tn5-mediated mutagenesis in vitro; TCCE 4. Screen clones for Tn5-interrupted target gene 5. Amplify interrupted target genes via PCR 6. Ligate interrupted genes into mating plasmid (pLD55); TCCE 7. Mate into T. crunogena

14. 1.E. coli (blue) carrying a plasmid (black), which carries a plasmid that contains a methyl-accepting chemotaxis gene interrupted by a transposon (yellow) that contains a kanamycin resistance gene (red), is mated with T. crunogena (pink) E. coliT. crunoE. coli 2. The transposon (yellow) cannot hop off the plasmid, as this plasmid does not express a transposase enzyme. Instead, the RecA protein catalyzes homologous recombination between the mutated gene on the plasmid and the wild-type gene on the chromosome, conferring kanamycin resistance on the recipient cell. T. cruno RecA

15. Second selection to remove wt copy Fusaric acid to impair Tet R cells – Selects for double recombinants

16. 3. Screening the phenotype of the 14 MCP mutant strains Redo the chemotaxis assay, but use the mutant strains of T. crunogena instead of wild-type Does chemotaxis change in any of the mutants? Can we correlate a nutrient to a particular MCP?

17. Next semester Pick up where we left off Mate into T. crunogena See if chemotaxis behavior changes Functional complementation in E. coli