1 / 17

Reverse genetics on a non-model organism

Reverse genetics on a non-model organism. Reverse genetics. Gene in hand. What’s its function?. Thiomicrospiras.

ciqala
Download Presentation

Reverse genetics on a non-model organism

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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.): Thiomicrospiracrunogena sp. nov., a colorless, sulfur-oxidizing bacterium from a deep-sea hydrothermal vent. Int. J. Syst. Bacteriol., 1985, 35, 422-424.

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

  5. Genetically manipulating a nonmodel organism • 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, 30oC • Mating interval (o/n) • Recovery interval (o/n) • Strength of initial counterselection • Antibiotic conc’n

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

  8. Our system: Thiomicrospiracrunogena Chemolithoautotroph *requirements for growth: O2, thiosulfate, CO2, 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 • Characterize chemotaxis in wild-type • Make 14 mutant strains • Screen the phenotype of the mutant strains • Write this up in a lab report.

  10. 1. Characterize chemotaxisin wild-type T. crunogena • We tested for chemotaxis toward: • High O2 • Low O2 • Thiosulfate • Phosphate • Nitrate • Ammonia • Bicarbonate

  11. Chemotaxis assay Cell suspension Chemotaxissolution

  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 2. Ligate PCR product intoworkhorse plasmid (pRC3.1); TCCE Amplify target genesfrom T. crunogenagDNAvia PCR 3. Subject plasmid to Tn5-mediatedmutagenesis in vitro; TCCE 7. Mate into T. crunogena 4. Screen clones for Tn5-interruptedtarget gene 5. Amplify interrupted target genes via PCR 6. Ligate interrupted genesinto mating plasmid (pLD55); TCCE

  14. E. coli T. cruno E. coli T. cruno • 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) RecA 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 kanamycinresistance on the recipient cell.

  15. Second selection to remove wt copy • Fusaric acid to impair TetR 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. crunogenainstead 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

More Related