1 / 13

Chemotaxis study

Chemotaxis study. Chemotaxis in (some) bacteria. J. S. Parkinson, 2003. Our system: Thiomicrospira crunogena. Chemolithoautotroph *requirements for growth: O 2 , thiosulfate , CO 2 , ammonia or nitrate, phosphate Motile

nelle-leblanc
Download Presentation

Chemotaxis study

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. Chemotaxisstudy

  2. Chemotaxis in (some) bacteria J. S. Parkinson, 2003

  3. Our system: Thiomicrospiracrunogena Chemolithoautotroph *requirements for growth: O2, thiosulfate, CO2, ammonia or nitrate, phosphate Motile 14 methyl-accepting chemotaxis protein chemoreceptor genes *14 ‘noses’ to sense nutrients or toxins *5 PAS/PAC domain CRs: redox?

  4. Objective • Identify what each CR binds Oxygen? Thiosulfate? Carbon dioxide? Ammonia? Nitrate? Phosphate? ??????? Parkinson lab, University of Utah

  5. How to test each CR individually • Put the gene encoding each CR into a nonchemotactic strain of E. coli • See whether the E. coli becomes sensitive to a particular nutrient • May be possible to ‘pin’ a nutrient on a particular CR

  6. Host and vector details • E. coli UU2612 host • All MCP genes gone • Motile but not chemotactic • pRR48 vector • AmpR • MCS behind a lacpromotor • ‘empty’ • ‘full’ • E. coli aer • T. cru

  7. Steps • Purify genomic DNA from T. crunogena • Amplify CR genes via PCR • Ligate genes into pRR48 • Transform E. coli UU2612 with plasmids • Observe whether E. coli becomes aerotactic

  8. PCR of target chemoreceptor genes

  9. Target genes and expression vector cut with restriction enzymes PCR product = copy of T. cru gene with sticky restriction enzyme-digested ends pRR48 expression plasmid

  10. Target genes and expression vectorligated together PCR product = copy of T. cru gene with sticky restriction enzyme-digested ends pRR48 expression plasmid

  11. Transformed competent E. coli with expression vector carrying target genes http://www.novasep.com/technologies/Upstream-expression-system.asp

  12. Next step • Screen the E. coli carrying T. crunogenagenes for aerotactic behavior Redox?

  13. Chemotaxis plates to assay motility • Details: • Low agar concentration • Succinate as sole carbon source

More Related