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Autoinducers

Autoinducers. 3OC6HSL is AI-1 which interacts with LuxR to activate pLuxCl, which activates Luxpr. 3OC12HSL is PAI-1, which interacts with LasR to activate pLAS, which activates pLAS Furanosyl borate diester is AI-2, which interacts with LuxQ to activate pLux, which activates pLuxpr.

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Autoinducers

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  1. Autoinducers • 3OC6HSL is AI-1 which interacts with LuxR to activate pLuxCl, which activates Luxpr. • 3OC12HSL is PAI-1, which interacts with LasR to activate pLAS, which activates pLAS • Furanosyl borate diester is AI-2, which interacts with LuxQ to activate pLux, which activates pLuxpr.

  2. We used 4, 2-Hybrid systems (alpha gal4) • AB -- ZiF • CD – T25 and T18 • EF • VW Apparently there is a 3 hybrid system. So we could use 3, 2-hybrid systems and 1, 3-hybrid system.

  3. Hin Recombinase • We found out this won’t work. • We can use small RNAs instead

  4. Gene of Interest PA1, PAL1, PA2 A 0 0 0 tet TET Gene A Luxpr pLaxCl B 0 0 1 Lux R tet R B C Spot42 RprA Gene B pLAS pLaS C 0 1 0 LAS R tet R A E Spot 42 rhyB Gene C pLUX pLux D 1 0 0 Lux Q tet R V D F RprA rhyB Gene D AB AB E 0 1 1 DsrA Gene E Spot 42 CD CD F 1 0 1 DsrA Gene F RprA EF EF G 1 1 0 DsrA Gene G Rhy B VW AB H 1 1 1 W DsrA Gene H

  5. Small RNAs in E. coli • All the ones in the following chart have a high efficiency • The following chart comes from “The Small RNA Regulators of Escherichia Coli: Roles and Mechanisms” by Susan Gottesman

  6. Quantification of Lux system • http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=176701&blobtype=pdf (This is not as applicable) • http://www.sciencedirect.com.proxy2.library.uiuc.edu/science?_ob=ArticleURL&_udi=B6WBK4PRHJ6K2&_user=571676&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000029040&_version=1&_urlVersion=0&_userid=571676&md5=ea6566137620b30f76b459cf252ad23a • (Log into the U of I library online database first)

  7. Efficiency of Autoinducers They used 3-oxo-hexanoyl-homoserine lactone (OHHL)

  8. Kinetics of Autoinducers We’re using a non-feedback system, so look at the triangles and the green.

  9. Yeast 2-Hybrid System 1 2 α Zif RNA Pol P(wk); weak Lac Promoter from E coli 10 bp 10 bp 10 bp 63 bp P(wk); weak Lac Promoter from E coli

  10. Fusion 1A; αGal4 protein Ala-Ala-Ala Linker 1 248 257 278 E. Coli RNA Polymerase Subunit A (residues 1-248) Yeast Gal4 protein (residues 58-92) On pACYC184 – derived pACL- αGal4 protein  1 PTG-inducible 1pp/lacUr5

  11. Fusion 2A; Gal 11P – Zif 123 AAAPVRTG Linker 1 89 113 207 Yeast Gal 11P (residues 263-352) *N341V mutation Zif 268 (residues 327-421) On pBR-GP-2123  Phagemid

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