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BioLogic

BioLogic. We’re going to use Autoinducers and the Lux system Small RNAs 2-Hybrid systems (and a 3-Hybrid system). Autoinducers. 3OC6HSL is AI-1 which interacts with LuxR to activate pLuxCl, which activates Luxpr.

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BioLogic

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  1. BioLogic • We’re going to use • Autoinducers and the Lux system • Small RNAs • 2-Hybrid systems (and a 3-Hybrid system)

  2. 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.

  3. Gene of Interest PA1, PAL1, PA2 A 0 0 0 tet TET Gene A Luxpr pLaxCl B 1 0 0 Lux R tet R B C Spot42 SgrS Gene B pLAS pLaS C 0 1 0 LAS R tet R A E OxyS GcvB Gene C pLUX pLux D 0 0 1 Lux Q tet R V D F MicC RhyB Gene D AB AB AB E 1 1 0 MicA Gene E Spot 42 GcvB CD CD CD F 1 0 1 MicA Gene F SgrS RhyB EF EF EF G 0 1 1 MicA Gene G MicC OxyS VW VW AB H 1 1 1 W MicA Gene H

  4. Gene of Interest Same deal with a 3 Hybrid System PA1, PAL1, PA2 A 0 0 0 tet TET Gene A Luxpr pLaxCl B 1 0 0 Lux R tet R X B C Spot42 SgrS Gene B pLAS pLaS C 0 1 0 LAS R tet R Y A E OxyS GcvB Gene C pLUX pLux D 0 0 1 Lux Q tet R Z D F MicC RhyB Gene D AB AB AB E 1 1 0 MicA Gene E Spot 42 GcvB CD CD CD F 1 0 1 MicA Gene F SgrS RhyB EF EF EF G 0 1 1 MicA Gene G MicC OxyS XYZ XYZ H 1 1 1 MicA 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 • Protocalls regarding working with sRNAs: Urban JH, Vogel J. Translational control and target recognition by Escherichiacoli small RNAs in vivo. Nucleic Acids Res. 2007;35(3):1018-37. Epub 2007 Jan 30.PubMed PMID: 17264113; PubMed Central PMCID: PMC1807950. • Another good Source: Regulatory RNAs in Bacteria by Gisela Storz ; http://www.sciencedirect.com.proxy2.library.uiuc.edu/science?_ob=ArticleURL&_udi=B6WSN-4VNHRSC-B&_user=571676&_coverDate=02%2F20%2F2009&_rdoc=10&_fmt=high&_orig=browse&_srch=doc-info(%23toc%237051%232009%23998639995%23933091%23FLA%23display%23Volume)&_cdi=7051&_sort=d&_docanchor=&_ct=22&_acct=C000029040&_version=1&_urlVersion=0&_userid=571676&md5=db7004c2567e64a29f9508281abc76ac

  6. sRNAs that we’re going to use:

  7. MicA secondarystructure and binding

  8. The part marked B in the upper left is the DNA sequence for the MicC gene.  The part marked A shows the binding site of MicC.

  9. RyhB Figure 2 Complementarity between the sdhCDAB operon and RyhB. Genes of the sdhCDAB operon are shown in A. Lines marked EM8 and EM9 show the position of the oligonucleotide probes used for Northern blots (Fig. 3). B shows the predicted interaction between RyhB and the sdhCDAB sense strand. The ribosome binding site for sdhD is underlined. The start codon for sdhD is shown underlined and in italics, and the stop codon for sdhC is shown in gray.

  10. OxyS It negatively controls oxidative stress response within the cell.

  11. Spot 42

  12. SgrS

  13. GcvB

  14. 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)

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

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

  17. Hybrid Systems • AB B2H1A+B2H1B • CD B2H2A+B2H2B • EF B2H3A+B2H3B • VW B2H4A+B2H4B • XYZ B3H1A+B3H1B+B3H1C Schematic of a 2-Hybrid System 1 2 α Zif RNA Pol Promoter

  18. 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

  19. B2H1A; 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

  20. B2H1B; 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

  21. Potential Problems • Lab conditions that need to be controlled limit the possible inputs. -inputs can’t alter Fe uptake, glucose uptake, pH, redox conditions, temperature, particular antibiotic susceptibility • 2-hybrid systems activate at different strengths • Time - there is a lot to do

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