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BioLogic

BioLogic. We’re going to use The Quorum System Small RNAs 2-Hybrid systems (and a 3-Hybrid system). Picture of sRNA system. Picture of the Lux Autoinducer system. Hybrid Systems. A+B B2H1A+B2H1B C+D B2H2A+B2H2B E+F B2H3A+B2H3B X+Y+Z B3H1A+B3H1B+B3H1C.

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BioLogic

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  1. BioLogic • We’re going to use • The Quorum System • Small RNAs • 2-Hybrid systems (and a 3-Hybrid system)

  2. Picture of sRNA system

  3. Picture of the Lux Autoinducer system

  4. Hybrid Systems • A+B B2H1A+B2H1B • C+D B2H2A+B2H2B • E+F B2H3A+B2H3B • X+Y+Z B3H1A+B3H1B+B3H1C Schematic of a 3-Hybrid System Schematic of a 2-Hybrid System

  5. Gene, Autoinducer(s) TET 1 - - - Gene 1 pLuxR pLuxI 2 + - - Lux R LuxI tet R X B C SgrS Gene 2 pRhiR pRhlI 3 - + - RhlR RhlI tet R Y A E SgrS Gene 3 ? ? 4 - - + ? ? tet R Z D F SgrS Gene 4 AB AB 5 + + - Spot42 Gene 5 SgrS CD CD 6 + - + Spot42 Gene 6 SgrS EF EF 7 - + + Spot42 Gene 7 SgrS XYZ XYZ 8 + + + Spot42 Gene 8

  6. Small RNAs in E. coli • We’re planning to use Spot 42 (which binds to the RBS) and SgrS (which binds to the 5’ UTR and recruits degradative enzymes) because there are professors on campus using them successfully. • We have access to strains of bacteria where the endogenous Spot 42 and SgrS systems have been knocked out. • Protocols 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.

  7. Autoinducers • We want to use autoinducers because of the quick reaction time. • We are planning to use LuxR and LuxI from Vibrio fisheri. This uses the autoinducer N-(3-Oxohexanoyl)-HSL. • Also the RhlI and RhlR system from Pseudomonas aeruginosa with the autoinducer N-(butyryl)-HSL • We have not characterized the final autoinducer system yet.

  8. Hybrid Systems • A+B B2H1A+B2H1B • C+D B2H2A+B2H2B • E+F B2H3A+B2H3B • X+Y+Z B3H1A+B3H1B+B3H1C Schematic of a 3-Hybrid System Schematic of a 2-Hybrid System

  9. Hybrid Systems Promoters 10 bp B2H1A+B2H1B B2H2A+B2H2B B2H3A+B2H3B B3H1A+B3H1B+B3H1C Zif269BS 63 bp P(wk); weak Lac Promoter from E coli 10 bp 63 bp TATA zifvar P(wk); weak Lac Promoter from E coli 10 bp P53zifvar 63 bp P(wk); weak Lac Promoter from E coli 10 bp OL2 62 bp P(wk); weak Lac Promoter from E coli

  10. B2H1 Ala-Ala-Ala Linker 1 248 257 278 B2H1A E. Coli RNA Polymerase Subunit A (residues 1-248) Yeast Gal4 protein (residues 58-92) AAAPVRTG Linker 1 89 113 207 B2H1B Yeast Gal 11P (residues 263-352) *N341V mutation Zif 268 (residues 327-421)

  11. B2H2 Ala-Ala-Ala Linker 1 248 257 823 B2H2A E. Coli RNA Polymerase Subunit A (residues 1-248) GacS (residues 253-819) AAAPVRTG Linker 1 89 113 B2H2B GacA TFIIB (DBD)

  12. B2H3 Ala-Ala-Ala Linker 1 248 257 B2H3A E. Coli RNA Polymerase Subunit A (residues 1-248) MavT AAAPVRTG Linker 1 62 86 B2H3B P53 (DBD) MavU (residues 1-62)

  13. B3H1 B3H1A FtsB B3H1B FtsL B3H1C CI (DBD) FtsW

  14. Pros Cons All the novel ideas makes it hard and complex. Time consuming. We need to characterize a third autoinducer. Each aspect of our project is a project within itself. There is a lot of data that we will need to reproduce. • It would be really cool and have lots interesting, novel aspects like the sRNA, hybrid systems, and autoinducer systems. • A quick reaction time. • We’d be introducing the hybrid system as a logic gate. • With sRNAs and hybrid system, you can create any comination of gates.

  15. Questions?!?!? • Time: • How much time would it take to make the 2:4? • How much time would it then take to complete the 3:8? • How practical is it to assume that we’ll be able to recreate the literature: • sRNAs • Hybrid Systems • Autoinducers • Are acyl-ACP and SAM naturally produced in E. coli?

  16. The End

  17. 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

  18. Gene of Interest System using 2 small RNAs PA1, PAL1, PA2 A 0 0 0 tet TET Gene A Luxpr pLaxCl B 1 0 0 Lux R tet R B C Spot 42 Gene B pLAS pLaS C 0 1 0 LAS R tet R A E Spot 42 Gene C pLUX pLux D 0 0 1 Lux Q tet R V D F Spot 42 Gene D AB AB E 1 1 0 MicA Gene E Spot 42 CD CD F 1 0 1 MicA Gene F Spot 42 EF EF G 0 1 1 MicA Gene G Spot 42 VW AB H 1 1 1 W Gene H

  19. 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 Spot 42 Gene B pLAS pLaS C 0 1 0 LAS R tet R Y A E Spot 42 Gene C pLUX pLux D 0 0 1 Lux Q tet R Z D F Spot 42 Gene D AB AB E 1 1 0 MicA Gene E Spot 42 CD CD F 1 0 1 MicA Gene F Spot 42 EF EF G 0 1 1 MicA Gene G Spot 42 XYZ XYZ H 1 1 1 MicA Gene H

  20. 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

  21. sRNAs that we’re going to use:

  22. MicA secondarystructure and binding

  23. 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.

  24. 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.

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

  26. Spot 42

  27. SgrS

  28. GcvB

  29. 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)

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

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

  32. 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.

  33. 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

  34. 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

  35. 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

  36. 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

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