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BioLogic We’re going to use Autoinducers and the Lux system Small RNAs
2-Hybrid systems (and a 3-Hybrid system)
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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. LasR PAI 1 3OC12HSL LuxR AI-1 3OC6HSL LuxQ AI-2 Furanosyl borate diester
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Gene of Interest PA1, PAL1, PA2 A 0 0 0 tet B 1 0 0 C 0 1 0 D 0 0 1
Gene A Luxpr pLaxCl B Lux R tet R B C Spot42 SgrS Gene B pLAS pLaS C LAS R tet R A E OxyS GcvB Gene C pLUX pLux D Lux Q tet R V D F MicC RhyB Gene D AB AB AB E Autoinducers (Acyl-homoserine Lactone) MicA Gene E Spot 42 GcvB CD CD CD F MicA Gene F SgrS RhyB EF EF EF G MicA Gene G MicC OxyS VW VW AB H W MicA Gene H
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Same deal with a 3 Hybrid System
Gene of Interest Same deal with a 3 Hybrid System PA1, PAL1, PA2 A TET tet Gene A Luxpr pLaxCl B Lux R tet R X B C Spot42 SgrS Gene B pLAS pLaS C LAS R tet R Y A E OxyS GcvB Gene C pLUX pLux D Lux Q tet R Z D F MicC RhyB Gene D AB AB AB E Autoinducers (Acyl-homoserine Lactone) MicA Gene E Spot 42 GcvB CD CD CD F MicA Gene F SgrS RhyB EF EF EF G MicA Gene G MicC OxyS XYZ XYZ H MicA Gene H
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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 Escherichia coli small RNAs in vivo. Nucleic Acids Res. 2007;35(3): Epub 2007 Jan 30. PubMed PMID: ; PubMed Central PMCID: PMC Another good Source: Regulatory RNAs in Bacteria by Gisela Storz ;
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Category Number Examples Size (nt) Mechanism/role Regulators/comments References Hfq-binding, antisense 22 DsrA 85 Stimulates rpoS Inhibits hns Low temp., LeuO 58, 75 RprA 105 RcsC/B phosphorelay 59 OxyS 109 Anti-rpoS, fhlA OxyR 2,3 RyhB/SraI 90 Anti-sdh, sodB Fur 61 Spot 42 Anti-galK CRP/cAMP 68 MicF 93 Anti-ompF SoxR/S MicC 108 Anti-ompC Inverse to MicF 19,19a DicF 56 Anti-ftsZ Phage promoter 10 Antisense 3 RyeA/SraC 275 Anti-RyeB Unknown 100
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sRNAs that we’re going to use:
SgrS Spot42 (sign into the U of I library) OxyS GcvB MicC RyhB MicA Access the most recent version at doi: /gad Genes Dev : Klas I. Udekwu, Fabien Darfeuille, Jörg Vogel, et al. antisense RNA Hfq-dependent regulation of OmpA synthesis is mediated by an
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MicA secondarystructure and binding
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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.
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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.
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OxyS It negatively controls oxidative stress response within the cell.
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Spot 42
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SgrS
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GcvB
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Quantification of Lux system
(This is not as applicable) (Log into the U of I library online database first)
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Efficiency of Autoinducers
They used 3-oxo-hexanoyl-homoserine lactone (OHHL)
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Kinetics of Autoinducers
We’re using a non-feedback system, so look at the triangles and the green.
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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
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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
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On pACYC184 – derived pACL- αGal4 protein 1 PTG-inducible 1pp/lacUr5
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
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B2H1B; Gal 11P – Zif 123 AAAPVRTG Linker 1 89 113 207 Yeast Gal 11P
(residues ) *N341V mutation Zif 268 (residues ) On pBR-GP-2123 Phagemid
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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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