October 6 th 2012 Technion – Israel Institute of Technology
Overview Reporters Riboswitch Phage Summary RNAPs Overview Technion – Israel Institute of Technology 2
Overview Overview Reporters Riboswitch Phage Summary RNAPs YES gates RNAPs Reporters Phage Summary Complex High 3D output specificity Yes No No No Analyze Environment Appropriate Response Technion – Israel Institute of Technology 3
Overview Reporters Riboswitch Phage Summary RNAPs Technion – Israel Institute of Technology 4
Overview Reporters Riboswitch Phage Summary RNAPs Lambda Phage Proof of concept E. coli Technion – Israel Institute of Technology
Overview Overview Reporters Riboswitch Phage Summary RNAPs YES gates RNAPs Reporters Phage Summary 3 different inducers Engineered insert in the phage genome Antibiotic resistance YES gates plasmid Proteins essential for lytic cycle Genes essential for lytic cycle Modified phage X X X genome Technion – Israel Institute of Technology 6
Overview Overview Reporters Riboswitch Phage Summary RNAPs YES gates RNAPs Reporters Phage Summary The YES gates The AND gate Technion – Israel Institute of Technology 7
Overview Overview Reporters Riboswitch Phage Summary RNAPs YES gates RNAPs Reporters Phage Summary RNA Novel Reporter Polymerases System The Phage 3 rd YES Gate and Riboswitch RNAP Technion – Israel Institute of Technology 8
RNAPs Overview Reporters Riboswitch Phage Summary RNAPs RNAP Technion – Israel Institute of Technology 9
RNAPs Overview Reporters Riboswitch Phage Summary The Requirements Host Tunable Orthogonality Compatibility Strength Technion – Israel Institute of Technology 10
RNAPs Overview Reporters Riboswitch Phage Summary The Sources Wild Type Engineered T7* T7*(T3) T7*(N4) T7*(K1F) T7 T3 SP6 Technion – Israel Institute of Technology 11
RNAPs Overview Reporters Riboswitch Phage Summary YES Regulatory Inducer gate element 1 P TetO Anhydrous tetracycline 2 P Lac/Ara Lactose+arabinose Technion – Israel Institute of Technology 12
RNAPs Overview Reporters Riboswitch Phage Summary RNAP Cloning RNAP+ Complete Still in YES gate Progress Terminator T7* T7*(N4) T7*(T3) T3 WT T7 WT T7*(K1F) SP6 WT Technion – Israel Institute of Technology 13
RNAPs Overview Reporters Riboswitch Phage Summary T7* RNAP Fluorescence phoA Technion – Israel Institute of Technology 14
RNAPs Reporters Overview Riboswitch Phage Summary Reporter Assay Technion – Israel Institute of Technology 15
RNAPs Reporters Overview Riboswitch Phage Summary Indicate and quantify RNA polymerase activity Test for cross reactivity phoA: PNPP+H 2 O PNP (yellow) +PO 4 2- xylE : catechol 2-Hydroxymuconic semialdehyde (yellow) Technion – Israel Institute of Technology 16
RNAPs Reporters Overview Riboswitch Phage Summary PET System Technion – Israel Institute of Technology 17
RNAPs Reporters Overview Riboswitch Phage Summary phoA xylE With Without With Without catechol catechol PNPP PNPP Technion – Israel Institute of Technology 18
RNAPs Reporters Overview Riboswitch Phage Summary pT7-phoA characterization pT7-xylE characterization Technion – Israel Institute of Technology 19
RNAPs Reporters Overview Riboswitch Phage Summary P T7 P T7 P T3 P K1F P T3 P SP6 phoA xylE P N4 P SP6 P N4 Technion – Israel Institute of Technology 20
Riboswitch Overview RNAPs Reporters Phage Summary Theophylline Riboswitch Technion – Israel Institute of Technology 21
Riboswitch Overview RNAPs Reporters Phage Summary YES Regulatory Inducer gate element 3 P lux +riboswitch 3OC 6 HSL+theophylline Technion – Israel Institute of Technology 22
Riboswitch Overview RNAPs Reporters Phage Summary = Theophylline Adopted from Lynch S. A., Gallivan J. P. 2009. Nucleic Acids Research 37 (1): 184-192 Technion – Israel Institute of Technology 23
Riboswitch Overview RNAPs Reporters Phage Summary Riboswitch+RNAP Fusions Initial Still in Standardized Fusion Progress T7 WT T7*(T3) T7* T7*(N4) T7*(K1F) T3 WT SP6 WT RFC10 Technion – Israel Institute of Technology 24
Riboswitch Overview RNAPs Reporters Phage Summary Riboswitch+mCherry High fluorescence Secondary No structure induction analysis Constitutive translation due to effects of the coding region Technion – Israel Institute of Technology 25
Riboswitch Overview RNAPs Reporters Phage Summary Riboswitch+T7*(N4) 0mM theophylline 1.97 negative control Technion – Israel Institute of Technology 26
Riboswitch Overview RNAPs Reporters Phage Summary Riboswitch+T7*(N4) 4.5 4 3.5 xyIE Absorbance 380nm 3 2.5 2 1.5 1 0.5 0 pN4 pK1F pT3 pT7 negative Promoter type Technion – Israel Institute of Technology 27
Phage Overview RNAPs Reporters Riboswitch Summary The Phage Technion – Israel Institute of Technology 28
Phage Overview RNAPs Reporters Riboswitch Summary λ ¡ phage strain - cI857s7 – heat inducible lysis Genetic manipulations Modified phage genome X X X Technion – Israel Institute of Technology 29
Phage Overview RNAPs Reporters Riboswitch Summary The Fragments Technion – Israel Institute of Technology 30
Phage Overview RNAPs Reporters Riboswitch Summary pSB1C3+MCS Technion – Israel Institute of Technology 31
Phage Overview RNAPs Reporters Riboswitch Summary The Fragments Technion – Israel Institute of Technology 32
Phage Overview RNAPs Reporters Riboswitch Summary Gibson Assembly Calibration Technion – Israel Institute of Technology 33
Phage Overview RNAPs Reporters Riboswitch Summary Three Fragments Technion – Israel Institute of Technology 34
Phage Overview RNAPs Reporters Riboswitch Summary Future Directions The Q gene Antibiotic resistance Technion – Israel Institute of Technology 35
Summary Overview RNAPs Reporters Riboswitch Phage Summary • Orthogonal RNAPs • RS+RNAP and RS+mCherry constructs • Novel reporter assay • Phage fragments cloning • Phage fragments assembly Technion – Israel Institute of Technology 36
Summary Overview RNAPs Reporters Riboswitch Phage Attributions • Ann K. Ganesan - WT T7 RNAP • Changwon Kang - WT SP6 RNAP • Christopher A. Voight - T7*, T7*(T3), T7*(N4), T7*(K1F) RNAPs with suitable induced promoters: pT7, pT3, pK1F, pN4 • Gallivan Lab (Gallivan Justin P., Lynch Sean A.) - Theophylline riboswitch and tac promoter. • Roee's Lab (Roee Amit) - Cerulean FP under control of pLac/Ara, mCitrin FP and mCherry under control of pTetO. Thanks • Advisers - Dr. Roee Amit, Dr. Orna Attar, Dr. Sharon Levi-Tal • Sarah, Aya, Michal and Slava from Roee’s lab • Dean of the faculty of Food and Biotechnology Engineering - Prof. Ben-Zion Levi Technion – Israel Institute of Technology 37
Summary Overview RNAPs Reporters Riboswitch Phage Sponsors Technion – Israel Institute of Technology 38
Summary Overview RNAPs Reporters Riboswitch Phage Questions Technion – Israel Institute of Technology 39
Summary Overview RNAPs Reporters Riboswitch Phage References 1. Milo R., et al. 2009. BioNumbers--the database of key numbers in molecular and cell biology. Nucleic Acids Research 38 (Database): D750-D753 . 2. Gibson D. G., et al. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6 (5): 343-345 . 3. Lynch S. A., Gallivan J. P. 2009. A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Research 37 (1): 184-192 . 4. Shaner N. C., Steinbach P. A., Tsien R. Y. 2005. A guide to choosing fluorescent proteins. Nature Methods 2 (12): 905-909 . 5. Shaner N. C., et al. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22 (12): 1567-1572 . 6. Lutz R., Bujard H. 1997. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research 25 (6): 1203-1210 . 7. Gottesman M. E., Weisberg R. A. 2004. Little Lambda, Who Made Thee? Microbiology and Molecular Biology Reviews 68 (4): 796-813 . Technion – Israel Institute of Technology 40
Summary Overview RNAPs Reporters Riboswitch Phage References 8. Temme K., et al. 2012. Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic Acids Research (advance access): 1-9 . 9. Shin I., Kang C. 2003. Mutational analysis and structure of the phage SP6 promoter. Methods in Enzymology 370 : 658-668 . 10. Zadeh J. N., et al. 2011. NUPACK: Analysis and design of nucleic acid systems. Journal of Computational Chemistry 32 (1): 170-173 . 11. Yon J., Fried M. 1989. Precise gene fusion by PCR. Nucleic Acids Research 17 (12 ). 12. Rajagopala S., Casjens S., Uetz P. 2011. The protein interaction map of bacteriophage lambda. BMC Microbiology 11 (1 ). 13. Moran Uri, Phillips Rob, Milo Ron. 2010. SnapShot: Key Numbers in Biology. Cell 141 (7): 1262-126 2. e1261 . 14. Suess B., et al. 2004. A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Research 32 (4): 1610-1614 . Technion – Israel Institute of Technology 41
Phage Overview RNAPs Reporters Riboswitch Summary Hidden slides Technion – Israel Institute of Technology 42
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