October 6 th 2012 Technion Israel Institute of Technology Overview - - PowerPoint PPT Presentation

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October 6 th 2012 Technion Israel Institute of Technology Overview - - PowerPoint PPT Presentation

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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

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Technion – Israel Institute of Technology

October 6th 2012

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Technion – Israel Institute of Technology

Overview

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Riboswitch Reporters Phage Summary RNAPs

Overview

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Technion – Israel Institute of Technology

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Overview

YES gates RNAPs Reporters Phage Summary

High specificity

No No No Yes

Complex 3D output

Analyze Environment  Appropriate Response Riboswitch Reporters Phage Summary RNAPs

Overview

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Technion – Israel Institute of Technology

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Riboswitch Reporters Phage Summary RNAPs

Overview

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Technion – Israel Institute of Technology

Proof of concept

  • E. coli

Lambda Phage

Riboswitch Reporters Phage Summary RNAPs

Overview

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Technion – Israel Institute of Technology YES gates plasmid Antibiotic resistance

X X X

Modified phage genome Engineered insert in the phage genome Proteins essential for lytic cycle Genes essential for lytic cycle

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Overview

YES gates RNAPs Reporters Phage Summary

3 different inducers

Riboswitch Reporters Phage Summary RNAPs

Overview

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Technion – Israel Institute of Technology The YES gates The AND gate

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Overview

YES gates RNAPs Reporters Phage Summary Riboswitch Reporters Phage Summary RNAPs

Overview

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Technion – Israel Institute of Technology

3rd YES Gate and Riboswitch RNA Polymerases Novel Reporter System The Phage

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Overview

YES gates RNAPs Reporters Phage Summary

RNAP

Riboswitch Reporters Phage Summary RNAPs

Overview

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Technion – Israel Institute of Technology

RNAPs

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RNAP

Overview Riboswitch

RNAPs

Reporters Phage Summary

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Technion – Israel Institute of Technology

The Requirements

Orthogonality Host Compatibility Tunable Strength

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Overview Riboswitch

RNAPs

Reporters Phage Summary

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Technion – Israel Institute of Technology

The Sources

Wild Type T7 T3 SP6 Engineered T7* T7*(T3) T7*(N4) T7*(K1F)

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Overview Riboswitch

RNAPs

Reporters Phage Summary

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Technion – Israel Institute of Technology

Inducer Regulatory element YES gate Anhydrous tetracycline PTetO 1 Lactose+arabinose PLac/Ara 2

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Overview Riboswitch

RNAPs

Reporters Phage Summary

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Technion – Israel Institute of Technology

Complete YES gate

T7*

RNAP+ Terminator

T7*(N4) T3 WT T7*(K1F)

Still in Progress

T7*(T3) T7 WT SP6 WT

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RNAP Cloning

Overview Riboswitch

RNAPs

Reporters Phage Summary

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Technion – Israel Institute of Technology

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Overview Riboswitch

RNAPs

Reporters Phage Summary

T7* RNAP

Fluorescence phoA

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Technion – Israel Institute of Technology

Reporter Assay

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Overview Riboswitch RNAPs Reporters Phage Summary

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Technion – Israel Institute of Technology

Indicate and quantify RNA polymerase activity Test for cross reactivity

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phoA: PNPP+H2OPNP(yellow)+PO4

2-

xylE : catechol  2-Hydroxymuconic semialdehyde (yellow)

Overview Riboswitch RNAPs Reporters Phage Summary

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Technion – Israel Institute of Technology

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PET System

Overview Riboswitch RNAPs Reporters Phage Summary

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Technion – Israel Institute of Technology

xylE

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Without catechol With catechol

phoA

Without PNPP With PNPP Overview Riboswitch RNAPs Reporters Phage Summary

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Technion – Israel Institute of Technology

pT7-phoA characterization pT7-xylE characterization

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Overview Riboswitch RNAPs Reporters Phage Summary

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Technion – Israel Institute of Technology

xylE

PT7 PK1F PSP6 PN4 PT3

phoA

PT7 PSP6 PN4 PT3

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Overview Riboswitch RNAPs Reporters Phage Summary

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Technion – Israel Institute of Technology

Theophylline Riboswitch

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Overview

Riboswitch

RNAPs Reporters Phage Summary

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Inducer Regulatory element YES gate 3OC6HSL+theophylline Plux+riboswitch 3 Overview

Riboswitch

RNAPs Reporters Phage Summary

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Technion – Israel Institute of Technology

= Theophylline

Adopted from Lynch S. A., Gallivan J. P. 2009. Nucleic Acids Research 37(1): 184-192

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Overview

Riboswitch

RNAPs Reporters Phage Summary

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Technion – Israel Institute of Technology RFC10

Standardized T7 WT T7*(N4) Initial Fusion T7*(T3) T7*(K1F) Still in Progress T7* T3 WT SP6 WT

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Riboswitch+RNAP Fusions

Overview

Riboswitch

RNAPs Reporters Phage Summary

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Technion – Israel Institute of Technology

Constitutive translation due to effects

  • f the coding

region

Secondary structure analysis High fluorescence No induction

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Overview

Riboswitch

RNAPs Reporters Phage Summary

Riboswitch+mCherry

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Technion – Israel Institute of Technology

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Overview

Riboswitch

RNAPs Reporters Phage Summary

Riboswitch+T7*(N4)

1.97 0mM theophylline negative control

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Technion – Israel Institute of Technology

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Overview

Riboswitch

RNAPs Reporters Phage Summary

Riboswitch+T7*(N4)

0.5 1 1.5 2 2.5 3 3.5 4 4.5 pN4 pK1F pT3 pT7 negative

xyIE Absorbance 380nm Promoter type

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Technion – Israel Institute of Technology

The Phage

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Overview

Phage

Summary RNAPs Riboswitch Reporters

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λ ¡phage strain - cI857s7 – heat inducible lysis Genetic manipulations

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Modified phage genome

Overview

Phage

Summary RNAPs Riboswitch Reporters

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Technion – Israel Institute of Technology

The Fragments

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Overview

Phage

Summary RNAPs Riboswitch Reporters

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pSB1C3+MCS

Overview

Phage

Summary RNAPs Riboswitch Reporters

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Technion – Israel Institute of Technology

The Fragments

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Overview

Phage

Summary RNAPs Riboswitch Reporters

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Gibson Assembly Calibration

Overview

Phage

Summary RNAPs Riboswitch Reporters

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Three Fragments

Overview

Phage

Summary RNAPs Riboswitch Reporters

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Technion – Israel Institute of Technology

Antibiotic resistance The Q gene

Future Directions

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Overview

Phage

Summary RNAPs Riboswitch Reporters

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Technion – Israel Institute of Technology

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Overview

Summary

Reporters Phage

Summary

  • Orthogonal RNAPs
  • RS+RNAP and RS+mCherry constructs
  • Novel reporter assay
  • Phage fragments cloning
  • Phage fragments assembly

RNAPs Riboswitch

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Technion – Israel Institute of Technology

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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

Overview

Summary

Reporters Phage RNAPs Riboswitch

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Technion – Israel Institute of Technology

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Sponsors

Overview

Summary

Reporters Phage RNAPs Riboswitch

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Questions

Overview

Summary

Reporters Phage RNAPs Riboswitch

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Technion – Israel Institute of Technology 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.

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References

Overview

Summary

Reporters Phage RNAPs Riboswitch

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Technion – Israel Institute of Technology 8. Temme K., et al. 2012. Modular control of multiple pathways using engineered

  • rthogonal 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

  • f 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): 1262-1262.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.

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References

Overview

Summary

Reporters Phage RNAPs Riboswitch

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Technion – Israel Institute of Technology

Hidden slides

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Overview

Phage

Summary RNAPs Riboswitch Reporters

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Technion – Israel Institute of Technology

Theophylline dose response

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1000 2000 3000 4000 5000 6000 7000 0.01 0.1 1 10 Fluorescence/O.D. Theophylline concentration (mM) RS+mChery RBS+mCherry

Overview Summary Reporters Phage RNAPs Riboswitch

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Technion – Israel Institute of Technology

Theophylline dose response

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Overview Summary Reporters Phage RNAPs Riboswitch

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Overview Summary Reporters Phage RNAPs Riboswitch

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Future Directions

  • Replace mCherry
  • Fusions to previously working constructs
  • Degradation tags

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Overview Summary Reporters Phage RNAPs Riboswitch

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Technion – Israel Institute of Technology

T C T A G AT C T A G AT C TA G A

XbaI BglII dam methylation

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Overview Summary Reporters Phage RNAPs Riboswitch

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Overview Summary Reporters Phage RNAPs Riboswitch

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Technion – Israel Institute of Technology 3’ 5’ 5’ 3’

5’ Exonuclease chews back 5’ ends and DNA fragments anneal

3’ 5’ 5’ 3’

T5 Exonuclease concentration Chew back Overlap

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Overview Summary Reporters Phage RNAPs Riboswitch

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Overview Summary Reporters Phage RNAPs Riboswitch

ALP vs. xyIE

ALP A homodimer A homotetramer

Escherichia coli. Organism_taxid: 562. Gene: phoa. Expressed in: escherichia coli. Expression_system_taxid: 562. Restriction

  • fragment. Other_details: lac promoter. For more information about the expression system consult mandecki et al. Gene 94, 103-
  • 107. (1990)

xyIE