De Novo Adaption of Streptococcus thermophilius CRISPR in - - PowerPoint PPT Presentation

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De Novo Adaption of Streptococcus thermophilius CRISPR in Escherichia coli Mitesh Agrawal, Calvin Goveia, Kettner Griswold Hannah Keith, Priya Kurani, Paul Sebexen GEORGIA INSTITUTE OF TECHNOLOGY INTRODUCTION Abstract GEORGIA INSTITUTE OF

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De Novo Adaption of Streptococcus thermophilius CRISPR in Escherichia coli

GEORGIA INSTITUTE OF TECHNOLOGY

Mitesh Agrawal, Calvin Goveia, Kettner Griswold Hannah Keith, Priya Kurani, Paul Sebexen

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INTRODUCTION

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Abstract

The ultimate goal of our project was to utilize the CRISPR system as a mobile gene targeting system capable of eliminating antibiotic resistance. The protoype construct, pSTINGER, was designed for quantitative modeling, proof of concept, and vaccination

  • n a single plasmid. As a result of experimental error,

intellectual property agreements, and material availability, our biobrick constructs remain experimentally theoretical, though we have rigorously modeled our system in various scenarios.

GEORGIA INSTITUTE OF TECHNOLOGY
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Objectives

  • Utilize the CRISPR system as a mobile

gene targeting system capable of eliminating antibiotic resistance

  • Rigorously model our system to prove its

functionality as a vaccination on a single plasmid

  • Further human health by providing a tool

in the fight against resistant strains

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CRISPR

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Components of the Locus

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  • Cas genes
  • Leader sequence
  • Repeats
  • Spacers
  • Terminator sequence
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Repeat

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CRISPR

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Streptococcus thermophilus

  • Possesses a well-documented CRISPR system
  • Lactic acid bacteria used in industrial fermentation to

produce yogurt

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Superbugs

  • Antibiotic susceptible

bacteria can gain immunity to antibiotics through horizontal gene transfer

  • Immunity is granted

through expression of an enzyme present on a plasmid

  • CRISPR can target this

plasmid and eliminate the resistance

  • Examples: MRSA, NDM-1

http://en.wikipedia.org/wiki/Super_bug_(bacteria)

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EXPERIMENTS

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Mobile CRISPR system from S. thermophilus DGCC 7710 Strain

Mobilize CRISPR system

  • n a plasmid

Biobrick Constructs Chromosomal Integration

  • f CRISPR1 locus from S.

thermophilus to B. subtilis Experiments and Future Applications

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Mobile CRISPR system from S. thermophilus DGCC 7710 Strain

  • S. thermophilus

DGCC 7710 strain

  • E. coli

CRISPR locus amplified from DGCC strain with kan spacer Shuttle vector with kanamycin resistance pNT1 vector with CRISPR locus No growth on kanamycin plates Expected outcome

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CRISPR from S. thermophilus LMD9 Strain and Biobricks

  • CRISPR sequence is analogous to the DGCC strain
  • Amplify only Spacer/Repeat Region and ligate it into the

pSB1C3 vector

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Bba_K582000

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CRISPR from S. thermophilus DGCC LMD9 Strain and Biobricks

  • cas9 itself is sufficient for the expression of

CRISPR

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Bba_K582003

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Chromosomal Integration of CRISPR in Bacillus Subtilis

  • B. subtilis is a model organism
  • Similar promoters present as in S. thermophilus
  • Integration vector ece113/pDG1662 used
  • S. thermophilus

DGCC 7710 strain CRISPR locus amplified from DGCC strain with kan spacer ece113 vector with CRISPR locus Bacillus Subtilis with CRISPR locus integrated in its chromosome Expected Outcome

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Results/Explanation

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FUTURE

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The Ideal Vaccine

  • Mechanism for rendering antibiotic

susceptibility

  • Method for introduction into population.
  • Mechanism for transfer across a population
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A Conjugating Vaccine

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Introduction to Population

Transformation Conjugation

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Rendering Antibiotic Susceptibility

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Mechanisms for delivery of pSTINGER

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Nonlysogenic Phage Delivery of Cosmid DNA

  • In vitro packaged lambda

phage

  • Common phage therapy

leads to resistant plasmids in solution.

  • Added cos sites for

packaging

  • Immune response to

phages has been experimentally low

  • Extremely host specific
  • Transforms E. Coli

Obligative Predatory Cells Bearing Cosmid DNA

  • Bdellovibrio

Bacteriovorus

  • Probiotic delivery in

chickens successful

  • Broad spectrum gram-

negative prey

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Mechansms for delivery of pSTINGER

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Lambda Phage Mechanisms

  • B. bacteriovorus

Mechanisms

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Methods for Transfer Across a Population

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Origins of Replication

  • pIP501 derived

transgenic Ori

  • pMB1 (puc18

derivative) Origins of Transfer

  • F-plasmid origin
  • IncP type
  • IncQ type
  • Pheremone

type

  • Conjugative

transposon Tn4555

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Rendering Antibiotic Susceptibility

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MODELING

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Population Model of E.coli

Consider 3 Sub-Populations

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Logistic Growth

  • Consider 3 Sub-Populations

Basic form: Specific Growth:

Image Credit: Public Domain

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Interaction Model

  • Fundamental System of form Y’=AY
  • Parameters expand into operators of form
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Model in Matrix Notation

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Rewrite as a System of Equations

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Numerical Solutions

MATLAB ode15s Solver

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Software Tool

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  • Standalone

Application

  • Source and

binaries maintained on Google Code SVN Repository

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Rendering Antibiotic Susceptibility

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References

  • Uri Alon. An Introduction to Systems Biology: Design Principles of Biological Circuits. Chapman & Hall /

CRC, 2007.

  • Z. Baharoglu, D. Bikard, and D. Mazel. Conjugative dna transfer induces the bacterial sos response and

promotes antibiotic resistance development through integron activation. PLoS Genetics, 2010.

  • Don B. Clewell. Nature of Col E1 plasmid replication in Escherichia coli in the presence of
  • chloramphenicol. Journal of Bacteriology, 1972.
  • H. Fujikawa, A. Kai, and S. Morozumi. A new logistic model for escherichia coli growth at constant and

dynamic temperatures. Food Microbiology, 2004.

  • Dianne Godwin and J. Howard Slater. The influence of the growth environment on the stability of a drug

resistance plasmid in Escherichia coli K12. Journal of General Microbiology, 1979.

  • Richard E. Lenski, Michael R. Rose, Suzanne C. Simpson, and Scott C. Tadler. Long-term experimental

evolution in eschericia coli. i. adaptation and divergence during 2,000 generations. American Naturalist, 1991.

  • Kirsten Skarstad, Harold B. Steen, and Erik Boye. Cell cycle parameters of slowly growing Escherichia coli

B/r studied by flow cytometry. Journal of Bacteriology, 1983.

  • P. Horvath, R. Barrangou. CRISPR/Cas, the immune system of bacteria and archaea. Science, 2010.
  • Blake Wiedenheft, G. C. L., Kaihong Zhou, Matthijs M. Jore, Stan J. J. Brouns, John van der Oost, Jennifer A.

Doudna & Eva Nogales (September 22, 2011). "Structures of the RNA-guided surveillance complex from a bacterial immune system." Nature 477: 4.

  • Banfield, A. F. A. a. J. F. (May 23, 2008). "Virus Population Dynamics and Acquired Virus Resistance in

Natural Microbial Communities." Science 320(5879): 4.