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 TECHNOLOGY 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 on 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 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
CRISPR GEORGIA INSTITUTE OF TECHNOLOGY
Components of the Locus GEORGIA INSTITUTE OF TECHNOLOGY • Cas genes • Leader sequence • Repeats • Spacers • Terminator sequence
Repeat GEORGIA INSTITUTE OF TECHNOLOGY
CRISPR GEORGIA INSTITUTE OF TECHNOLOGY
Streptococcus thermophilus GEORGIA INSTITUTE OF TECHNOLOGY • Possesses a well-documented CRISPR system • Lactic acid bacteria used in industrial fermentation to produce yogurt
Superbugs GEORGIA INSTITUTE OF TECHNOLOGY • 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)
EXPERIMENTS
Mobile CRISPR system from S. thermophilus GEORGIA INSTITUTE OF TECHNOLOGY DGCC 7710 Strain Experiments and Future Applications Chromosomal Integration of CRISPR1 locus from S. thermophilus to B. subtilis Biobrick Constructs Mobilize CRISPR system on a plasmid
Mobile CRISPR system from S. thermophilus GEORGIA INSTITUTE OF TECHNOLOGY DGCC 7710 Strain S. thermophilus DGCC 7710 strain Shuttle vector with kanamycin CRISPR locus amplified from DGCC strain with kan resistance spacer Expected outcome E. coli No growth on kanamycin plates pNT1 vector with CRISPR locus
CRISPR from S. thermophilus LMD9 GEORGIA INSTITUTE OF TECHNOLOGY Strain and Biobricks • CRISPR sequence is analogous to the DGCC strain • Amplify only Spacer/Repeat Region and ligate it into the pSB1C3 vector Bba_K582000
CRISPR from S. thermophilus DGCC LMD9 GEORGIA INSTITUTE OF TECHNOLOGY Strain and Biobricks • cas9 itself is sufficient for the expression of CRISPR Bba_K582003
Chromosomal Integration of CRISPR GEORGIA INSTITUTE OF TECHNOLOGY 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 CRISPR locus amplified from DGCC strain with kan strain spacer Bacillus Subtilis with Expected Outcome CRISPR locus ece113 integrated in its vector with chromosome CRISPR locus
Results/Explanation GEORGIA INSTITUTE OF TECHNOLOGY
FUTURE
The Ideal Vaccine GEORGIA INSTITUTE OF TECHNOLOGY • Mechanism for rendering antibiotic susceptibility • Method for introduction into population. • Mechanism for transfer across a population
A Conjugating Vaccine GEORGIA INSTITUTE OF TECHNOLOGY Introduction to Population Conjugation Transformation
Rendering Antibiotic Susceptibility GEORGIA INSTITUTE OF TECHNOLOGY
Mechanisms for delivery of GEORGIA INSTITUTE OF TECHNOLOGY pSTINGER Nonlysogenic Phage Obligative Predatory Cells Delivery of Cosmid DNA Bearing Cosmid DNA • • Bdellovibrio In vitro packaged lambda phage Bacteriovorus • Common phage therapy • Probiotic delivery in leads to resistant plasmids chickens successful in solution. • Broad spectrum gram- • Added cos sites for negative prey packaging • Immune response to phages has been experimentally low • Extremely host specific • Transforms E. Coli
Mechansms for delivery of GEORGIA INSTITUTE OF TECHNOLOGY pSTINGER Lambda Phage B. bacteriovorus Mechanisms Mechanisms
Methods for Transfer Across GEORGIA INSTITUTE OF TECHNOLOGY a Population Origins of Origins of Transfer Replication • F-plasmid origin • pIP501 derived • IncP type transgenic Ori • IncQ type • pMB1 (puc18 • Pheremone derivative) type • Conjugative transposon Tn4555
Rendering Antibiotic Susceptibility GEORGIA INSTITUTE OF TECHNOLOGY
MODELING
Population Model of E.coli GEORGIA INSTITUTE OF TECHNOLOGY Consider 3 Sub-Populations
Logistic Growth GEORGIA INSTITUTE OF TECHNOLOGY • Consider 3 Sub-Populations Basic form: Specific Growth: Image Credit: Public Domain
Interaction Model GEORGIA INSTITUTE OF TECHNOLOGY •Fundamental System of form Y’=AY • Parameters expand into operators of form
Model in Matrix Notation GEORGIA INSTITUTE OF TECHNOLOGY
Rewrite as a System of Equations GEORGIA INSTITUTE OF TECHNOLOGY
Numerical Solutions GEORGIA INSTITUTE OF TECHNOLOGY MATLAB ode15s Solver
Software Tool GEORGIA INSTITUTE OF TECHNOLOGY • Standalone Application • Source and binaries maintained on Google Code SVN Repository
Rendering Antibiotic Susceptibility GEORGIA INSTITUTE OF TECHNOLOGY
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.
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