Difference between revisions of "Team:Stanford-Brown/CRATER"

Revision as of 04:55, 14 September 2015

SB iGEM 2015

Welcome to CRATER
Method for More Efficient Bacterial Transformations

Abstract

The CRISPR/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be applied to facilitate efficient plasmid selection for transformation as well as selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts after restriction enzyme digestion and ligation. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes unwanted ligation byproducts and increases transformation efficiency of desired inserts from 20% up to 97% ± 3%. This CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient method for obtaining specific cloning products.

See our BioBricks

Introduction

We have developed a novel method to increase transformation efficiency.

Molecular cloning is a fundamental technique in molecular biology to produce plasmid constructs. Several methods currently exist to minimize or select against unwanted plasmid products created during ligation of inserts into vectors, including cross-incompatible sticky ends, X-gal blue/white screening, dephosphorylation of backbone sticky ends, the addition of antibiotics, and agarose electrophoresis/gel extraction. However, in special circumstances existing methods may be insufficient to quickly, cheaply, and effectively screen for specific cloning products. The use of antibiotics requires a host strain lacking resistance and the inclusion of genes conferring antibiotic resistance. Genes of interest may include restriction sites that would otherwise be used to create incompatible sticky ends. A plasmid vector also may simply not include multiple restriction sites with incompatible sticky ends. Unwanted byproducts are also difficult to control in situations where blunt ends are used.

We developed a new method for degrading unwanted ligation products using the Cas9 nuclease from Streptococcus pyogenes in a one-pot reaction, enhancing transformation efficiency from 20% up to 97% ± 3%. The Cas9 protein is a component of the clustered, regularly interspaced, short palindromic repeats (CRISPR) system. The CRISPR/CRISPR-associated (Cas) system provides bacteria with acquired immunity by incorporating fragments of foreign DNA and using the transcribed CRISPR-RNA (crRNA) to guide the cleavage of matching dsDNA sequences (Bhaya, Wiedenheft). In type II CRISPR systems, a ternary complex of Cas9, crRNA, and trans-activating crRNA (tracrRNA) binds to and cleaves dsDNA sequences that match the crRNA and include a short protospacer-adjacent motif (PAM) recognized by Cas9 (Gasiunas, Jinek). In type II systems, the crRNA and tracrRNA can be combined into a single guide-RNA (sgRNA) that is sufficient to lead Cas9 to its target (Jinek). Further, the PAM sequence recognized by the S. pyogenes Cas9 is only three nucleotides in length (NGG), allowing this system to be easily adapted to recognize and cut a desired sequence (Mojica).

With the knowledge that Cas9 can be used to cleave short (~24 bp) sequences, we investigated whether this system could be adapted to cleave unwanted ligation byproducts. We used the the RFP BioBrick plasmid (pSB1C3) as a starting vector and replaced the RFP insert with various genes of interest using restriction enzyme digestion and ligation, before transforming into Escherichia coli. We then quantified insertion efficiency based on the presence of fluorescent proteins in colonies and culture. We show, for the first time to our knowledge, that Cas9 and sgRNAs can be used to increase molecular cloning efficiency by cleavage of specific ligation byproducts; we call this novel technique CRISPR/Cas9-assisted transformation-efficient reaction (CRATER).

Experiment Engineering E. coli to produce polystyrene

Donec ullamcorper nulla non metus auctor fringilla. Vestibulum id ligula porta felis euismod semper. Praesent commodo cursus magna, vel scelerisque nisl consectetur. Fusce dapibus, tellus ac cursus commodo.

Donec tincidunt aliquet justo, sit amet mollis purus varius ac. Quisque ac sapien eu ante convallis cursus congue vel odio. Sed efficitur sapien ut eros sodales ornare. Vestibulum pellentesque lorem sed nulla interdum, non tincidunt velit sagittis. Vestibulum cursus, enim eu porta euismod, enim lectus facilisis diam, at sodales metus ligula sit amet eros. Sed ullamcorper, mauris nec mollis pretium, justo ligula dapibus nulla, non elementum nisl libero ut elit. Proin mi urna, finibus at scelerisque quis, porttitor at mauris. Nulla laoreet venenatis cursus. Vivamus et pellentesque quam, eget malesuada ex. Quisque eu massa ligula. Nam interdum dui sed laoreet efficitur. Aliquam sed vulputate orci. Pellentesque sed sollicitudin lectus. Vivamus nec tortor risus. Vestibulum malesuada feugiat lorem a dignissim. In diam mauris, venenatis at vulputate eget, venenatis sit amet metus. Suspendisse ut mi in ipsum sagittis malesuada at nec erat. Etiam volutpat risus quis nisi hendrerit porttitor vel eu tortor. Donec venenatis, risus sit amet ullamcorper scelerisque, tellus erat consequat nibh, vel dictum velit augue id leo. In eleifend tristique ipsum sed dignissim. Duis mattis, ipsum nec aliquet varius, turpis orci tempus nulla, in sodales libero massa at diam. Nulla maximus eros sed venenatis congue. Phasellus diam nunc, ullamcorper vitae tempor eget, sagittis eu odio. Praesent a mauris porttitor, mattis sem a, sodales massa. Proin et justo lectus. Proin varius magna ac leo ullamcorper accumsan. Proin id diam eget dolor vulputate mattis. Suspendisse pellentesque, nunc sit amet blandit feugiat, risus eros egestas massa, nec condimentum ante sapien ac velit. Vivamus efficitur justo dolor, at gravida lorem venenatis at. Aenean at ligula sapien. Mauris eget eleifend justo, eget faucibus ante. Ut mattis ante vitae dignissim maximus. Integer feugiat arcu purus, a viverra dui elementum vitae. Phasellus mattis porttitor iaculis. In eu nisi eu augue lacinia fringilla venenatis at nunc. Nam est erat, hendrerit ac dignissim sed, mollis eu eros. Morbi vel egestas dui, consectetur posuere nisi. Aliquam vitae tortor vulputate, fringilla est vel, faucibus diam. Suspendisse potenti. Donec sed commodo nulla. Duis feugiat, diam eu pulvinar rhoncus, arcu erat pretium orci, ut porta diam elit eu mi. Etiam eros massa, egestas eu mattis id, hendrerit at ligula. Duis placerat felis nec risus volutpat lobortis. Sed elementum, dolor non feugiat placerat, libero sapien pharetra diam, sed faucibus est ex tristique sem. Vivamus rutrum libero eget mollis sodales. Pellentesque vel scelerisque felis, a imperdiet erat. Fusce quis nisl magna. Sed non libero ultrices sapien hendrerit suscipit aliquet convallis leo. Quisque nec aliquam libero, in commodo ex. In eget nulla consequat, commodo quam id, hendrerit velit. Vestibulum non interdum enim. Ut elit justo, suscipit vel pretium vitae, rutrum sed dui. Donec vehicula sit amet ex ac finibus. Donec ultrices tellus et laoreet dictum.

Data and Results Optimizing the production of biological PHA

See our Picture Gallery!

Protocols

Vestibulum nec nisl eu ex ullamcorper mattis ac vel tortor.

Duis nec nibh non nisl tristique condimentum quis eu leo.

Sed venenatis massa in tortor gravida dictum.

Nam sollicitudin enim ac egestas fermentum. Suspendisse tempor urna vel mollis mollis. Proin ac mauris facilisis sapien maximus suscipit nec eget felis. Fusce ac urna sit amet nunc condimentum gravida. Aenean commodo nunc et tempus egestas. Suspendisse cursus quam placerat, vestibulum nunc non, imperdiet felis. Curabitur et erat non justo eleifend commodo. In sit amet sem vitae eros placerat facilisis. Quisque eget ligula vel tellus fermentum vestibulum. Curabitur eu ligula non lorem pulvinar posuere ac commodo ante. Sed convallis quam ut risus dignissim, nec pellentesque risus malesuada. Vestibulum vel sem eu tortor ornare consequat ac eget ligula. Suspendisse eu lacus ut nisi aliquet mollis id nec eros. Integer vulputate sem sed massa porta, eget dapibus odio pellentesque. Morbi sit amet lacus quis urna mattis elementum.

See our Lab Notebook!

References

[1] Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu. Rev. Genet . 45, 273–297(2011).

[2] Wiedenheft, B., Sternberg, S.H. & Doudna, J.A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012).

[3] Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109 , E2579–E2586 (2012).

[4] Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

[5] F. J. Mojica, C. Díez-Villaseñor, J. García-Martínez, C. Almendros, Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733 (2009).

[6] Conley E C, Saunders V, Saunders J R. Deletion and rearrangements of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules. Nucleic Acids Res. 1986;14:8905–8917.

@@ Line 198: / Line 198: @@
        <p>
-         Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. <i>Annu. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp Rev. Genet </i>. 45, 273–297(2011). <br> <br>
+         [1] Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. <i>Annu. Rev. Genet </i>. 45, 273–297(2011). <br> <br>
-         Wiedenheft, B., Sternberg, S.H. & Doudna, J.A. RNA-guided genetic silencing systems in bacteria and archaea. <i>Nature </i> <b> 482</b>, 331–338 (2012). <br> <br>
+         [2] Wiedenheft, B., Sternberg, S.H. & Doudna, J.A. RNA-guided genetic silencing systems in bacteria and archaea. <i>Nature </i> <b> 482</b>, 331–338 (2012). <br> <br>
-         Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; bacteria. Proc. Natl. <i>Acad. Sci. USA </i> <b> 109 </b>, E2579–E2586 (2012). <br> <br>
+         [3] Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. <i>Acad. Sci. USA </i> <b> 109 </b>, E2579–E2586 (2012). <br> <br>
-         Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. <i>Science</i> <b> 337</b>, 816–821 (2012). <br> <br>
+         [4] Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. <i>Science</i> <b> 337</b>, 816–821 (2012). <br> <br>
-         F. J. Mojica, C. Díez-Villaseñor, J. García-Martínez, C. Almendros, Short motif sequences determine the targets of the prokaryotic CRISPR defence &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; system. <i>Microbiology</i> 155, 733 (2009). <br> <br>
+         [5] F. J. Mojica, C. Díez-Villaseñor, J. García-Martínez, C. Almendros, Short motif sequences determine the targets of the prokaryotic CRISPR defence system. <i>Microbiology</i> 155, 733 (2009). <br> <br>
-         Conley E C, Saunders V, Saunders J R. Deletion and rearrangements of plasmid DNA during transformation of <i>Escherichia coli</i> with linear plasmid &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; molecules. <i>Nucleic Acids Res.</i> 1986;14:8905–8917.
+         [6] Conley E C, Saunders V, Saunders J R. Deletion and rearrangements of plasmid DNA during transformation of <i>Escherichia coli</i> with linear plasmid molecules. <i>Nucleic Acids Res.</i> 1986;14:8905–8917.
          </p>