Team:Calgary/TestPage

From 2013.igem.org

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<h2>Week 5: May 27 - May 31</h2>
<h2>Week 5: May 27 - May 31</h2>
<p>We continued our literature search for a unique and stable pathogenic <i>E. coli</i> marker for the TALEs to bind to. Potential pathogenic O157:H7 <i>E. coli</i> markers: <i>stx1</i>, <i>stx2</i> (Yoshitomi et al., 2012), <i>uidA</i> (Yoshitomi et al., 2003 and Feng et al., 1994), <i>eaeA</i>, <i>hly</i>, <i>rbfE</i> (Fortin et al., 2000), and <i>Z3276</i> (Li & Chen, 2012). However, BLAST searches determined that the use of only one 18 bp sequence is not sufficiently specific and sensitive to detect pathogenic markers. Should we continue to search for only one marker or more than one or expand the detection to any toxic Shiga producing strains?</p><p>In addition to searching for a pathogenic <i>E. coli</i> marker, we also started working on the TALE sequence and its construction. The TALE sequence chosen has a truncation in both N- and C-terminus. The truncated N-terminus has 111 residues and the C-terminus has 42 residues (Meckler et al., 2013). The sequence was codon optimized for <i>E.coli</i>. <a href="http://tools.neb.com/NEBcutter2/" >NEBCutter 2.0</a> was used to screen restriction sites for PstI, EcoRI, NotI, XbaI, SpeI, NgoMIV and AgeI, all present in the RFC 25. Cut sites for BsaI and Esp3I were also removed to pave the road for Golden Gate Assembly method. The original TALE sequence has 11 cut sites: BsaI (3), PstI (1), EcoRI (1), Esp3I (4), AgeI (2). The restriction sites were then eliminated with silent mutations, based on a <i>E. coli</i> codon usage table (San Diego State University, 2012 modified from Maloy, Stewart & Taylor, 1996).</p><p>The large size of the TALE prevents simple cloning, therefore we looked to Golden Gate Assembly. Golden Gate Assembly is a subcloning strategy that uses type IIs restriction enzymes, which cut outside of their recognition sequence allowing ligation into a final product that lacks any specific scar sites (Engler, Kandzia & Marillonnet, 2008).</p>
<p>We continued our literature search for a unique and stable pathogenic <i>E. coli</i> marker for the TALEs to bind to. Potential pathogenic O157:H7 <i>E. coli</i> markers: <i>stx1</i>, <i>stx2</i> (Yoshitomi et al., 2012), <i>uidA</i> (Yoshitomi et al., 2003 and Feng et al., 1994), <i>eaeA</i>, <i>hly</i>, <i>rbfE</i> (Fortin et al., 2000), and <i>Z3276</i> (Li & Chen, 2012). However, BLAST searches determined that the use of only one 18 bp sequence is not sufficiently specific and sensitive to detect pathogenic markers. Should we continue to search for only one marker or more than one or expand the detection to any toxic Shiga producing strains?</p><p>In addition to searching for a pathogenic <i>E. coli</i> marker, we also started working on the TALE sequence and its construction. The TALE sequence chosen has a truncation in both N- and C-terminus. The truncated N-terminus has 111 residues and the C-terminus has 42 residues (Meckler et al., 2013). The sequence was codon optimized for <i>E.coli</i>. <a href="http://tools.neb.com/NEBcutter2/" >NEBCutter 2.0</a> was used to screen restriction sites for PstI, EcoRI, NotI, XbaI, SpeI, NgoMIV and AgeI, all present in the RFC 25. Cut sites for BsaI and Esp3I were also removed to pave the road for Golden Gate Assembly method. The original TALE sequence has 11 cut sites: BsaI (3), PstI (1), EcoRI (1), Esp3I (4), AgeI (2). The restriction sites were then eliminated with silent mutations, based on a <i>E. coli</i> codon usage table (San Diego State University, 2012 modified from Maloy, Stewart & Taylor, 1996).</p><p>The large size of the TALE prevents simple cloning, therefore we looked to Golden Gate Assembly. Golden Gate Assembly is a subcloning strategy that uses type IIs restriction enzymes, which cut outside of their recognition sequence allowing ligation into a final product that lacks any specific scar sites (Engler, Kandzia & Marillonnet, 2008).</p>
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<h2>Week 6: June 3 - June 7</h2>
 
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<p>This week also mostly spent on literature research. </p>
 
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                <p>The next step after optimization of TALEs and their target sequence is to locate this system’s niche in the beef industry. Many of the current high fidelity methods for pathogenic <i>E. coli</i> detection require cell cultures, which increases turnaround time up to 4 days (Blais et al., 2012 & Gill et al., 2013).</p>
 
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              <p>A 2013 paper by Jeon et al. explores the genetic and physiological effects of the cattle on the amount of the O157 it excretes. According to this paper, 90% of the bacteria excreted from the cattle comes from the super-shedders. Super-shedders are cattle that excrete more than more than 10 000 CFU/g and have long term recto anal junction colonization. Higher excretion is detected in bulls, pen raised cattle, and certain breeds with a seasonal peak between late spring and early fall. Preharvest detection of pathogenic <i>E. coli</i> decreases cross contamination during meat processing.</p>
 
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                <p>Shiga toxin 1 and 2 have 55% homology and are composed of 5 identical highly conserved B subunits that bind to the Gb3 host receptor cells and a single A subunit which enters the host target cell and cleaves rRNA. Cattle do not have Gb3, consequently, O157:H7 infected cattle are asymptomatic and difficult to detect. More than 200 serotypes of <i>E.coli</i> contain Stx, of these, some have LEE (locus of enterocyte) which does not directly indicate pathogenicity. Other strains of pathogenic shiga toxin producing <i>E. coli</i> recognized by the USDA as harmful are: O26:H11, O103:H2, O111:H8, O118:H16, O121:H19, and O145:H28 (USDA  Laboratory Guidebook, 2012). These strains are also known as the Big Six. Currently, the USDA targets both the <i>eae</i> and <i>stx</i> genes in the PCR. When these genes are being used in identification, if the sample tests positive for both <i>eae</i> and <i>stx</i>, the sample undergoes further testing. If it only has one or neither of the genes, the sample is considered safe and testing stops (USDA  Laboratory Guidebook, 2012).</p>
 
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                <p>Regarding our TALE construct, we compared 2 standard construction strategies for multiple DNA fragments: Golden Gate and Gibson Assembly. Gibson Assembly is a one-step isothermal reaction involving an exonuclease, a DNA polymerase and a DNA ligase. All enzymes are put together in one reaction along with DNA fragments and the vector and incubated at 50°C for 15 to 60 min. (Gibson et al., 2009)</p>
 
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<figure>
 
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<img src="https://static.igem.org/mediawiki/2013/thumb/6/6a/2013_Gibson_assembly_vs_golden_gate.jpg/524px-2013_Gibson_assembly_vs_golden_gate.jpg">
 
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</figure>
 
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<h2>Week 7: June 10 - June 14</h2>
 
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<p>This week, we had the opportunity to tour Cargill Meat Solutions’ High River beef plant and experience firsthand the stringency and efficiency of the meat processing facility. Global prevalence of O157:H7 in cattle farms is 20% (Chase-Topping et al., 2008) and the presence of super shedders is influenced by the <i>E. coli</i> strain and host dependent factors (breed, sex, reproductivity, stress, movement) independent of the external farm environment (Chase-Topping et al., 2007). These few super shedders are important factors to pre-harvest contamination as the source of 80% of O157:H7 strains isolated from beef carcasses are from the high cattle density and rapid turnover environment of the processing plant and transport trailers, not the original feedlot (Arthur, 2009).</p><p>We also met with and discussed <i>E. coli</i> markers with Dr. Glen Armstrong, professor and head of the Department of Microbiology, Immunology & Infectious Diseases at the University of Calgary. Dr. Armstrong suggested that we implement multiple TALEs to target different markers to identify <i>E. coli</i> serotypes.</p><p>Primers containing the target sequences of TALE A (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K782004" >BBa_K782004</a>), TALE B(<a href="http://parts.igem.org/Part:BBa_K782006" >BBa_K782006</a>), and variations of the target sequences (with point mutations in different positions) were used to perform a PCR on RFP generator (<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>); the product of the PCR would be the target sequence of the TALE and a part of the RFP gene which is 660 base paris (figures 1, 2 and 3). The PCR products were purified and digested with XbaI and PstI and inserted into  a vector; RFP generator was also digested with XbaI and PstI to prepare the vector. </p>
 
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<p>IPTG inducible promoter with RBS (<a href="http://parts.igem.org/Part:BBa_J04500">BBa_J04500</a>) was transformed with competent cells and plated on Amp and Chlor.</p>
 
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<figure>
 
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<img src="https://static.igem.org/mediawiki/2013/d/db/YYC2013_TALE_June_12_DNA_gel_001_PCR_products.jpg">
 
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<figcaption>
 
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<p><b>Figure 1.</b>PCR products of mutated TALE B target sequences, which were inserted in the RFP generator gene. Bands were expected at 660bp.</p>
 
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</figcaption>
 
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</figure>
 
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<figure>
 
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<img src="https://static.igem.org/mediawiki/2013/c/c2/YYC2013_TALE_June_12_DNA_gel_002_PCR_products.jpg">
 
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<figcaption>
 
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<p><b>Figure 2.</b>PCR products of intact and mutated TALE B target sequences, which were inserted in the RFP generator gene. Bands were expected at 660bp.</p>
 
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</figcaption>
 
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</figure>
 
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<figure>
 
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<img src="https://static.igem.org/mediawiki/2013/2/21/YYC2013_TALE_June_12_DNA_gel_003_PCR_products.jpg">
 
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<figcaption>
 
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<p><b>Figure 3.</b>PCR products of mutated TALE B target sequence and intact TALE A target sequence, which were inserted in the RFP generator gene. Bands were expected at 660bp.</p>
 
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</figcaption>
 
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</figure>
 
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<p>The parts received from the registry - TALE A (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K782004" >BBa_K782004</a>), TALE B (<a href="http://parts.igem.org/Part:BBa_K782006" >BBa_K782006</a>), TALE D (<a href="http://parts.igem.org/Part:BBa_K782005" >BBa_K782005</a>), NicTAL 12 (<a href="http://parts.igem.org/Part:BBa_K782007" >BBa_K782007</a>) and GFP in RFC 25 Standard (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K648013" >BBa_K648013</a>)- were transformed. Subsequently, a overnight cultures of the colonies were made and a plasmid purification was performed. Digestion of the purified plasmids with NotI and running the products on the gel produced the expected results (figure 4).  TALE A (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K782004" >BBa_K782004</a>), TALE B (<a href="http://parts.igem.org/Part:BBa_K782006" >BBa_K782006</a>) and TALE D (<a href="http://parts.igem.org/Part:BBa_K782005" >BBa_K782005</a>)were sent for sequencing.</p>
 
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<figure>
 
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<img src="https://static.igem.org/mediawiki/2013/6/63/YYC2013_TALE_June_14_DNA_gel_001_Verification_Digest.jpg">
 
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<figcaption>
 
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<p><b>Figure 4.</b>Verification digest of TALEs A (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K782004" >BBa_K782004</a>), B  (<a href="http://parts.igem.org/Part:BBa_K782006" >BBa_K782006</a>) and D (<a href="http://parts.igem.org/Part:BBa_K782005" >BBa_K782005</a>) and GFP (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K648013" >BBa_K648013</a>) with NotI.</p>
 
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</figcaption>
 
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</figure>
 
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Revision as of 04:10, 23 September 2013

Detector Journal

Week 1: May 1 - May 3

This week we attended courses required by our University to work in the lab. Besides that, the undergraduate supervisors presented about the principles of Genetics and Synthetic Biology to the new team members.

Week 2: May 6 - May 10

This week we attended a workshop based on general molecular biology techniques.

Week 3: May 13 - May 17

We continued the molecular biology workshop. Also, the team members were assigned in different parts of the project such as TALEs as our detector, Ferritin as our reporter, Linker, Human Practices, Modelling and Business.

Week 4: May 20 - May 24

We did our literature search for optimization of TALE binding affinity. There were conflicting results for the optimal truncation of the N and C terminus. On N terminus, 152 (Miller et al., 2011), 147 (Mussolini et al., 2011), 186 (Slovenia’s TALEs), and 158 or 186 (Mercer et al., 2012) residues were retained. On C terminus, 28 or 63 (Miller et al., 2011), and 76 (Slovenia’s TALEs) residues were retained.

We learned about three very important TALE characteristics: Firstly, TALEs demonstrate polarity. In other words, they interact more strongly with DNA at the N terminus compared to the C terminus (Meckler et al., 2013). Secondly, the Thymidine nucleotide at the zero repeat is very important to the binding affinity of the TALE (Meckler et al., 2013). Thirdly, the binding affinity of TALE varies based on the types of the nucleotides present in its target site. The relative affinities are: NG(1)>NN (0.18)!HD (0.16) " NI (0.0016)>NK (0.00016) (Meckler et al., 2013). All these points must be carefully considered while designing the TALE to maximize its binding affinity and specificity.

The four TALEs designed by Slovenia 2012 iGEM team, were ordered from the registry (TALE A (BBa_K782004), TALE B (BBa_K782006) and TALE D (BBa_K782005)). Primers were designed to incorporate the the target sequences of TALE A (BBa_K782004) and TALE B (BBa_K782006) ([A] and [B]) in the RFP generator. This plasmid will be used as a target sequence. Primers were also designed that incorporate selected point mutations of [A] and [B] into a biobrick. Once these plasmids are made they will be used to determine how the TALEs affinity is altered by mutations. In addition, two other TALEs were ordered from the the authors of the Meckler et al. 2013 paper.

Week 5: May 27 - May 31

We continued our literature search for a unique and stable pathogenic E. coli marker for the TALEs to bind to. Potential pathogenic O157:H7 E. coli markers: stx1, stx2 (Yoshitomi et al., 2012), uidA (Yoshitomi et al., 2003 and Feng et al., 1994), eaeA, hly, rbfE (Fortin et al., 2000), and Z3276 (Li & Chen, 2012). However, BLAST searches determined that the use of only one 18 bp sequence is not sufficiently specific and sensitive to detect pathogenic markers. Should we continue to search for only one marker or more than one or expand the detection to any toxic Shiga producing strains?

In addition to searching for a pathogenic E. coli marker, we also started working on the TALE sequence and its construction. The TALE sequence chosen has a truncation in both N- and C-terminus. The truncated N-terminus has 111 residues and the C-terminus has 42 residues (Meckler et al., 2013). The sequence was codon optimized for E.coli. NEBCutter 2.0 was used to screen restriction sites for PstI, EcoRI, NotI, XbaI, SpeI, NgoMIV and AgeI, all present in the RFC 25. Cut sites for BsaI and Esp3I were also removed to pave the road for Golden Gate Assembly method. The original TALE sequence has 11 cut sites: BsaI (3), PstI (1), EcoRI (1), Esp3I (4), AgeI (2). The restriction sites were then eliminated with silent mutations, based on a E. coli codon usage table (San Diego State University, 2012 modified from Maloy, Stewart & Taylor, 1996).

The large size of the TALE prevents simple cloning, therefore we looked to Golden Gate Assembly. Golden Gate Assembly is a subcloning strategy that uses type IIs restriction enzymes, which cut outside of their recognition sequence allowing ligation into a final product that lacks any specific scar sites (Engler, Kandzia & Marillonnet, 2008).

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