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2012-10-04T02:43:58Z
<p>Sgenyk: </p>
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Lab&nbsp;Notebook </h2><!-- .postitle --><br />
<br />
<p><span style="color:#000000;"><strong>5/21/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan transformed casBCED12 with Amp resistance, casBCED12 with Cm resistance, pSBIC3_RFP, pSBIK3_RFP, and pSBIA3_RFP</span><br /><br />
<span style="color:#000000;"> -successful results: pSBIC3_RFP and pSBIK3_RFP.</span><br /><br />
<span style="color:#000000;"> -Luke inoculated pSBIC3_RFP and pSBIK3_RFP</span><br /><br />
<span style="color:#000000;"> -Stephan miniprepped pSBIK3_RFP, and pSBIT3_RFP</span><br /><br />
<span style="color:#000000;"> -all very low concentration</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped pSBIC3_RFP, pSBIA3_RFP</span></p><br />
<p><span style="color:#000000;">-concentration (got 98 and 120 ug/ml, respectively)<a href="http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg"><img class="alignright size-medium wp-image-190" title="DSC_0028" src="http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg?w=442&h=292" alt="" width="442" height="292" /></a></span></p><br />
<p><span style="color:#000000;">-Rachel digested pSBIC3 from pSBIC3_RFP, pSBIK3 from pSBIK3_RFP</span><br /><br />
<span style="color:#000000;"> -Eric transformed pSBIT3_RFP, pSBIA3_RFP, casBCDE12 on Amp</span><br /><br />
<span style="color:#000000;"> -Stephan digested <em>casABCDE123</em>, tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Stephan digated <em>casA</em>,3 with tetO-GFP, with pSBIA3 or pSBIC3</span><br /><br />
<span style="color:#000000;"> -Eric digated casBCDE12 with pSBIA3 or PSBIC3</span></p><br />
<p><span style="color:#000000;"> <strong>5/28/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan inoculated (2x) 5mL psB1A3_RFP and psB1T3_RFP.</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed psB1C3_RFP, tetO_GFP, and psB3T5_RFP.</span><br /><br />
<span style="color:#000000;"> -Rebecca miniprepped psB1C3_RFP and psB1K3_RFP</span><br /><br />
<span style="color:#000000;"> -Ellen transformed psB1C3-RFP, psB3T5-RFP, tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Rachel PCRed <em>fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</em></span><br /><br />
<span style="color:#000000;"> -Luke gel extracted and digested the genes with EP or EX</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped pSB3T5_RFP (3) and tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Rachel digested tetO-GFP 2</span></p><br />
<p><span style="color:#000000;"> <strong>6/4/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Stephan PCRed genes using BL21 and HT115</span><br /><br />
<span style="color:#000000;"> -Stephan gel extracted and digested </span><br /><br />
<span style="color:#000000;"> -Ellen digated the genes to corresponding psb1C3_RFP vectors</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -successful: 4-12, 3-8D, 3-2H, 1-9F, 3-2J. (very low efficiency)</span><br /><br />
<span style="color:#000000;"> -Rachel transformed <em>cheB, cheC, cheY, cheZ, luxS, lsrK, lsrR, flaU, flbA, flhB, flhD, flgJ, flgK, fliA, fliC, fliD, fliF, fliG, fliH, fliM</em></span><br /><br />
<span style="color:#000000;"> -Rachel inoculated 4-12A, 3-8D, 3-2H, 1-9F, 3-2J</span><br /><br />
<span style="color:#000000;"> -Luke inoculated HT115 (w/ 5uL tet) and HB101 (w/o antibiotics)</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed <em>fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</em></span></p><br />
<p><span style="color:#000000;"> <strong>6/11/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen digested <em>fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</em></span><br /><br />
<span style="color:#000000;"> -Luke gel extracted <em>fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</em></span><br /><br />
<span style="color:#000000;"> -Rebecca PCRed <em>fliC</em> with DH5alpha, BL21, HT115</span><br /><br />
<span style="color:#000000;"> -Rachel ligated cm1, cm2, rfp1, rfp2</span><br /><br />
<span style="color:#000000;"> -Eric gel purified cm1, cm2, rfp1, rfp2</span><br /><br />
<span style="color:#000000;"> -Megan transformed <em>fliA, cheZ, cheY, fliH, fliG, flgJ, flihB, flgK, fliD,fliM</em>, 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -successful: 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -Stephan digested cm vector with EP</span><br /><br />
<span style="color:#000000;"> -Ellen gel extracted cm vectors</span><br /><br />
<span style="color:#000000;"> -Ellen inoculated 3-8D, 1-9F, 4-12A</span><br /><br />
<span style="color:#000000;"> -Eric PCRed <em>chA, cheB, cheC, cheY, cheZ</em></span><br /><br />
<span style="color:#000000;"> -Luke transformed ligation products: <em>fliG, flgJ, cheY, cheZ, fliD, flhB, fliM, fliH, flgK</em></span><br /><br />
<span style="color:#000000;"> -Rachel ligated <em>fliM, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2 to Cm vector, fliH</em></span><br /><br />
<span style="color:#000000;"> -Megan miniprepped the innoculations (3-8D, 1-9F, 4-12A)</span><br /><br />
<span style="color:#000000;"> -low concentrations</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed <em>fliH, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2</em>, cm vector</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped 1-9F(1), 1-9F(2), 3-8D(1), 3-8D(2), 4-12A(1), 4-12A(2)</span><br /><br />
<span style="color:#000000;"> -low concentrations</span></p><br />
<p><span style="color:#000000;"><a href="http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg"><img class="alignright size-medium wp-image-165" title="_MG_4058" src="http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg?w=412&h=274" alt="" width="412" height="274" /></a><strong>6/18/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel PCRed <em>lsrR</em>(BL21), <em>lsrR</em>(HT115), <em>lrsK</em>(BL21), <em>lsrK</em>(HT115)</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -low concentrations</span><br /><br />
<span style="color:#000000;"> -Luke transformed J23100, RBS B0034, <em>luxS</em>, GFP E0040</span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated J23100, RBS B0034, <em>luxS</em>, GFP E0040</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed <em>cheA, cheB, cheY, cheZ</em> using HTB genomic DNA</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed <em>fliC</em>(1), <em>motA</em>(2), <em>motB</em>(3) using DH5alpha</span><br /><br />
<span style="color:#000000;"> -Luke miniprepped 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Megan digested <em>cheA, cheB, cheY, cheZ</em> and gel purified</span><br /><br />
<span style="color:#000000;"> -Luke ligated <em>cheA, cheB, cheY, cheZ</em></span><br /><br />
<span style="color:#000000;"> -Megan transformed <em>cheA, cheB, cheY, cheZ</em></span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Eric digested psB1C3 with X and X/S</span><br /><br />
<span style="color:#000000;"> -Rachel digested <em>fliC, motA, motB</em></span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped J23100, GFP E0040, <em>luxS</em>, RBS Boo34</span><br /><br />
<span style="color:#000000;"> -low concentrations</span></p><br />
<p><span style="color:#000000;"> <strong>6/25/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen transformed <em>motA, motB</em>, and control</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated T7 promoter and <em>cheY</em></span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed 20 colonies from <em>motA, motB</em></span><br /><br />
<span style="color:#000000;"> -Stephan inoculated <em>motA, motB, cheA, cheB, cheY, cheZ</em></span><br /><br />
<span style="color:#000000;"> -Luke miniprepped <em>cheZ, cheY, cheA, cheB, motA, motB</em></span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rachel glycerol stock: <em>motA</em> 6-14, <em>cheZ, cheY, cheA, cheB, motA, motB</em></span><br /><br />
<span style="color:#000000;"> -Luke transformed <em>cheZ</em>, pLaeI+RBS+<em>cheZ</em>, RBS+<em>cheZ</em>+dbl terminator, <em>cheZ</em> w/ H13 tag+dbl terminator, <em>cheZ</em> w/ H13 tag, strong promoter w/RBS</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated <em>cheZ</em>, pLaeI+RBS+<em>cheZ</em>, RBS+<em>cheZ</em>+dbl terminator, <em>cheZ</em> w/ H13 tag+dbl terminator, <em>cheZ</em> w/ H13 tag, strong promoter w/RBS</span><br /><br />
<span style="color:#000000;"> -Stephan PCR miniprep of <em>cheA</em>(7,16), <em>cheB</em>(14), <em>cheZ</em>(14), <em>motA</em>(15)</span></p><br />
<p><span style="color:#000000;"> <strong>7/2/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan miniprepped <em>cheZ</em> w/ histag, <em>cheZ</em>, <em>cheZ</em> w/ RBS + dbl termination, pLacI w/ RBS + <em>cheZ</em>, <em>cheZ</em> w/ Histag + dbl termination, strong promoter w/ RBS (cm)</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Megan inoculated <em>cheZ</em> w/ histag (amp), <em>cheZ</em> (amp), <em>cheZ</em> w/ RBS, double termination (amp), pLacI w/ RBS, <em>cheZ</em> (amp), <em>cheZ</em> w/ histag, double termination (amp), strong promoter w/ RBS (cm)</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped: psB1C3RFP (5 times), <em>cheZ</em>, strong promoter+RBS+cm, RBS+<em>cheZ</em>+dbl term site, pLacI+RBS+<em>cheZ</em>, <em>cheZ</em>+Hist+dbl, <em>cheZ</em>+Histag</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span></p><br />
<p><span style="color:#000000;"> <strong>7/9/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen digested and gel extracted <em>fliC</em> and <em>GFP</em></span><br /><br />
<span style="color:#000000;"> -Rachel ligated fliC</span><br /><br />
<span style="color:#000000;"> -Luke transformed fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Rebecca received and plated strains: BW30044, BW30045, K-12 wildtype strain, strain B wildtype, JW1871-1, JW1879-2, JW2662-1, MG1655 (motile), W3110 </span></p><br />
<p><span style="color:#000000;"> <strong>7/16/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel made competent cells MG1655 </span><br /><br />
<span style="color:#000000;"> -Ellen digested GFP vector for control using XbaI<a href="http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg"><img class="alignright size-medium wp-image-187" title="DSC_0026" src="http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg?w=512&h=338" alt="" width="512" height="338" /></a></span><br /><br />
<span style="color:#000000;"> -Luke colony PCRed the fliC GFP </span><br /><br />
<span style="color:#000000;"> -primer dimers. unsuccessful-</span></p><br />
<p><span style="color:#000000;"> <strong>7/23/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen PCRed fliC w/ RBS</span><br /><br />
<span style="color:#000000;"> -Stephan transformed GFP-DH5a, GFP-MG1655 </span><br /><br />
<span style="color:#000000;"> -Megan inoculated w/ 50uL transformation product</span><br /><br />
<span style="color:#000000;"> -Stephan plated GFP-DH5a, GFP-MG1655</span><br /><br />
<span style="color:#000000;"> -Rachel PCRed fliC w/ RBS</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped GFP-DH5a (x2), GFP-MG1655 (x2)</span></p><br />
<p><span style="color:#000000;">-acceptable concentrations</span></p><br />
<p><span style="color:#000000;">-Digested fliC and GFP w/ XbaI and gel purified</span><br /><br />
<span style="color:#000000;"> -Eric ligated fliC-GFP and GFP w/ H2O (control)</span><br /><br />
<span style="color:#000000;"> -Eric transformed 1hour ligation product</span><br /><br />
<span style="color:#000000;"> -unsuccessul</span><br /><br />
<span style="color:#000000;"> -Stephan made control and cheY mutagenesis solutions</span><br /><br />
<span style="color:#000000;"> -Megan transformed fliC-GFP and GFP w/ H2O control into DH5a cells</span><br /><br />
<span style="color:#000000;"> -successful: GFP + H2O control and fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Luke quickchange mutagenesis</span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCR fliC-GFP</span></p><br />
<p><span style="color:#000000;"> <strong>7/30/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan miniprepped fliC-GFP</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rachel digested miniprepped products</span><br /><br />
<span style="color:#000000;"> -Eric quickchange PCRed fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Stephan digested quickchange PCR products with DPN1</span><br /><br />
<span style="color:#000000;"> -Megan digested fliC minipreps with Spe1 </span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Megan transformed 43, 44, quickchange digests</span></p><br />
<p><span style="color:#000000;"> <strong>8/6/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rebecca digested cheY</span><br /><br />
<span style="color:#000000;"> -Rebecca quickchange PCRed fliC and cheY with control</span><br /><br />
<span style="color:#000000;"> -Rebecca digested fliC-GFP, cheY, control</span><br /><br />
<span style="color:#000000;"> -Stephan plated fliC, cheY, and pUC18</span><br /><br />
<span style="color:#000000;"> -Rachel inoculated cheY mut colonies</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped cheY mut</span><br /><br />
<span style="color:#000000;"> -Stephan glycerol stocks of cheY mut</span><br /><br />
<span style="color:#000000;"> -Luke PCRed cheY mut</span><br /><br />
<span style="color:#000000;"> -Luke digested cheY mut</span></p><br />
<p><span style="color:#000000;"> <strong>8/13/12 week</strong></span><br /><br />
<span style="color:#000000;"> -PCRed fliA, fliD, fliE, fliF, fliG, fliH, fliM, flhB, flhC, flhD, flgA, flgB, flgJ, flgK, fliC-GFP, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Ellen digested and gel extracted the flagella genes</span><br /><br />
<span style="color:#000000;"> -Stephan transformed fliA, fliD,fliF, fliM, flhB, flgB, flgJ, flhD, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Megan digested fliC-GFP with DpnI, BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed pSB1C3 and fliC-GFP mut</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated pSB1C3-RFP, BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped pSB1C3 and BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Luke digested pSB1C3 fliF, fliD, flhB, flhD, flgJ, fliM, fliA</span><br /><br />
<span style="color:#000000;"> -Eric ligated fliF, fliD, flhB, flhD, flgJ, fliM, fliA with pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>8/20/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed fliA, fliM, fliD, fliF, flgJ, flhB, flhD</span><br /><br />
<span style="color:#000000;"> -Rachel imaged MG1655 in various conditions: pH 5, 6, 8, 9; NaCl 50mM, 100mM, 200mM, 400mM, 500mM; temperature 15C, 20C, 42C; glucose 10mM, 20mM, 50mM</span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped fliC-GFP mut, cheY mut</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Stephan glycerol stocks of fliC-GFP, cheY </span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed flgJ, flhD, fliA, flhB, cheY</span><br /><br />
<span style="color:#000000;"> -Megan inoculated successful colonies</span><br /><br />
<span style="color:#000000;"> -Rachel colony PCRed fliM, fliF, fliD</span><br /><br />
<span style="color:#000000;"> -Rebecca glycerol stocks of fliA, flhB, flgJ, flhD</span><br /><br />
<span style="color:#000000;"> -Eric imaging of MG1655</span><br /><br />
<span style="color:#000000;"> -Luke miniprepped fliA, flhB, flgJ, flhD</span><br /><br />
<span style="color:#000000;"> -Luke colony PCRed fliM, fliD, fliF</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated fliM </span><br /><br />
<span style="color:#000000;"> -Stephan PCRed flhD, flhB, flgJ, fliA to test directionality</span></p><br />
<p><span style="color:#000000;"> <strong>8/27/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR</span><br /><br />
<span style="color:#000000;"> -Eric digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR</span><br /><br />
<span style="color:#000000;"> -all successful except ompR, metR, araC</span><br /><br />
<span style="color:#000000;"> -Stephan digested pSB1C3 </span><br /><br />
<span style="color:#000000;"> -Megan ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter to pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>9/3/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns </span><br /><br />
<span style="color:#000000;"> -Eric transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Stephan digested pSB1C3</span><br /><br />
<span style="color:#000000;"> -Luke ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns with pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>9/10/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan transformed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated transformations </span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns</span><br /><br />
<span style="color:#000000;"> -Ellen transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406 </span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Rachel transformed BBa_K398108, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Luke inoculated BBa_K398108, BBa_K398406, fliH</span><br /><br />
<span style="color:#000000;"> -Eric miniprepped BBa_K398108, BBa_K398406, fliH</span></p><br />
<p><span style="color:#000000;">-acceptable concentrations<a href="http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg"><img class="alignright size-medium wp-image-162" title="_MG_4057" src="http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg?w=496&h=331" alt="" width="496" height="331" /></a></span></p><br />
<p><span style="color:#000000;">-Eric colony PCRed BBa_K398108, BBa_K398406, fliH </span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped flhD</span></p><br />
<p><span style="color:#000000;"> <strong>9/17/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Stephan transformed arabinose</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated cheY, T7</span><br /><br />
<span style="color:#000000;"> -Rachel digested cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Luke ligated cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped T7, cheY</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed T7/p.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed cheZ RBS, cheZ T7, p.cola cheY</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated cheZ RBS, cheZ T7, p.cola cheY</span><br /><br />
<span style="color:#000000;"> -Stephan miniprepped cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Megan transformed cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Sean inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Megan imaged cheZ, cheZ T7, cheZ RBS T7, cheZ T7</span><br /><br />
<span style="color:#000000;"> -Ellen glycerol stocks of cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Eric imaging of pcola cheZ A, cheZ RBS T7, cheZ RBS T7, cheZ T7, pcdf cheY A</span><br /><br />
<span style="color:#000000;"> -Luke inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span></p><br />
<p><span style="color:#000000;"> <strong>9/24/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rebecca imaged BBa_K398108, cheZ RBS T7, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Stephan imaged cheZ RBS T7, cheZ T7, pcdf cheY, control</span><br /><br />
<span style="color:#000000;"> -Luke digested cheY p.cola, pcdf cheY, pcdf cheZ, pcola cheZ, cheZ T7 RBS</span><br /><br />
<span style="color:#000000;"> -Megan transformed T7 RBS cheZ </span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated salt biobricks from frozen stocks</span><br /><br />
<span style="color:#000000;"> -Rachel colony PCRed T7 RBS cheZ </span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped cheZ RBS T7</span></p><br />
<p><span style="color:#000000;"> <strong>10/1/12 week</strong></span></p><br />
<p><span style="color:#000000;">-Eric miniprepped cheA, cheB, cheY, cheZ, motA. motB, T7 RBS cheZ, T7 RBS cheY, flgJ, fliA, fliH, fliC-GFP, flhB, flhD</span></p><br />
<p>-Western blot for T7 RBS cheY, T7 RBS cheZ</p><br />
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Sgenyk
http://2012.igem.org/Team:USC/Notebook
Team:USC/Notebook
2012-10-04T02:42:55Z
<p>Sgenyk: </p>
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Lab&nbsp;Notebook </h2><!-- .postitle --><br />
<br />
<p><span style="color:#000000;"><strong>5/21/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan transformed casBCED12 with Amp resistance, casBCED12 with Cm resistance, pSBIC3_RFP, pSBIK3_RFP, and pSBIA3_RFP</span><br /><br />
<span style="color:#000000;"> -successful results: pSBIC3_RFP and pSBIK3_RFP.</span><br /><br />
<span style="color:#000000;"> -Luke inoculated pSBIC3_RFP and pSBIK3_RFP</span><br /><br />
<span style="color:#000000;"> -Stephan miniprepped pSBIK3_RFP, and pSBIT3_RFP</span><br /><br />
<span style="color:#000000;"> -all very low concentration</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped pSBIC3_RFP, pSBIA3_RFP</span></p><br />
<p><span style="color:#000000;">-concentration (got 98 and 120 ug/ml, respectively)<a href="http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg"><img class="alignright size-medium wp-image-190" title="DSC_0028" src="http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg?w=442&h=292" alt="" width="442" height="292" /></a></span></p><br />
<p><span style="color:#000000;">-Rachel digested pSBIC3 from pSBIC3_RFP, pSBIK3 from pSBIK3_RFP</span><br /><br />
<span style="color:#000000;"> -Eric transformed pSBIT3_RFP, pSBIA3_RFP, casBCDE12 on Amp</span><br /><br />
<span style="color:#000000;"> -Stephan digested <em>casABCDE123</em>, tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Stephan digated <em>casA</em>,3 with tetO-GFP, with pSBIA3 or pSBIC3</span><br /><br />
<span style="color:#000000;"> -Eric digated casBCDE12 with pSBIA3 or PSBIC3</span></p><br />
<p><span style="color:#000000;"> <strong>5/28/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan inoculated (2x) 5mL psB1A3_RFP and psB1T3_RFP.</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed psB1C3_RFP, tetO_GFP, and psB3T5_RFP.</span><br /><br />
<span style="color:#000000;"> -Rebecca miniprepped psB1C3_RFP and psB1K3_RFP</span><br /><br />
<span style="color:#000000;"> -Ellen transformed psB1C3-RFP, psB3T5-RFP, tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Rachel PCRed <em>fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</em></span><br /><br />
<span style="color:#000000;"> -Luke gel extracted and digested the genes with EP or EX</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped pSB3T5_RFP (3) and tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Rachel digested tetO-GFP 2</span></p><br />
<p><span style="color:#000000;"> <strong>6/4/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Stephan PCRed genes using BL21 and HT115</span><br /><br />
<span style="color:#000000;"> -Stephan gel extracted and digested </span><br /><br />
<span style="color:#000000;"> -Ellen digated the genes to corresponding psb1C3_RFP vectors</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -successful: 4-12, 3-8D, 3-2H, 1-9F, 3-2J. (very low efficiency)</span><br /><br />
<span style="color:#000000;"> -Rachel transformed <em>cheB, cheC, cheY, cheZ, luxS, lsrK, lsrR, flaU, flbA, flhB, flhD, flgJ, flgK, fliA, fliC, fliD, fliF, fliG, fliH, fliM</em></span><br /><br />
<span style="color:#000000;"> -Rachel inoculated 4-12A, 3-8D, 3-2H, 1-9F, 3-2J</span><br /><br />
<span style="color:#000000;"> -Luke inoculated HT115 (w/ 5uL tet) and HB101 (w/o antibiotics)</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed <em>fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</em></span></p><br />
<p><span style="color:#000000;"> <strong>6/11/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen digested <em>fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</em></span><br /><br />
<span style="color:#000000;"> -Luke gel extracted <em>fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</em></span><br /><br />
<span style="color:#000000;"> -Rebecca PCRed <em>fliC</em> with DH5alpha, BL21, HT115</span><br /><br />
<span style="color:#000000;"> -Rachel ligated cm1, cm2, rfp1, rfp2</span><br /><br />
<span style="color:#000000;"> -Eric gel purified cm1, cm2, rfp1, rfp2</span><br /><br />
<span style="color:#000000;"> -Megan transformed <em>fliA, cheZ, cheY, fliH, fliG, flgJ, flihB, flgK, fliD,fliM</em>, 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -successful: 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -Stephan digested cm vector with EP</span><br /><br />
<span style="color:#000000;"> -Ellen gel extracted cm vectors</span><br /><br />
<span style="color:#000000;"> -Ellen inoculated 3-8D, 1-9F, 4-12A</span><br /><br />
<span style="color:#000000;"> -Eric PCRed <em>chA, cheB, cheC, cheY, cheZ</em></span><br /><br />
<span style="color:#000000;"> -Luke transformed ligation products: <em>fliG, flgJ, cheY, cheZ, fliD, flhB, fliM, fliH, flgK</em></span><br /><br />
<span style="color:#000000;"> -Rachel ligated <em>fliM, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2 to Cm vector, fliH</em></span><br /><br />
<span style="color:#000000;"> -Megan miniprepped the innoculations (3-8D, 1-9F, 4-12A)</span><br /><br />
<span style="color:#000000;"> -low concentrations</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed <em>fliH, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2</em>, cm vector</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped 1-9F(1), 1-9F(2), 3-8D(1), 3-8D(2), 4-12A(1), 4-12A(2)</span><br /><br />
<span style="color:#000000;"> -low concentrations</span></p><br />
<p><span style="color:#000000;"><a href="http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg"><img class="alignright size-medium wp-image-165" title="_MG_4058" src="http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg?w=412&h=274" alt="" width="412" height="274" /></a><strong>6/18/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel PCRed <em>lsrR</em>(BL21), <em>lsrR</em>(HT115), <em>lrsK</em>(BL21), <em>lsrK</em>(HT115)</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -low concentrations</span><br /><br />
<span style="color:#000000;"> -Luke transformed J23100, RBS B0034, <em>luxS</em>, GFP E0040</span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated J23100, RBS B0034, <em>luxS</em>, GFP E0040</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed <em>cheA, cheB, cheY, cheZ</em> using HTB genomic DNA</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed <em>fliC</em>(1), <em>motA</em>(2), <em>motB</em>(3) using DH5alpha</span><br /><br />
<span style="color:#000000;"> -Luke miniprepped 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Megan digested <em>cheA, cheB, cheY, cheZ</em> and gel purified</span><br /><br />
<span style="color:#000000;"> -Luke ligated <em>cheA, cheB, cheY, cheZ</em></span><br /><br />
<span style="color:#000000;"> -Megan transformed <em>cheA, cheB, cheY, cheZ</em></span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Eric digested psB1C3 with X and X/S</span><br /><br />
<span style="color:#000000;"> -Rachel digested <em>fliC, motA, motB</em></span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped J23100, GFP E0040, <em>luxS</em>, RBS Boo34</span><br /><br />
<span style="color:#000000;"> -low concentrations</span></p><br />
<p><span style="color:#000000;"> <strong>6/25/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen transformed <em>motA, motB</em>, and control</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated T7 promoter and <em>cheY</em></span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed 20 colonies from <em>motA, motB</em></span><br /><br />
<span style="color:#000000;"> -Stephan inoculated <em>motA, motB, cheA, cheB, cheY, cheZ</em></span><br /><br />
<span style="color:#000000;"> -Luke miniprepped <em>cheZ, cheY, cheA, cheB, motA, motB</em></span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rachel glycerol stock: <em>motA</em> 6-14, <em>cheZ, cheY, cheA, cheB, motA, motB</em></span><br /><br />
<span style="color:#000000;"> -Luke transformed <em>cheZ</em>, pLaeI+RBS+<em>cheZ</em>, RBS+<em>cheZ</em>+dbl terminator, <em>cheZ</em> w/ H13 tag+dbl terminator, <em>cheZ</em> w/ H13 tag, strong promoter w/RBS</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated <em>cheZ</em>, pLaeI+RBS+<em>cheZ</em>, RBS+<em>cheZ</em>+dbl terminator, <em>cheZ</em> w/ H13 tag+dbl terminator, <em>cheZ</em> w/ H13 tag, strong promoter w/RBS</span><br /><br />
<span style="color:#000000;"> -Stephan PCR miniprep of <em>cheA</em>(7,16), <em>cheB</em>(14), <em>cheZ</em>(14), <em>motA</em>(15)</span></p><br />
<p><span style="color:#000000;"> <strong>7/2/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan miniprepped <em>cheZ</em> w/ histag, <em>cheZ</em>, <em>cheZ</em> w/ RBS + dbl termination, pLacI w/ RBS + <em>cheZ</em>, <em>cheZ</em> w/ Histag + dbl termination, strong promoter w/ RBS (cm)</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Megan inoculated <em>cheZ</em> w/ histag (amp), <em>cheZ</em> (amp), <em>cheZ</em> w/ RBS, double termination (amp), pLacI w/ RBS, <em>cheZ</em> (amp), <em>cheZ</em> w/ histag, double termination (amp), strong promoter w/ RBS (cm)</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped: psB1C3RFP (5 times), <em>cheZ</em>, strong promoter+RBS+cm, RBS+<em>cheZ</em>+dbl term site, pLacI+RBS+<em>cheZ</em>, <em>cheZ</em>+Hist+dbl, <em>cheZ</em>+Histag</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span></p><br />
<p><span style="color:#000000;"> <strong>7/9/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen digested and gel extracted <em>fliC</qem> and <em>GFP</em></span><br /><br />
<span style="color:#000000;"> -Rachel ligated fliC</span><br /><br />
<span style="color:#000000;"> -Luke transformed fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Rebecca received and plated strains: BW30044, BW30045, K-12 wildtype strain, strain B wildtype, JW1871-1, JW1879-2, JW2662-1, MG1655 (motile), W3110 </span></p><br />
<p><span style="color:#000000;"> <strong>7/16/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel made competent cells MG1655 </span><br /><br />
<span style="color:#000000;"> -Ellen digested GFP vector for control using XbaI<a href="http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg"><img class="alignright size-medium wp-image-187" title="DSC_0026" src="http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg?w=512&h=338" alt="" width="512" height="338" /></a></span><br /><br />
<span style="color:#000000;"> -Luke colony PCRed the fliC GFP </span><br /><br />
<span style="color:#000000;"> -primer dimers. unsuccessful-</span></p><br />
<p><span style="color:#000000;"> <strong>7/23/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen PCRed fliC w/ RBS</span><br /><br />
<span style="color:#000000;"> -Stephan transformed GFP-DH5a, GFP-MG1655 </span><br /><br />
<span style="color:#000000;"> -Megan inoculated w/ 50uL transformation product</span><br /><br />
<span style="color:#000000;"> -Stephan plated GFP-DH5a, GFP-MG1655</span><br /><br />
<span style="color:#000000;"> -Rachel PCRed fliC w/ RBS</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped GFP-DH5a (x2), GFP-MG1655 (x2)</span></p><br />
<p><span style="color:#000000;">-acceptable concentrations</span></p><br />
<p><span style="color:#000000;">-Digested fliC and GFP w/ XbaI and gel purified</span><br /><br />
<span style="color:#000000;"> -Eric ligated fliC-GFP and GFP w/ H2O (control)</span><br /><br />
<span style="color:#000000;"> -Eric transformed 1hour ligation product</span><br /><br />
<span style="color:#000000;"> -unsuccessul</span><br /><br />
<span style="color:#000000;"> -Stephan made control and cheY mutagenesis solutions</span><br /><br />
<span style="color:#000000;"> -Megan transformed fliC-GFP and GFP w/ H2O control into DH5a cells</span><br /><br />
<span style="color:#000000;"> -successful: GFP + H2O control and fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Luke quickchange mutagenesis</span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCR fliC-GFP</span></p><br />
<p><span style="color:#000000;"> <strong>7/30/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan miniprepped fliC-GFP</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rachel digested miniprepped products</span><br /><br />
<span style="color:#000000;"> -Eric quickchange PCRed fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Stephan digested quickchange PCR products with DPN1</span><br /><br />
<span style="color:#000000;"> -Megan digested fliC minipreps with Spe1 </span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Megan transformed 43, 44, quickchange digests</span></p><br />
<p><span style="color:#000000;"> <strong>8/6/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rebecca digested cheY</span><br /><br />
<span style="color:#000000;"> -Rebecca quickchange PCRed fliC and cheY with control</span><br /><br />
<span style="color:#000000;"> -Rebecca digested fliC-GFP, cheY, control</span><br /><br />
<span style="color:#000000;"> -Stephan plated fliC, cheY, and pUC18</span><br /><br />
<span style="color:#000000;"> -Rachel inoculated cheY mut colonies</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped cheY mut</span><br /><br />
<span style="color:#000000;"> -Stephan glycerol stocks of cheY mut</span><br /><br />
<span style="color:#000000;"> -Luke PCRed cheY mut</span><br /><br />
<span style="color:#000000;"> -Luke digested cheY mut</span></p><br />
<p><span style="color:#000000;"> <strong>8/13/12 week</strong></span><br /><br />
<span style="color:#000000;"> -PCRed fliA, fliD, fliE, fliF, fliG, fliH, fliM, flhB, flhC, flhD, flgA, flgB, flgJ, flgK, fliC-GFP, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Ellen digested and gel extracted the flagella genes</span><br /><br />
<span style="color:#000000;"> -Stephan transformed fliA, fliD,fliF, fliM, flhB, flgB, flgJ, flhD, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Megan digested fliC-GFP with DpnI, BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed pSB1C3 and fliC-GFP mut</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated pSB1C3-RFP, BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped pSB1C3 and BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Luke digested pSB1C3 fliF, fliD, flhB, flhD, flgJ, fliM, fliA</span><br /><br />
<span style="color:#000000;"> -Eric ligated fliF, fliD, flhB, flhD, flgJ, fliM, fliA with pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>8/20/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed fliA, fliM, fliD, fliF, flgJ, flhB, flhD</span><br /><br />
<span style="color:#000000;"> -Rachel imaged MG1655 in various conditions: pH 5, 6, 8, 9; NaCl 50mM, 100mM, 200mM, 400mM, 500mM; temperature 15C, 20C, 42C; glucose 10mM, 20mM, 50mM</span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped fliC-GFP mut, cheY mut</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Stephan glycerol stocks of fliC-GFP, cheY </span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed flgJ, flhD, fliA, flhB, cheY</span><br /><br />
<span style="color:#000000;"> -Megan inoculated successful colonies</span><br /><br />
<span style="color:#000000;"> -Rachel colony PCRed fliM, fliF, fliD</span><br /><br />
<span style="color:#000000;"> -Rebecca glycerol stocks of fliA, flhB, flgJ, flhD</span><br /><br />
<span style="color:#000000;"> -Eric imaging of MG1655</span><br /><br />
<span style="color:#000000;"> -Luke miniprepped fliA, flhB, flgJ, flhD</span><br /><br />
<span style="color:#000000;"> -Luke colony PCRed fliM, fliD, fliF</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated fliM </span><br /><br />
<span style="color:#000000;"> -Stephan PCRed flhD, flhB, flgJ, fliA to test directionality</span></p><br />
<p><span style="color:#000000;"> <strong>8/27/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR</span><br /><br />
<span style="color:#000000;"> -Eric digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR</span><br /><br />
<span style="color:#000000;"> -all successful except ompR, metR, araC</span><br /><br />
<span style="color:#000000;"> -Stephan digested pSB1C3 </span><br /><br />
<span style="color:#000000;"> -Megan ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter to pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>9/3/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns </span><br /><br />
<span style="color:#000000;"> -Eric transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Stephan digested pSB1C3</span><br /><br />
<span style="color:#000000;"> -Luke ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns with pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>9/10/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan transformed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated transformations </span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns</span><br /><br />
<span style="color:#000000;"> -Ellen transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406 </span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Rachel transformed BBa_K398108, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Luke inoculated BBa_K398108, BBa_K398406, fliH</span><br /><br />
<span style="color:#000000;"> -Eric miniprepped BBa_K398108, BBa_K398406, fliH</span></p><br />
<p><span style="color:#000000;">-acceptable concentrations<a href="http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg"><img class="alignright size-medium wp-image-162" title="_MG_4057" src="http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg?w=496&h=331" alt="" width="496" height="331" /></a></span></p><br />
<p><span style="color:#000000;">-Eric colony PCRed BBa_K398108, BBa_K398406, fliH </span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped flhD</span></p><br />
<p><span style="color:#000000;"> <strong>9/17/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Stephan transformed arabinose</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated cheY, T7</span><br /><br />
<span style="color:#000000;"> -Rachel digested cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Luke ligated cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped T7, cheY</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed T7/p.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed cheZ RBS, cheZ T7, p.cola cheY</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated cheZ RBS, cheZ T7, p.cola cheY</span><br /><br />
<span style="color:#000000;"> -Stephan miniprepped cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Megan transformed cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Sean inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Megan imaged cheZ, cheZ T7, cheZ RBS T7, cheZ T7</span><br /><br />
<span style="color:#000000;"> -Ellen glycerol stocks of cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Eric imaging of pcola cheZ A, cheZ RBS T7, cheZ RBS T7, cheZ T7, pcdf cheY A</span><br /><br />
<span style="color:#000000;"> -Luke inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span></p><br />
<p><span style="color:#000000;"> <strong>9/24/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rebecca imaged BBa_K398108, cheZ RBS T7, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Stephan imaged cheZ RBS T7, cheZ T7, pcdf cheY, control</span><br /><br />
<span style="color:#000000;"> -Luke digested cheY p.cola, pcdf cheY, pcdf cheZ, pcola cheZ, cheZ T7 RBS</span><br /><br />
<span style="color:#000000;"> -Megan transformed T7 RBS cheZ </span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated salt biobricks from frozen stocks</span><br /><br />
<span style="color:#000000;"> -Rachel colony PCRed T7 RBS cheZ </span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped cheZ RBS T7</span></p><br />
<p><span style="color:#000000;"> <strong>10/1/12 week</strong></span></p><br />
<p><span style="color:#000000;">-Eric miniprepped cheA, cheB, cheY, cheZ, motA. motB, T7 RBS cheZ, T7 RBS cheY, flgJ, fliA, fliH, fliC-GFP, flhB, flhD</span></p><br />
<p>-Western blot for T7 RBS cheY, T7 RBS cheZ</p><br />
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Sgenyk
http://2012.igem.org/Team:USC/Notebook
Team:USC/Notebook
2012-10-04T02:27:41Z
<p>Sgenyk: </p>
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Lab&nbsp;Notebook </h2><!-- .postitle --><br />
<br />
<p><span style="color:#000000;"><strong>5/21/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan transformed casBCED12 with Amp resistance, casBCED12 with Cm resistance, pSBIC3_RFP, pSBIK3_RFP, and pSBIA3_RFP</span><br /><br />
<span style="color:#000000;"> -successful results: pSBIC3_RFP and pSBIK3_RFP.</span><br /><br />
<span style="color:#000000;"> -Luke inoculated pSBIC3_RFP and pSBIK3_RFP</span><br /><br />
<span style="color:#000000;"> -Stephan miniprepped pSBIK3_RFP, and pSBIT3_RFP</span><br /><br />
<span style="color:#000000;"> -all very low concentration</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped pSBIC3_RFP, pSBIA3_RFP</span></p><br />
<p><span style="color:#000000;">-concentration (got 98 and 120 ug/ml, respectively)<a href="http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg"><img class="alignright size-medium wp-image-190" title="DSC_0028" src="http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg?w=442&h=292" alt="" width="442" height="292" /></a></span></p><br />
<p><span style="color:#000000;">-Rachel digested pSBIC3 from pSBIC3_RFP, pSBIK3 from pSBIK3_RFP</span><br /><br />
<span style="color:#000000;"> -Eric transformed pSBIT3_RFP, pSBIA3_RFP, casBCDE12 on Amp</span><br /><br />
<span style="color:#000000;"> -Stephan digested <em>casABCDE123</em>, tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Stephan digated <em>casA</em>,3 with tetO-GFP, with pSBIA3 or pSBIC3</span><br /><br />
<span style="color:#000000;"> -Eric digated casBCDE12 with pSBIA3 or PSBIC3</span></p><br />
<p><span style="color:#000000;"> <strong>5/28/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan inoculated (2x) 5mL psB1A3_RFP and psB1T3_RFP.</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed psB1C3_RFP, tetO_GFP, and psB3T5_RFP.</span><br /><br />
<span style="color:#000000;"> -Rebecca miniprepped psB1C3_RFP and psB1K3_RFP</span><br /><br />
<span style="color:#000000;"> -Ellen transformed psB1C3-RFP, psB3T5-RFP, tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Rachel PCRed <em>fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</em></span><br /><br />
<span style="color:#000000;"> -Luke gel extracted and digested the genes with EP or EX</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped pSB3T5_RFP (3) and tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Rachel digested tetO-GFP 2</span></p><br />
<p><span style="color:#000000;"> <strong>6/4/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Stephan PCRed genes using BL21 and HT115</span><br /><br />
<span style="color:#000000;"> -Stephan gel extracted and digested </span><br /><br />
<span style="color:#000000;"> -Ellen digated the genes to corresponding psb1C3_RFP vectors</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -successful: 4-12, 3-8D, 3-2H, 1-9F, 3-2J. (very low efficiency)</span><br /><br />
<span style="color:#000000;"> -Rachel transformed <em>cheB, cheC, cheY, cheZ, luxS, lsrK, lsrR, flaU, flbA, flhB, flhD, flgJ, flgK, fliA, fliC, fliD, fliF, fliG, fliH, fliM</em></span><br /><br />
<span style="color:#000000;"> -Rachel inoculated 4-12A, 3-8D, 3-2H, 1-9F, 3-2J</span><br /><br />
<span style="color:#000000;"> -Luke inoculated HT115 (w/ 5uL tet) and HB101 (w/o antibiotics)</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed <em>fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</em></span></p><br />
<p><span style="color:#000000;"> <strong>6/11/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen digested fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Luke gel extracted fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Rebecca PCRed fliC with DH5alpha, BL21, HT115</span><br /><br />
<span style="color:#000000;"> -Rachel ligated cm1, cm2, rfp1, rfp2</span><br /><br />
<span style="color:#000000;"> -Eric gel purified cm1, cm2, rfp1, rfp2</span><br /><br />
<span style="color:#000000;"> -Megan transformed fliA, cheZ, cheY, fliH, fliG, flgJ, flihB, flgK, fliD,fliM, 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -successful: 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -Stephan digested cm vector with EP</span><br /><br />
<span style="color:#000000;"> -Ellen gel extracted cm vectors</span><br /><br />
<span style="color:#000000;"> -Ellen inoculated 3-8D, 1-9F, 4-12A</span><br /><br />
<span style="color:#000000;"> -Eric PCRed chA, cheB, cheC, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -Luke transformed ligation products: fliG, flgJ, cheY, cheZ, fliD, flhB, fliM, fliH, flgK</span><br /><br />
<span style="color:#000000;"> -Rachel ligated fliM, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2 to Cm vector, fliH</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped the innoculations (3-8D, 1-9F, 4-12A)</span><br /><br />
<span style="color:#000000;"> -low concentrations</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed fliH, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2, cm vector</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped 1-9F(1), 1-9F(2), 3-8D(1), 3-8D(2), 4-12A(1), 4-12A(2)</span><br /><br />
<span style="color:#000000;"> -low concentrations</span></p><br />
<p><span style="color:#000000;"><a href="http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg"><img class="alignright size-medium wp-image-165" title="_MG_4058" src="http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg?w=412&h=274" alt="" width="412" height="274" /></a><strong>6/18/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel PCRed lsrR(BL21), lsrR(HT115), lrsK(BL21), lsrK(HT115)</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -low concentrations</span><br /><br />
<span style="color:#000000;"> -Luke transformed J23100, RBS B0034, luxS, GFP E0040</span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated J23100, RBS B0034, luxS, GFP E0040</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed cheA, cheB, cheY, cheZ using HTB genomic DNA</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed fliC(1), motA(2), motB(3) using DH5alpha</span><br /><br />
<span style="color:#000000;"> -Luke miniprepped 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Megan digested cheA, cheB, cheY, cheZ and gel purified</span><br /><br />
<span style="color:#000000;"> -Luke ligated cheA, cheB, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -Megan transformed cheA, cheB, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Eric digested psB1C3 with X and X/S</span><br /><br />
<span style="color:#000000;"> -Rachel digested fliC, motA, motB</span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped J23100, GFP E0040, luxS, RBS Boo34</span><br /><br />
<span style="color:#000000;"> -low concentrations</span></p><br />
<p><span style="color:#000000;"> <strong>6/25/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen transformed motA, motB, and control</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated T7 promoter and cheY</span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed 20 colonies from motA, motB</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated motA, motB, cheA, cheB, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -Luke miniprepped cheZ, cheY, cheA, cheB, motA, motB</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rachel glycerol stock: motA 6-14, cheZ, cheY, cheA, cheB, motA, motB</span><br /><br />
<span style="color:#000000;"> -Luke transformed cheZ, pLaeI+RBS+cheZ, RBS+cheZ+dbl terminator, cheZ w/ H13 tag+dbl terminator, cheZ w/ H13 tag, strong promoter w/RBS</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated cheZ, pLaeI+RBS+cheZ, RBS+cheZ+dbl terminator, cheZ w/ H13 tag+dbl terminator, cheZ w/ H13 tag, strong promoter w/RBS</span><br /><br />
<span style="color:#000000;"> -Stephan PCR miniprep of cheA(7,16), cheB(14), cheZ(14), motA(15)</span></p><br />
<p><span style="color:#000000;"> <strong>7/2/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan miniprepped cheZ w/ histag, cheZ, cheZ w/ RBS + dbl termination, pLacI w/ RBS + cheZ, cheZ w/ Histag + dbl termination, strong promoter w/ RBS (cm)</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Megan inoculated cheZ w/ histag (amp), cheZ (amp), cheZ w/ RBS, double termination (amp), pLacI w/ RBS, cheZ (amp), cheZ w/ histag, double termination (amp), strong promoter w/ RBS (cm)</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped: psB1C3RFP (5 times), cheZ, strong promoter+RBS+cm, RBS+cheZ+dbl term site, pLacI+RBS+cheZ, cheZ+Hist+dbl, cheZ+Histag</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span></p><br />
<p><span style="color:#000000;"> <strong>7/9/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen digested and gel extracted fliC and GFP</span><br /><br />
<span style="color:#000000;"> -Rachel ligated fliC</span><br /><br />
<span style="color:#000000;"> -Luke transformed fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Rebecca received and plated strains: BW30044, BW30045, K-12 wildtype strain, strain B wildtype, JW1871-1, JW1879-2, JW2662-1, MG1655 (motile), W3110 </span></p><br />
<p><span style="color:#000000;"> <strong>7/16/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel made competent cells MG1655 </span><br /><br />
<span style="color:#000000;"> -Ellen digested GFP vector for control using XbaI<a href="http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg"><img class="alignright size-medium wp-image-187" title="DSC_0026" src="http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg?w=512&h=338" alt="" width="512" height="338" /></a></span><br /><br />
<span style="color:#000000;"> -Luke colony PCRed the fliC GFP </span><br /><br />
<span style="color:#000000;"> -primer dimers. unsuccessful-</span></p><br />
<p><span style="color:#000000;"> <strong>7/23/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen PCRed fliC w/ RBS</span><br /><br />
<span style="color:#000000;"> -Stephan transformed GFP-DH5a, GFP-MG1655 </span><br /><br />
<span style="color:#000000;"> -Megan inoculated w/ 50uL transformation product</span><br /><br />
<span style="color:#000000;"> -Stephan plated GFP-DH5a, GFP-MG1655</span><br /><br />
<span style="color:#000000;"> -Rachel PCRed fliC w/ RBS</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped GFP-DH5a (x2), GFP-MG1655 (x2)</span></p><br />
<p><span style="color:#000000;">-acceptable concentrations</span></p><br />
<p><span style="color:#000000;">-Digested fliC and GFP w/ XbaI and gel purified</span><br /><br />
<span style="color:#000000;"> -Eric ligated fliC-GFP and GFP w/ H2O (control)</span><br /><br />
<span style="color:#000000;"> -Eric transformed 1hour ligation product</span><br /><br />
<span style="color:#000000;"> -unsuccessul</span><br /><br />
<span style="color:#000000;"> -Stephan made control and cheY mutagenesis solutions</span><br /><br />
<span style="color:#000000;"> -Megan transformed fliC-GFP and GFP w/ H2O control into DH5a cells</span><br /><br />
<span style="color:#000000;"> -successful: GFP + H2O control and fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Luke quickchange mutagenesis</span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCR fliC-GFP</span></p><br />
<p><span style="color:#000000;"> <strong>7/30/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan miniprepped fliC-GFP</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rachel digested miniprepped products</span><br /><br />
<span style="color:#000000;"> -Eric quickchange PCRed fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Stephan digested quickchange PCR products with DPN1</span><br /><br />
<span style="color:#000000;"> -Megan digested fliC minipreps with Spe1 </span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Megan transformed 43, 44, quickchange digests</span></p><br />
<p><span style="color:#000000;"> <strong>8/6/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rebecca digested cheY</span><br /><br />
<span style="color:#000000;"> -Rebecca quickchange PCRed fliC and cheY with control</span><br /><br />
<span style="color:#000000;"> -Rebecca digested fliC-GFP, cheY, control</span><br /><br />
<span style="color:#000000;"> -Stephan plated fliC, cheY, and pUC18</span><br /><br />
<span style="color:#000000;"> -Rachel inoculated cheY mut colonies</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped cheY mut</span><br /><br />
<span style="color:#000000;"> -Stephan glycerol stocks of cheY mut</span><br /><br />
<span style="color:#000000;"> -Luke PCRed cheY mut</span><br /><br />
<span style="color:#000000;"> -Luke digested cheY mut</span></p><br />
<p><span style="color:#000000;"> <strong>8/13/12 week</strong></span><br /><br />
<span style="color:#000000;"> -PCRed fliA, fliD, fliE, fliF, fliG, fliH, fliM, flhB, flhC, flhD, flgA, flgB, flgJ, flgK, fliC-GFP, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Ellen digested and gel extracted the flagella genes</span><br /><br />
<span style="color:#000000;"> -Stephan transformed fliA, fliD,fliF, fliM, flhB, flgB, flgJ, flhD, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Megan digested fliC-GFP with DpnI, BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed pSB1C3 and fliC-GFP mut</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated pSB1C3-RFP, BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped pSB1C3 and BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Luke digested pSB1C3 fliF, fliD, flhB, flhD, flgJ, fliM, fliA</span><br /><br />
<span style="color:#000000;"> -Eric ligated fliF, fliD, flhB, flhD, flgJ, fliM, fliA with pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>8/20/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed fliA, fliM, fliD, fliF, flgJ, flhB, flhD</span><br /><br />
<span style="color:#000000;"> -Rachel imaged MG1655 in various conditions: pH 5, 6, 8, 9; NaCl 50mM, 100mM, 200mM, 400mM, 500mM; temperature 15C, 20C, 42C; glucose 10mM, 20mM, 50mM</span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped fliC-GFP mut, cheY mut</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Stephan glycerol stocks of fliC-GFP, cheY </span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed flgJ, flhD, fliA, flhB, cheY</span><br /><br />
<span style="color:#000000;"> -Megan inoculated successful colonies</span><br /><br />
<span style="color:#000000;"> -Rachel colony PCRed fliM, fliF, fliD</span><br /><br />
<span style="color:#000000;"> -Rebecca glycerol stocks of fliA, flhB, flgJ, flhD</span><br /><br />
<span style="color:#000000;"> -Eric imaging of MG1655</span><br /><br />
<span style="color:#000000;"> -Luke miniprepped fliA, flhB, flgJ, flhD</span><br /><br />
<span style="color:#000000;"> -Luke colony PCRed fliM, fliD, fliF</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated fliM </span><br /><br />
<span style="color:#000000;"> -Stephan PCRed flhD, flhB, flgJ, fliA to test directionality</span></p><br />
<p><span style="color:#000000;"> <strong>8/27/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR</span><br /><br />
<span style="color:#000000;"> -Eric digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR</span><br /><br />
<span style="color:#000000;"> -all successful except ompR, metR, araC</span><br /><br />
<span style="color:#000000;"> -Stephan digested pSB1C3 </span><br /><br />
<span style="color:#000000;"> -Megan ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter to pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>9/3/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns </span><br /><br />
<span style="color:#000000;"> -Eric transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Stephan digested pSB1C3</span><br /><br />
<span style="color:#000000;"> -Luke ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns with pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>9/10/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan transformed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated transformations </span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns</span><br /><br />
<span style="color:#000000;"> -Ellen transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406 </span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Rachel transformed BBa_K398108, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Luke inoculated BBa_K398108, BBa_K398406, fliH</span><br /><br />
<span style="color:#000000;"> -Eric miniprepped BBa_K398108, BBa_K398406, fliH</span></p><br />
<p><span style="color:#000000;">-acceptable concentrations<a href="http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg"><img class="alignright size-medium wp-image-162" title="_MG_4057" src="http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg?w=496&h=331" alt="" width="496" height="331" /></a></span></p><br />
<p><span style="color:#000000;">-Eric colony PCRed BBa_K398108, BBa_K398406, fliH </span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped flhD</span></p><br />
<p><span style="color:#000000;"> <strong>9/17/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Stephan transformed arabinose</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated cheY, T7</span><br /><br />
<span style="color:#000000;"> -Rachel digested cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Luke ligated cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped T7, cheY</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed T7/p.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed cheZ RBS, cheZ T7, p.cola cheY</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated cheZ RBS, cheZ T7, p.cola cheY</span><br /><br />
<span style="color:#000000;"> -Stephan miniprepped cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Megan transformed cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Sean inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Megan imaged cheZ, cheZ T7, cheZ RBS T7, cheZ T7</span><br /><br />
<span style="color:#000000;"> -Ellen glycerol stocks of cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Eric imaging of pcola cheZ A, cheZ RBS T7, cheZ RBS T7, cheZ T7, pcdf cheY A</span><br /><br />
<span style="color:#000000;"> -Luke inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span></p><br />
<p><span style="color:#000000;"> <strong>9/24/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rebecca imaged BBa_K398108, cheZ RBS T7, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Stephan imaged cheZ RBS T7, cheZ T7, pcdf cheY, control</span><br /><br />
<span style="color:#000000;"> -Luke digested cheY p.cola, pcdf cheY, pcdf cheZ, pcola cheZ, cheZ T7 RBS</span><br /><br />
<span style="color:#000000;"> -Megan transformed T7 RBS cheZ </span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated salt biobricks from frozen stocks</span><br /><br />
<span style="color:#000000;"> -Rachel colony PCRed T7 RBS cheZ </span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped cheZ RBS T7</span></p><br />
<p><span style="color:#000000;"> <strong>10/1/12 week</strong></span></p><br />
<p><span style="color:#000000;">-Eric miniprepped cheA, cheB, cheY, cheZ, motA. motB, T7 RBS cheZ, T7 RBS cheY, flgJ, fliA, fliH, fliC-GFP, flhB, flhD</span></p><br />
<p>-Western blot for T7 RBS cheY, T7 RBS cheZ</p><br />
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Sgenyk
http://2012.igem.org/Team:USC/Notebook
Team:USC/Notebook
2012-10-04T02:21:21Z
<p>Sgenyk: </p>
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<meta name="description" content="5/21/12 week -Transformed casBCED12 with Amp resistance, casBCED12 with Cm resistance, pSBIC3_RFP, pSBIK3_RFP, and pSBIA3_RFP -successful results: pSBIC3_RFP and pSBIK3_RFP. -inoculated pSBIC3_RFP and pSBIK3_RFP -miniprepped pSBIK3_RFP, and pSBIT3_RFP -all very low&hellip; Read More &rarr;" /><br />
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Lab&nbsp;Notebook </h2><!-- .postitle --><br />
<br />
<p><span style="color:#000000;"><strong>5/21/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan transformed casBCED12 with Amp resistance, casBCED12 with Cm resistance, pSBIC3_RFP, pSBIK3_RFP, and pSBIA3_RFP</span><br /><br />
<span style="color:#000000;"> -successful results: pSBIC3_RFP and pSBIK3_RFP.</span><br /><br />
<span style="color:#000000;"> -Luke inoculated pSBIC3_RFP and pSBIK3_RFP</span><br /><br />
<span style="color:#000000;"> -Stephan miniprepped pSBIK3_RFP, and pSBIT3_RFP</span><br /><br />
<span style="color:#000000;"> -all very low concentration</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped pSBIC3_RFP, pSBIA3_RFP</span></p><br />
<p><span style="color:#000000;">-concentration (got 98 and 120 ug/ml, respectively)<a href="http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg"><img class="alignright size-medium wp-image-190" title="DSC_0028" src="http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg?w=442&h=292" alt="" width="442" height="292" /></a></span></p><br />
<p><span style="color:#000000;">-Rachel digested pSBIC3 from pSBIC3_RFP, pSBIK3 from pSBIK3_RFP</span><br /><br />
<span style="color:#000000;"> -Eric transformed pSBIT3_RFP, pSBIA3_RFP, casBCDE12 on Amp</span><br /><br />
<span style="color:#000000;"> -Stephan digested casABCDE123, tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Stephan digated casA,3 with tetO-GFP, with pSBIA3 or pSBIC3</span><br /><br />
<span style="color:#000000;"> -Eric digated casBCDE12 with pSBIA3 or PSBIC3</span></p><br />
<p><span style="color:#000000;"> <strong>5/28/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan inoculated (2x) 5mL psB1A3_RFP and psB1T3_RFP.</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed psB1C3_RFP, tetO_GFP, and psB3T5_RFP.</span><br /><br />
<span style="color:#000000;"> -Rebecca miniprepped psB1C3_RFP and psB1K3_RFP</span><br /><br />
<span style="color:#000000;"> -Ellen transformed psB1C3-RFP, psB3T5-RFP, tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Rachel PCRed fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Luke gel extracted and digested the genes with EP or EX</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped pSB3T5_RFP (3) and tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Rachel digested tetO-GFP 2</span></p><br />
<p><span style="color:#000000;"> <strong>6/4/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Stephan PCRed genes using BL21 and HT115</span><br /><br />
<span style="color:#000000;"> -Stephan gel extracted and digested </span><br /><br />
<span style="color:#000000;"> -Ellen digated the genes to corresponding psb1C3_RFP vectors</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -successful: 4-12, 3-8D, 3-2H, 1-9F, 3-2J. (very low efficiency)</span><br /><br />
<span style="color:#000000;"> -Rachel transformed cheB, cheC, cheY, cheZ, luxS, lsrK, lsrR, flaU, flbA, flhB, flhD, flgJ, flgK, fliA, fliC, fliD, fliF, fliG, fliH, fliM</span><br /><br />
<span style="color:#000000;"> -Rachel inoculated 4-12A, 3-8D, 3-2H, 1-9F, 3-2J</span><br /><br />
<span style="color:#000000;"> -Luke inoculated HT115 (w/ 5uL tet) and HB101 (w/o antibiotics)</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</span></p><br />
<p><span style="color:#000000;"> <strong>6/11/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen digested fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Luke gel extracted fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Rebecca PCRed fliC with DH5alpha, BL21, HT115</span><br /><br />
<span style="color:#000000;"> -Rachel ligated cm1, cm2, rfp1, rfp2</span><br /><br />
<span style="color:#000000;"> -Eric gel purified cm1, cm2, rfp1, rfp2</span><br /><br />
<span style="color:#000000;"> -Megan transformed fliA, cheZ, cheY, fliH, fliG, flgJ, flihB, flgK, fliD,fliM, 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -successful: 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -Stephan digested cm vector with EP</span><br /><br />
<span style="color:#000000;"> -Ellen gel extracted cm vectors</span><br /><br />
<span style="color:#000000;"> -Ellen inoculated 3-8D, 1-9F, 4-12A</span><br /><br />
<span style="color:#000000;"> -Eric PCRed chA, cheB, cheC, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -Luke transformed ligation products: fliG, flgJ, cheY, cheZ, fliD, flhB, fliM, fliH, flgK</span><br /><br />
<span style="color:#000000;"> -Rachel ligated fliM, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2 to Cm vector, fliH</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped the innoculations (3-8D, 1-9F, 4-12A)</span><br /><br />
<span style="color:#000000;"> -low concentrations</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed fliH, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2, cm vector</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped 1-9F(1), 1-9F(2), 3-8D(1), 3-8D(2), 4-12A(1), 4-12A(2)</span><br /><br />
<span style="color:#000000;"> -low concentrations</span></p><br />
<p><span style="color:#000000;"><a href="http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg"><img class="alignright size-medium wp-image-165" title="_MG_4058" src="http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg?w=412&h=274" alt="" width="412" height="274" /></a><strong>6/18/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel PCRed lsrR(BL21), lsrR(HT115), lrsK(BL21), lsrK(HT115)</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -low concentrations</span><br /><br />
<span style="color:#000000;"> -Luke transformed J23100, RBS B0034, luxS, GFP E0040</span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated J23100, RBS B0034, luxS, GFP E0040</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed cheA, cheB, cheY, cheZ using HTB genomic DNA</span><br /><br />
<span style="color:#000000;"> -Stephan PCRed fliC(1), motA(2), motB(3) using DH5alpha</span><br /><br />
<span style="color:#000000;"> -Luke miniprepped 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Megan digested cheA, cheB, cheY, cheZ and gel purified</span><br /><br />
<span style="color:#000000;"> -Luke ligated cheA, cheB, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -Megan transformed cheA, cheB, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Eric digested psB1C3 with X and X/S</span><br /><br />
<span style="color:#000000;"> -Rachel digested fliC, motA, motB</span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped J23100, GFP E0040, luxS, RBS Boo34</span><br /><br />
<span style="color:#000000;"> -low concentrations</span></p><br />
<p><span style="color:#000000;"> <strong>6/25/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen transformed motA, motB, and control</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated T7 promoter and cheY</span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed 20 colonies from motA, motB</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated motA, motB, cheA, cheB, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -Luke miniprepped cheZ, cheY, cheA, cheB, motA, motB</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rachel glycerol stock: motA 6-14, cheZ, cheY, cheA, cheB, motA, motB</span><br /><br />
<span style="color:#000000;"> -Luke transformed cheZ, pLaeI+RBS+cheZ, RBS+cheZ+dbl terminator, cheZ w/ H13 tag+dbl terminator, cheZ w/ H13 tag, strong promoter w/RBS</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated cheZ, pLaeI+RBS+cheZ, RBS+cheZ+dbl terminator, cheZ w/ H13 tag+dbl terminator, cheZ w/ H13 tag, strong promoter w/RBS</span><br /><br />
<span style="color:#000000;"> -Stephan PCR miniprep of cheA(7,16), cheB(14), cheZ(14), motA(15)</span></p><br />
<p><span style="color:#000000;"> <strong>7/2/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan miniprepped cheZ w/ histag, cheZ, cheZ w/ RBS + dbl termination, pLacI w/ RBS + cheZ, cheZ w/ Histag + dbl termination, strong promoter w/ RBS (cm)</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Megan inoculated cheZ w/ histag (amp), cheZ (amp), cheZ w/ RBS, double termination (amp), pLacI w/ RBS, cheZ (amp), cheZ w/ histag, double termination (amp), strong promoter w/ RBS (cm)</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped: psB1C3RFP (5 times), cheZ, strong promoter+RBS+cm, RBS+cheZ+dbl term site, pLacI+RBS+cheZ, cheZ+Hist+dbl, cheZ+Histag</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span></p><br />
<p><span style="color:#000000;"> <strong>7/9/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen digested and gel extracted fliC and GFP</span><br /><br />
<span style="color:#000000;"> -Rachel ligated fliC</span><br /><br />
<span style="color:#000000;"> -Luke transformed fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Rebecca received and plated strains: BW30044, BW30045, K-12 wildtype strain, strain B wildtype, JW1871-1, JW1879-2, JW2662-1, MG1655 (motile), W3110 </span></p><br />
<p><span style="color:#000000;"> <strong>7/16/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel made competent cells MG1655 </span><br /><br />
<span style="color:#000000;"> -Ellen digested GFP vector for control using XbaI<a href="http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg"><img class="alignright size-medium wp-image-187" title="DSC_0026" src="http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg?w=512&h=338" alt="" width="512" height="338" /></a></span><br /><br />
<span style="color:#000000;"> -Luke colony PCRed the fliC GFP </span><br /><br />
<span style="color:#000000;"> -primer dimers. unsuccessful-</span></p><br />
<p><span style="color:#000000;"> <strong>7/23/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen PCRed fliC w/ RBS</span><br /><br />
<span style="color:#000000;"> -Stephan transformed GFP-DH5a, GFP-MG1655 </span><br /><br />
<span style="color:#000000;"> -Megan inoculated w/ 50uL transformation product</span><br /><br />
<span style="color:#000000;"> -Stephan plated GFP-DH5a, GFP-MG1655</span><br /><br />
<span style="color:#000000;"> -Rachel PCRed fliC w/ RBS</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped GFP-DH5a (x2), GFP-MG1655 (x2)</span></p><br />
<p><span style="color:#000000;">-acceptable concentrations</span></p><br />
<p><span style="color:#000000;">-Digested fliC and GFP w/ XbaI and gel purified</span><br /><br />
<span style="color:#000000;"> -Eric ligated fliC-GFP and GFP w/ H2O (control)</span><br /><br />
<span style="color:#000000;"> -Eric transformed 1hour ligation product</span><br /><br />
<span style="color:#000000;"> -unsuccessul</span><br /><br />
<span style="color:#000000;"> -Stephan made control and cheY mutagenesis solutions</span><br /><br />
<span style="color:#000000;"> -Megan transformed fliC-GFP and GFP w/ H2O control into DH5a cells</span><br /><br />
<span style="color:#000000;"> -successful: GFP + H2O control and fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Luke quickchange mutagenesis</span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCR fliC-GFP</span></p><br />
<p><span style="color:#000000;"> <strong>7/30/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan miniprepped fliC-GFP</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rachel digested miniprepped products</span><br /><br />
<span style="color:#000000;"> -Eric quickchange PCRed fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Stephan digested quickchange PCR products with DPN1</span><br /><br />
<span style="color:#000000;"> -Megan digested fliC minipreps with Spe1 </span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Megan transformed 43, 44, quickchange digests</span></p><br />
<p><span style="color:#000000;"> <strong>8/6/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rebecca digested cheY</span><br /><br />
<span style="color:#000000;"> -Rebecca quickchange PCRed fliC and cheY with control</span><br /><br />
<span style="color:#000000;"> -Rebecca digested fliC-GFP, cheY, control</span><br /><br />
<span style="color:#000000;"> -Stephan plated fliC, cheY, and pUC18</span><br /><br />
<span style="color:#000000;"> -Rachel inoculated cheY mut colonies</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped cheY mut</span><br /><br />
<span style="color:#000000;"> -Stephan glycerol stocks of cheY mut</span><br /><br />
<span style="color:#000000;"> -Luke PCRed cheY mut</span><br /><br />
<span style="color:#000000;"> -Luke digested cheY mut</span></p><br />
<p><span style="color:#000000;"> <strong>8/13/12 week</strong></span><br /><br />
<span style="color:#000000;"> -PCRed fliA, fliD, fliE, fliF, fliG, fliH, fliM, flhB, flhC, flhD, flgA, flgB, flgJ, flgK, fliC-GFP, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Ellen digested and gel extracted the flagella genes</span><br /><br />
<span style="color:#000000;"> -Stephan transformed fliA, fliD,fliF, fliM, flhB, flgB, flgJ, flhD, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Megan digested fliC-GFP with DpnI, BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed pSB1C3 and fliC-GFP mut</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated pSB1C3-RFP, BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped pSB1C3 and BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Luke digested pSB1C3 fliF, fliD, flhB, flhD, flgJ, fliM, fliA</span><br /><br />
<span style="color:#000000;"> -Eric ligated fliF, fliD, flhB, flhD, flgJ, fliM, fliA with pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>8/20/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed fliA, fliM, fliD, fliF, flgJ, flhB, flhD</span><br /><br />
<span style="color:#000000;"> -Rachel imaged MG1655 in various conditions: pH 5, 6, 8, 9; NaCl 50mM, 100mM, 200mM, 400mM, 500mM; temperature 15C, 20C, 42C; glucose 10mM, 20mM, 50mM</span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped fliC-GFP mut, cheY mut</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Stephan glycerol stocks of fliC-GFP, cheY </span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed flgJ, flhD, fliA, flhB, cheY</span><br /><br />
<span style="color:#000000;"> -Megan inoculated successful colonies</span><br /><br />
<span style="color:#000000;"> -Rachel colony PCRed fliM, fliF, fliD</span><br /><br />
<span style="color:#000000;"> -Rebecca glycerol stocks of fliA, flhB, flgJ, flhD</span><br /><br />
<span style="color:#000000;"> -Eric imaging of MG1655</span><br /><br />
<span style="color:#000000;"> -Luke miniprepped fliA, flhB, flgJ, flhD</span><br /><br />
<span style="color:#000000;"> -Luke colony PCRed fliM, fliD, fliF</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated fliM </span><br /><br />
<span style="color:#000000;"> -Stephan PCRed flhD, flhB, flgJ, fliA to test directionality</span></p><br />
<p><span style="color:#000000;"> <strong>8/27/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Ellen PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR</span><br /><br />
<span style="color:#000000;"> -Eric digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR</span><br /><br />
<span style="color:#000000;"> -all successful except ompR, metR, araC</span><br /><br />
<span style="color:#000000;"> -Stephan digested pSB1C3 </span><br /><br />
<span style="color:#000000;"> -Megan ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter to pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>9/3/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rachel digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns </span><br /><br />
<span style="color:#000000;"> -Eric transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Stephan digested pSB1C3</span><br /><br />
<span style="color:#000000;"> -Luke ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns with pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>9/10/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan transformed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated transformations </span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns</span><br /><br />
<span style="color:#000000;"> -Ellen transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406 </span><br /><br />
<span style="color:#000000;"> -Megan colony PCRed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Rachel transformed BBa_K398108, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Luke inoculated BBa_K398108, BBa_K398406, fliH</span><br /><br />
<span style="color:#000000;"> -Eric miniprepped BBa_K398108, BBa_K398406, fliH</span></p><br />
<p><span style="color:#000000;">-acceptable concentrations<a href="http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg"><img class="alignright size-medium wp-image-162" title="_MG_4057" src="http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg?w=496&h=331" alt="" width="496" height="331" /></a></span></p><br />
<p><span style="color:#000000;">-Eric colony PCRed BBa_K398108, BBa_K398406, fliH </span><br /><br />
<span style="color:#000000;"> -Rachel miniprepped flhD</span></p><br />
<p><span style="color:#000000;"> <strong>9/17/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Stephan transformed arabinose</span><br /><br />
<span style="color:#000000;"> -Stephan inoculated cheY, T7</span><br /><br />
<span style="color:#000000;"> -Rachel digested cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Luke ligated cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Megan miniprepped T7, cheY</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Rebecca transformed T7/p.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Rebecca colony PCRed cheZ RBS, cheZ T7, p.cola cheY</span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated cheZ RBS, cheZ T7, p.cola cheY</span><br /><br />
<span style="color:#000000;"> -Stephan miniprepped cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Megan transformed cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Sean inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Megan imaged cheZ, cheZ T7, cheZ RBS T7, cheZ T7</span><br /><br />
<span style="color:#000000;"> -Ellen glycerol stocks of cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Eric imaging of pcola cheZ A, cheZ RBS T7, cheZ RBS T7, cheZ T7, pcdf cheY A</span><br /><br />
<span style="color:#000000;"> -Luke inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span></p><br />
<p><span style="color:#000000;"> <strong>9/24/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Rebecca imaged BBa_K398108, cheZ RBS T7, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Stephan imaged cheZ RBS T7, cheZ T7, pcdf cheY, control</span><br /><br />
<span style="color:#000000;"> -Luke digested cheY p.cola, pcdf cheY, pcdf cheZ, pcola cheZ, cheZ T7 RBS</span><br /><br />
<span style="color:#000000;"> -Megan transformed T7 RBS cheZ </span><br /><br />
<span style="color:#000000;"> -Rebecca inoculated salt biobricks from frozen stocks</span><br /><br />
<span style="color:#000000;"> -Rachel colony PCRed T7 RBS cheZ </span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped cheZ RBS T7</span></p><br />
<p><span style="color:#000000;"> <strong>10/1/12 week</strong></span></p><br />
<p><span style="color:#000000;">-Eric miniprepped cheA, cheB, cheY, cheZ, motA. motB, T7 RBS cheZ, T7 RBS cheY, flgJ, fliA, fliH, fliC-GFP, flhB, flhD</span></p><br />
<p>-Western blot for T7 RBS cheY, T7 RBS cheZ</p><br />
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Sgenyk
http://2012.igem.org/Team:USC/Notebook
Team:USC/Notebook
2012-10-04T02:00:36Z
<p>Sgenyk: </p>
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<meta name="description" content="5/21/12 week -Transformed casBCED12 with Amp resistance, casBCED12 with Cm resistance, pSBIC3_RFP, pSBIK3_RFP, and pSBIA3_RFP -successful results: pSBIC3_RFP and pSBIK3_RFP. -inoculated pSBIC3_RFP and pSBIK3_RFP -miniprepped pSBIK3_RFP, and pSBIT3_RFP -all very low&hellip; Read More &rarr;" /><br />
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Lab&nbsp;Notebook </h2><!-- .postitle --><br />
<br />
<p><span style="color:#000000;"><strong>5/21/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Megan transformed casBCED12 with Amp resistance, casBCED12 with Cm resistance, pSBIC3_RFP, pSBIK3_RFP, and pSBIA3_RFP</span><br /><br />
<span style="color:#000000;"> -successful results: pSBIC3_RFP and pSBIK3_RFP.</span><br /><br />
<span style="color:#000000;"> -Luke inoculated pSBIC3_RFP and pSBIK3_RFP</span><br /><br />
<span style="color:#000000;"> -Stephan miniprepped pSBIK3_RFP, and pSBIT3_RFP</span><br /><br />
<span style="color:#000000;"> -all very low concentration</span><br /><br />
<span style="color:#000000;"> -Ellen miniprepped pSBIC3_RFP, pSBIA3_RFP</span></p><br />
<p><span style="color:#000000;">-concentration (got 98 and 120 ug/ml, respectively)<a href="http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg"><img class="alignright size-medium wp-image-190" title="DSC_0028" src="http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg?w=442&h=292" alt="" width="442" height="292" /></a></span></p><br />
<p><span style="color:#000000;">-Rachel digested pSBIC3 from pSBIC3_RFP, pSBIK3 from pSBIK3_RFP</span><br /><br />
<span style="color:#000000;"> -Transformed pSBIT3_RFP, pSBIA3_RFP, casBCDE12 on Amp</span><br /><br />
<span style="color:#000000;"> -Digested casABCDE123, tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Ligated casA,3 with tetO-GFP, with pSBIA3 or pSBIC3</span><br /><br />
<span style="color:#000000;"> -Ligated casBCDE12 with pSBIA3 or PSBIC3</span></p><br />
<p><span style="color:#000000;"> <strong>5/28/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Inoculated (2x) 5mL psB1A3_RFP and psB1T3_RFP.</span><br /><br />
<span style="color:#000000;"> -Transformed psB1C3_RFP, tetO_GFP, and psB3T5_RFP.</span><br /><br />
<span style="color:#000000;"> -Miniprepped psB1C3_RFP and psB1K3_RFP</span><br /><br />
<span style="color:#000000;"> -Transformed psB1C3-RFP, psB3T5-RFP, tetO-GFP</span><br /><br />
<span style="color:#000000;"> -PCRed fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Gel extracted and digested the genes with EP or EX</span><br /><br />
<span style="color:#000000;"> -Miniprepped pSB3T5_RFP (3) and tetO-GFP</span><br /><br />
<span style="color:#000000;"> -Digested tetO-GFP 2</span></p><br />
<p><span style="color:#000000;"> <strong>6/4/12 week</strong></span><br /><br />
<span style="color:#000000;"> -PCRed genes using BL21 and HT115</span><br /><br />
<span style="color:#000000;"> -Gel extracted and digested </span><br /><br />
<span style="color:#000000;"> -Ligated the genes to corresponding psb1C3_RFP vectors</span><br /><br />
<span style="color:#000000;"> -Transformed 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -successful: 4-12, 3-8D, 3-2H, 1-9F, 3-2J. (very low efficiency)</span><br /><br />
<span style="color:#000000;"> -Transformed cheB, cheC, cheY, cheZ, luxS, lsrK, lsrR, flaU, flbA, flhB, flhD, flgJ, flgK, fliA, fliC, fliD, fliF, fliG, fliH, fliM</span><br /><br />
<span style="color:#000000;"> -Inoculated 4-12A, 3-8D, 3-2H, 1-9F, 3-2J</span><br /><br />
<span style="color:#000000;"> -Inoculated HT115 (w/ 5uL tet) and HB101 (w/o antibiotics)</span><br /><br />
<span style="color:#000000;"> -PCRed fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</span></p><br />
<p><span style="color:#000000;"> <strong>6/11/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Digested fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Gel extracted fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -PCRed fliC with DH5alpha, BL21, HT115</span><br /><br />
<span style="color:#000000;"> -Ligated cm1, cm2, rfp1, rfp2</span><br /><br />
<span style="color:#000000;"> -Gel purified cm1, cm2, rfp1, rfp2</span><br /><br />
<span style="color:#000000;"> -Transformed fliA, cheZ, cheY, fliH, fliG, flgJ, flihB, flgK, fliD,fliM, 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -successful: 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -Digested cm vector with EP</span><br /><br />
<span style="color:#000000;"> -Gel extracted cm vectors</span><br /><br />
<span style="color:#000000;"> -Inoculated 3-8D, 1-9F, 4-12A</span><br /><br />
<span style="color:#000000;"> -PCRed chA, cheB, cheC, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -Transformed ligation products: fliG, flgJ, cheY, cheZ, fliD, flhB, fliM, fliH, flgK</span><br /><br />
<span style="color:#000000;"> -Ligated fliM, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2 to Cm vector, fliH</span><br /><br />
<span style="color:#000000;"> -Miniprepped the innoculations (3-8D, 1-9F, 4-12A)</span><br /><br />
<span style="color:#000000;"> -low concentrations</span><br /><br />
<span style="color:#000000;"> -Transformed fliH, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2, cm vector</span><br /><br />
<span style="color:#000000;"> -Miniprepped 1-9F(1), 1-9F(2), 3-8D(1), 3-8D(2), 4-12A(1), 4-12A(2)</span><br /><br />
<span style="color:#000000;"> -low concentrations</span></p><br />
<p><span style="color:#000000;"><a href="http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg"><img class="alignright size-medium wp-image-165" title="_MG_4058" src="http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg?w=412&h=274" alt="" width="412" height="274" /></a><strong>6/18/12 week</strong></span><br /><br />
<span style="color:#000000;"> -PCRed lsrR(BL21), lsrR(HT115), lrsK(BL21), lsrK(HT115)</span><br /><br />
<span style="color:#000000;"> -Inoculated 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -Miniprepped 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -low concentrations</span><br /><br />
<span style="color:#000000;"> -Transformed J23100, RBS B0034, luxS, GFP E0040</span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Inoculated J23100, RBS B0034, luxS, GFP E0040</span><br /><br />
<span style="color:#000000;"> -PCRed cheA, cheB, cheY, cheZ using HTB genomic DNA</span><br /><br />
<span style="color:#000000;"> -PCRed fliC(1), motA(2), motB(3) using DH5alpha</span><br /><br />
<span style="color:#000000;"> -Miniprepped 1-9F, 3-8D, 4-12A</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Digested cheA, cheB, cheY, cheZ and gel purified</span><br /><br />
<span style="color:#000000;"> -Ligated cheA, cheB, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -Transformed cheA, cheB, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Digested psB1C3 with X and X/S</span><br /><br />
<span style="color:#000000;"> -Digested fliC, motA, motB</span><br /><br />
<span style="color:#000000;"> -Miniprepped J23100, GFP E0040, luxS, RBS Boo34</span><br /><br />
<span style="color:#000000;"> -low concentrations</span></p><br />
<p><span style="color:#000000;"> <strong>6/25/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Transformed motA, motB, and control</span><br /><br />
<span style="color:#000000;"> -Inoculated T7 promoter and cheY</span><br /><br />
<span style="color:#000000;"> -Colony PCRed 20 colonies from motA, motB</span><br /><br />
<span style="color:#000000;"> -Inoculated motA, motB, cheA, cheB, cheY, cheZ</span><br /><br />
<span style="color:#000000;"> -Miniprepped cheZ, cheY, cheA, cheB, motA, motB</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Glycerol stock: motA 6-14, cheZ, cheY, cheA, cheB, motA, motB</span><br /><br />
<span style="color:#000000;"> -Transformed cheZ, pLaeI+RBS+cheZ, RBS+cheZ+dbl terminator, cheZ w/ H13 tag+dbl terminator, cheZ w/ H13 tag, strong promoter w/RBS</span><br /><br />
<span style="color:#000000;"> -Inoculated cheZ, pLaeI+RBS+cheZ, RBS+cheZ+dbl terminator, cheZ w/ H13 tag+dbl terminator, cheZ w/ H13 tag, strong promoter w/RBS</span><br /><br />
<span style="color:#000000;"> -PCR miniprep of cheA(7,16), cheB(14), cheZ(14), motA(15)</span></p><br />
<p><span style="color:#000000;"> <strong>7/2/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Miniprepped cheZ w/ histag, cheZ, cheZ w/ RBS + dbl termination, pLacI w/ RBS + cheZ, cheZ w/ Histag + dbl termination, strong promoter w/ RBS (cm)</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Inoculated cheZ w/ histag (amp), cheZ (amp), cheZ w/ RBS, double termination (amp), pLacI w/ RBS, cheZ (amp), cheZ w/ histag, double termination (amp), strong promoter w/ RBS (cm)</span><br /><br />
<span style="color:#000000;"> -Miniprepped: psB1C3RFP (5 times), cheZ, strong promoter+RBS+cm, RBS+cheZ+dbl term site, pLacI+RBS+cheZ, cheZ+Hist+dbl, cheZ+Histag</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span></p><br />
<p><span style="color:#000000;"> <strong>7/9/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Digested and gel extracted fliC and GFP</span><br /><br />
<span style="color:#000000;"> -Ligated fliC</span><br /><br />
<span style="color:#000000;"> -Transformed fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Received and plated strains: BW30044, BW30045, K-12 wildtype strain, strain B wildtype, JW1871-1, JW1879-2, JW2662-1, MG1655 (motile), W3110 </span></p><br />
<p><span style="color:#000000;"> <strong>7/16/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Made competent cells MG1655 </span><br /><br />
<span style="color:#000000;"> -digested GFP vector for control using XbaI<a href="http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg"><img class="alignright size-medium wp-image-187" title="DSC_0026" src="http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg?w=512&h=338" alt="" width="512" height="338" /></a></span><br /><br />
<span style="color:#000000;"> -Colony PCRed the fliC GFP </span><br /><br />
<span style="color:#000000;"> -primer dimers. unsuccessful-</span></p><br />
<p><span style="color:#000000;"> <strong>7/23/12 week</strong></span><br /><br />
<span style="color:#000000;"> -PCRed fliC w/ RBS</span><br /><br />
<span style="color:#000000;"> -Transformed GFP-DH5a, GFP-MG1655 </span><br /><br />
<span style="color:#000000;"> -Inoculated w/ 50uL transformation product</span><br /><br />
<span style="color:#000000;"> -Plated GFP-DH5a, GFP-MG1655</span><br /><br />
<span style="color:#000000;"> -PCRed fliC w/ RBS</span><br /><br />
<span style="color:#000000;"> -Miniprepped GFP-DH5a (x2), GFP-MG1655 (x2)</span></p><br />
<p><span style="color:#000000;">-acceptable concentrations</span></p><br />
<p><span style="color:#000000;">-Digested fliC and GFP w/ XbaI and gel purified</span><br /><br />
<span style="color:#000000;"> -Ligated fliC-GFP and GFP w/ H2O (control)</span><br /><br />
<span style="color:#000000;"> -Transformed 1hour ligation product</span><br /><br />
<span style="color:#000000;"> -unsuccessul</span><br /><br />
<span style="color:#000000;"> -Made control and cheY mutagenesis solutions</span><br /><br />
<span style="color:#000000;"> -Transformed fliC-GFP and GFP w/ H2O control into DH5a cells</span><br /><br />
<span style="color:#000000;"> -successful: GFP + H2O control and fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Quickchange mutagenesis</span><br /><br />
<span style="color:#000000;"> -Colony PCR fliC-GFP</span></p><br />
<p><span style="color:#000000;"> <strong>7/30/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Miniprepped fliC-GFP</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Digested miniprepped products</span><br /><br />
<span style="color:#000000;"> -Quickchange PCRed fliC-GFP</span><br /><br />
<span style="color:#000000;"> -Digested quickchange PCR products with DPN1</span><br /><br />
<span style="color:#000000;"> -Digested fliC minipreps with Spe1 </span><br /><br />
<span style="color:#000000;"> -successful</span><br /><br />
<span style="color:#000000;"> -Transformed 43, 44, quickchange digests</span></p><br />
<p><span style="color:#000000;"> <strong>8/6/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Digested cheY</span><br /><br />
<span style="color:#000000;"> -Quickchange PCRed fliC and cheY with control</span><br /><br />
<span style="color:#000000;"> -Digested fliC-GFP, cheY, control</span><br /><br />
<span style="color:#000000;"> -Plated fliC, cheY, and pUC18</span><br /><br />
<span style="color:#000000;"> -Inoculated cheY mut colonies</span><br /><br />
<span style="color:#000000;"> -Miniprepped cheY mut</span><br /><br />
<span style="color:#000000;"> -Glycerol stocks of cheY mut</span><br /><br />
<span style="color:#000000;"> -PCRed cheY mut</span><br /><br />
<span style="color:#000000;"> -Digested cheY mut</span></p><br />
<p><span style="color:#000000;"> <strong>8/13/12 week</strong></span><br /><br />
<span style="color:#000000;"> -PCRed fliA, fliD, fliE, fliF, fliG, fliH, fliM, flhB, flhC, flhD, flgA, flgB, flgJ, flgK, fliC-GFP, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Digested and gel extracted the flagella genes</span><br /><br />
<span style="color:#000000;"> -Transformed fliA, fliD,fliF, fliM, flhB, flgB, flgJ, flhD, lsrR, lsrK</span><br /><br />
<span style="color:#000000;"> -Digested fliC-GFP with DpnI, BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -Transformed pSB1C3 and fliC-GFP mut</span><br /><br />
<span style="color:#000000;"> -Inoculated pSB1C3-RFP, BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -Miniprepped pSB1C3 and BBa_K318512, BBa_K338001, BBa_K216005</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Digested pSB1C3 fliF, fliD, flhB, flhD, flgJ, fliM, fliA</span><br /><br />
<span style="color:#000000;"> -Ligated fliF, fliD, flhB, flhD, flgJ, fliM, fliA with pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>8/20/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Colony PCRed fliA, fliM, fliD, fliF, flgJ, flhB, flhD</span><br /><br />
<span style="color:#000000;"> -Imaged MG1655 in various conditions: pH 5, 6, 8, 9; NaCl 50mM, 100mM, 200mM, 400mM, 500mM; temperature 15C, 20C, 42C; glucose 10mM, 20mM, 50mM</span><br /><br />
<span style="color:#000000;"> -Miniprepped fliC-GFP mut, cheY mut</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Glycerol stocks of fliC-GFP, cheY </span><br /><br />
<span style="color:#000000;"> -Colony PCRed flgJ, flhD, fliA, flhB, cheY</span><br /><br />
<span style="color:#000000;"> -Inoculated successful colonies</span><br /><br />
<span style="color:#000000;"> -Colony PCRed fliM, fliF, fliD</span><br /><br />
<span style="color:#000000;"> -Glycerol stocks of fliA, flhB, flgJ, flhD</span><br /><br />
<span style="color:#000000;"> -Imaging of MG1655</span><br /><br />
<span style="color:#000000;"> -Miniprepped fliA, flhB, flgJ, flhD</span><br /><br />
<span style="color:#000000;"> -Colony PCRed fliM, fliD, fliF</span><br /><br />
<span style="color:#000000;"> -Inoculated fliM </span><br /><br />
<span style="color:#000000;"> -PCRed flhD, flhB, flgJ, fliA to test directionality</span></p><br />
<p><span style="color:#000000;"> <strong>8/27/12 week</strong></span><br /><br />
<span style="color:#000000;"> -PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR</span><br /><br />
<span style="color:#000000;"> -Digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR</span><br /><br />
<span style="color:#000000;"> -all successful except ompR, metR, araC</span><br /><br />
<span style="color:#000000;"> -Digested pSB1C3 </span><br /><br />
<span style="color:#000000;"> -Ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter to pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>9/3/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns </span><br /><br />
<span style="color:#000000;"> -Transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Digested pSB1C3</span><br /><br />
<span style="color:#000000;"> -Ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns with pSB1C3</span></p><br />
<p><span style="color:#000000;"> <strong>9/10/12 week</strong></span><br /><br />
<span style="color:#000000;"> Transformed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns</span><br /><br />
<span style="color:#000000;"> -Inoculated transformations </span><br /><br />
<span style="color:#000000;"> -Colony PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns</span><br /><br />
<span style="color:#000000;"> -Transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406 </span><br /><br />
<span style="color:#000000;"> -Colony PCRed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Transformed BBa_K398108, BBa_K398406</span><br /><br />
<span style="color:#000000;"> -Inoculated BBa_K398108, BBa_K398406, fliH</span><br /><br />
<span style="color:#000000;"> -Miniprepped BBa_K398108, BBa_K398406, fliH</span></p><br />
<p><span style="color:#000000;">-acceptable concentrations<a href="http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg"><img class="alignright size-medium wp-image-162" title="_MG_4057" src="http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg?w=496&h=331" alt="" width="496" height="331" /></a></span></p><br />
<p><span style="color:#000000;">-Colony PCRed BBa_K398108, BBa_K398406, fliH </span><br /><br />
<span style="color:#000000;"> -Miniprepped flhD</span></p><br />
<p><span style="color:#000000;"> <strong>9/17/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Transformed arabinose</span><br /><br />
<span style="color:#000000;"> Inoculated cheY, T7</span><br /><br />
<span style="color:#000000;"> -Digested cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Ligated cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Miniprepped T7, cheY</span><br /><br />
<span style="color:#000000;"> -acceptable concentrations</span><br /><br />
<span style="color:#000000;"> -Transformed T7/p.cola, pACYC</span><br /><br />
<span style="color:#000000;"> -Colony PCRed cheZ RBS, cheZ T7, p.cola cheY</span><br /><br />
<span style="color:#000000;"> -Inoculated cheZ RBS, cheZ T7, p.cola cheY</span><br /><br />
<span style="color:#000000;"> -Miniprepped cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Transformed cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Imaged cheZ, cheZ T7, cheZ RBS T7, cheZ T7</span><br /><br />
<span style="color:#000000;"> -Glycerol stocks of cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Imaging of pcola cheZ A, cheZ RBS T7, cheZ RBS T7, cheZ T7, pcdf cheY A</span><br /><br />
<span style="color:#000000;"> -Inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span></p><br />
<p><span style="color:#000000;"> <strong>9/24/12 week</strong></span><br /><br />
<span style="color:#000000;"> -Imaged BBa_K398108, cheZ RBS T7, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ</span><br /><br />
<span style="color:#000000;"> -Imaged cheZ RBS T7, cheZ T7, pcdf cheY, control</span><br /><br />
<span style="color:#000000;"> -Digested cheY p.cola, pcdf cheY, pcdf cheZ, pcola cheZ, cheZ T7 RBS</span><br /><br />
<span style="color:#000000;"> -Transformed T7 RBS cheZ </span><br /><br />
<span style="color:#000000;"> -Inoculated salt biobricks from frozen stocks</span><br /><br />
<span style="color:#000000;"> -Colony PCRed T7 RBS cheZ </span><br /><br />
<span style="color:#000000;"> -Miniprepped cheZ RBS T7</span></p><br />
<p><span style="color:#000000;"> <strong>10/1/12 week</strong></span></p><br />
<p><span style="color:#000000;">-Miniprepped cheA, cheB, cheY, cheZ, motA. motB, T7 RBS cheZ, T7 RBS cheY, flgJ, fliA, fliH, fliC-GFP, flhB, flhD</span></p><br />
<p>-Western blot for T7 RBS cheY, T7 RBS cheZ</p><br />
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Sgenyk
http://2012.igem.org/Team:USC/Parts
Team:USC/Parts
2012-10-04T01:48:44Z
<p>Sgenyk: </p>
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<a href="https://2012.igem.org/Team:USC" title="E. musici" rel="home">E. musici</a><br />
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<h1><br />
<a href="http://uscigem2012.wordpress.com/">E. musici</a><br />
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University of Southern California iGEM 2012 </div><!-- .desc --><br />
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<h2 class="postitle"><br />
Our&nbsp;BioBricks </h2><!-- .postitle --><br />
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<p style="text-align:center;"><span style="color:#000000;">Below are the BioBrick parts our team created and submitted to the iGEM Registry.</span></p><br />
<p style="text-align:center;"><span style="color:#000000;">Click images to enlarge. Featured BioBricks are highlighted in yellow.</span></p><br />
<p><span style="color:#000000;"><span style="color:#000000;"><a href="http://uscigem2012.files.wordpress.com/2012/07/biobrick-gene-pathway.gif"><img class="aligncenter" title="BioBrick-Gene-Pathway" src="http://uscigem2012.files.wordpress.com/2012/07/biobrick-gene-pathway.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></span></p><br />
<table width="918" border="0" cellspacing="0" cellpadding="0"><br />
<tbody><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chea1.gif"><img class="aligncenter size-medium wp-image-205" title="cheA" src="http://uscigem2012.files.wordpress.com/2012/07/chea1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842006</strong></span></p><br />
<p><span style="color:#000000;"><em>cheA</em></span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;"><em>cheA</em> is a regulatory protein that becomes activated when the methyl-accepting chemotaxis protein (MCP) is not bound to any attractant. The activated cheA self-phosphorylates itself by hydrolyzing ATP in order to gain a phosphate group. <em>cheA</em> then donates its phosphate group to activate <em>cheB</em> or <em>cheA</em>.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis protein CheA &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P07363</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/cheb1.gif"><img class="aligncenter size-medium wp-image-206" title="cheB" src="http://uscigem2012.files.wordpress.com/2012/07/cheb1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842007</strong></span></p><br />
<p><span style="color:#000000;"><em>cheB</em></span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;"><em>CheB</em> removes the methyl groups that are attached to the gamma-glutamyl methyl ester residues located in the methyl-accepting chemotaxis protein (MCP). This allows for the bacteria to remain alert to the changes in the environment.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842007"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842007</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella. </span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis response regulator protein-glutamate methylesterase &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P04042</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif"><img class="aligncenter size-medium wp-image-208" title="cheY" src="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842009</strong></span></p><br />
<p><span style="color:#000000;">cheY</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">CheY is a regulatory protein that reverses the direction of flagella rotation from counter-clockwise to clockwise. When the methyl-accepting chemotaxis protein (MCP) is not bound to any attractant, cheA is activated and told to self-phosphorylate itself by hydrolyzing ATP. Once the cheA is phosphorylated, it transfers its phosphate group to cheY, therefore activating it. CheY then moves to the cytoplasmic side of the flagella apparatus and binds to it. When flagella is not bound to cheY, it rotates in a counter-clockwise direction, which allows the bacteria to move in a “forward” direction. Flagella that is bound to phosphorylated cheY switches its rotation to clockwise, which induces tumbling.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842009"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842009</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis protein CheY &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0A2D5</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chez1.gif"><img class="aligncenter size-medium wp-image-209" title="cheZ" src="http://uscigem2012.files.wordpress.com/2012/07/chez1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842010</strong></span></p><br />
<p><span style="color:#000000;">cheZ</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">CheZ is a regulatory protein that constantly dephosphorylates cheY that is attached to a flagellum. The incessant dephosphorylation is what causes bacteria to run, stop, and tumble when in an environment that has a concentration gradient and remain responsive to any changes in chemical concentration.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Protein phosphatase CheZ &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P07800</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/mota1.gif"><img class="aligncenter size-medium wp-image-214" title="motA" src="http://uscigem2012.files.wordpress.com/2012/07/mota1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842011</strong></span></p><br />
<p><span style="color:#000000;">motA</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">motA is a structural protein for the stationary element of the flagella motor. The protein works in conjunction with motB to form this structure, which then in turn controls the flagella’s rotation.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. The protein works in conjunction with motB to form the stationary base of the flagella motor, which then in turn controls the flagella’s rotation. This part can be used in the complete recreation of an <em>E. coli’s</em> genetic flagellar pathway.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Motility protein A &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P09348</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/motb1.gif"><img class="aligncenter size-medium wp-image-215" title="motB" src="http://uscigem2012.files.wordpress.com/2012/07/motb1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842012</strong></span></p><br />
<p><span style="color:#000000;">motB</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">motB is a structural protein for the stationary element of the flagella motor. The protein works in conjunction with motA to form this structure, which then in turn controls the flagella’s rotation. It is also believed that motB is responsible for forming the structure that connects the flagella motor to the cell wall.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. The protein works in conjunction with motA to form the stationary base of the flagella motor, which then in turn controls the flagella’s rotation. This part can be used in the complete recreation of an <em>E. coli’s</em> genetic flagellar pathway.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Motility protein B &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0AF06</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/t7_rbs_chez.gif"><img class="aligncenter size-medium wp-image-217" title="T7_RBS_CheZ" src="http://uscigem2012.files.wordpress.com/2012/07/t7_rbs_chez.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842014</strong></span></p><br />
<p><span style="color:#000000;">T7 RBS cheZ</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">T7 serves as a promoter that is stimulated by IPTG. RBS is a sequence in DNA located upstream of the start codon. It affects the rate at which the open reading frame is translated. CheZ is a regulatory protein that constantly dephosphorylates cheY that is attached to a flagellum. The incessant dephosphorylation is what causes bacteria to run, stop, and tumble when in an environment that has a concentration gradient and remain responsive to any changes in chemical concentration. Together, cheZ is induced by the presence of IPTG.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Protein phosphatase CheZ &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P07800</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif"><img class="aligncenter size-medium wp-image-208" title="cheY" src="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842015</strong></span></p><br />
<p><span style="color:#000000;">T7 RBS cheY</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">T7 serves as a promoter that is stimulated by IPTG. RBS is a sequence in DNA located upstream of the start codon. It affects the rate at which the open reading frame is translated. CheY is regulatory protein that reverses the direction of flagella rotation from counter-clockwise to clockwise. When the methyl-accepting chemotaxis protein (MCP) is not bound to any attractant, cheA is activated and told to self-phosphorylate itself by hydrolyzing ATP. Once the cheA is phosphorylated, it transfers its phosphate group to cheY, therefore activating it. CheY then moves to the cytoplasmic side of the flagella apparatus and binds to it. When flagella are not bound to cheY, it rotates in a counter-clockwise direction, which allows the bacteria to move in a “forward” direction. Flagella that are bound to phosphorylated cheY switches its rotation to clockwise, which induces tumbling.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842015"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842015</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis protein CheY &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0A2D5</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flgj.gif"><img class="aligncenter size-medium wp-image-220" title="flgJ" src="http://uscigem2012.files.wordpress.com/2012/07/flgj.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842002</strong></span></p><br />
<p><span style="color:#000000;">flgJ</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">flgJ transcribes a flagellum specific muramidase which hydrolyzes the peptidoglycan layer in the cell membrane. This creates a small hole in the side of the bacterium in which the flagellar rod is formed. It is most commonly believed that flgJ is exported via the flagellum specific exportation system, making it a class 3 flagella gene.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used in conjunction with the entire flagella synthesis genetic pathway to reproduce a complete flagella apparatus. Additionally, if this part is used separate from the flagellar genetic pathway and connected to a gene promoter, it will continually react with the cell wall until the peptidoglycan layer of the cell membrane can no longer sustain the structure of the bacterium.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Nambu, T., Minamino, T., Macnab, R., &amp; Kutsukake, K. (1999). Peptidoglycan-Hydrolyzing Activity of the FlgJ Protein, Essential for Flagellar Rod Formation in Salmonella typhimurium. <em>Journal of Bacteriology</em>, <em>181</em>(5), 1555-1561. Retrieved October 1, 2012, from http://jb.asm.org/content/181/5/1555</span></p><br />
<p><span style="color:#000000;">Peptidoglycan hydrolase flgJ &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P75942</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flia.gif"><img class="aligncenter size-medium wp-image-222" title="fliA" src="http://uscigem2012.files.wordpress.com/2012/07/flia.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842003</strong></span></p><br />
<p><span style="color:#000000;">fliA</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">fliA is an alternate sigma factor for the class 3 flagella operons. When transcribed, it forms a component of RNA polymerase sigma 28.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used in conjunction with the entire flagella synthesis genetic pathway to reproduce a complete flagella apparatus.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">fliA:Quickview &#8211; EcoliWiki. (n.d.). <em>EcoliWiki</em>. Retrieved October 1, 2012, from http://ecoliwiki.net/colipedia/index.php/fliA</span></p><br />
<p><span style="color:#000000;">Ide, N., &amp; Kutsukake, K. (n.d.). Identification of a novel Escherichia coli gene whose e&#8230; [Gene. 1997] &#8211; PubMed &#8211; NCBI. <em>National Center for Biotechnology Information</em>. Retrieved October 1, 2012, from http://www.ncbi.nlm.nih.gov/pubmed/9358034</span></p><br />
<p><span style="color:#000000;">Claret, L., Miquel, S., Vieille, N., Ryjenkov, D., Gomelsky, M., &amp; Darfeuille-Michaud, A. A. (2007). The Flagellar Sigma Factor FliA Regulates Adhesion and Invasion of Crohn Disease-associated Escherichia coli via a Cyclic Dimeric GMP-dependent Pathway. <em>The Journal Of Biological Chemistry</em>, <em>282</em>(46), 33275-33283. Retrieved October 1, 2012, from http://www.jbc.org/content/282/46/33275.full.pdf</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flih1.gif"><img class="aligncenter size-medium wp-image-213" title="fliH" src="http://uscigem2012.files.wordpress.com/2012/07/flih1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842005</strong></span></p><br />
<p><span style="color:#000000;">fliH</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">FliH functions as part of the flagella specific export system and is necessary for flagellar growth and assembly. It is believed to be part of a two-component system consisting of fliH and fliL that hydrolyses ATP until construction of the flagella apparatus is completed.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used in conjunction with the entire flagella synthesis genetic pathway to reproduce a complete flagella apparatus. </span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Minamino, T., &amp; MacNab, R. (n.d.). FliH, a soluble component of the type III flag&#8230; [Mol Microbiol. 2000] &#8211; PubMed &#8211; NCBI. <em>National Center for Biotechnology Information</em>. Retrieved October 1, 2012, from http://www.ncbi.nlm.nih.gov/pubmed/10998179</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flic_gfp.gif"><img class="aligncenter size-medium wp-image-212" title="fliC_GFP" src="http://uscigem2012.files.wordpress.com/2012/07/flic_gfp.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842013</strong></span></p><br />
<p><span style="color:#000000;">fliC-GFP</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">When transcribed, RBS-fliC-GFP generates the flagellum protein bonded with a green fluorescent protein. This structure would then be exported to the growing flagella apparatus and used in the construction of the filament. When strongly expressed, this structure would directly result in the flagella filament's containing GFP, allowing for the flagella to be detectable. Unfortunately we were unable to promote the sequence enough to test whether this construct actually functions in this manner.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">The fliC gene was cloned via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. Due to the fact that fliC has multiple S and P restriction enzyme sites located within its DNA sequence, it was generated with XbaI sites on the forward and reverse primer. This is so as to allow for the placement of a gene promoter in the E-X sites upstream of the construct in an iGEM biobrick vector. It is important to note though that the current structure consists of the following so as to avoid the accidental loss of a gene during the cloning procedure.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;">EcoRI-XbaI-RBS-fliC-XbaI-GFP-SpeI-PstI</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">fliC:Quickview &#8211; EcoliWiki. (n.d.). <em>EcoliWiki</em>. Retrieved October 1, 2012, from http://ecoliwiki.net/colipedia/index.php/fliC</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flhb1.gif"><img class="aligncenter size-medium wp-image-210" title="flhB" src="http://uscigem2012.files.wordpress.com/2012/07/flhb1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842000</strong></span></p><br />
<p><span style="color:#000000;">flhB</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">Class 2 flagella operon. When transcribed, flhB is one of three flagella genes responsible for the generation of proteins that export flagellin to the flagella apparatus. The flagellin is then directly used in the synthesis of the structure's basal body. Additionally, flhB directly transcribes the operon flhBAE.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842000"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842000</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">flhB was use generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is directly downstream of the class 1 flagella operons flhD and flhC. It also works in conjunction with fliL and fliH to make up the flagellin export system. This part can be used either to generate a flagella in an <em>E. coli</em> strain in which they are not already present, or to promote further formation of pre-existing flagella.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Flagellar biosynthetic protein flhB &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P76299</span></p><br />
<p><span style="color:#000000;">flhB:Quickview &#8211; EcoliWiki. (n.d.). <em>EcoliWiki</em>. Retrieved October 1, 2012, from http://ecoliwiki.net/colipedia/index.php/flhB:Quickview</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flhd.gif"><img class="aligncenter size-medium wp-image-221" title="flhD" src="http://uscigem2012.files.wordpress.com/2012/07/flhd.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842001</strong></span></p><br />
<p><span style="color:#000000;">flhD</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">flhD along with flhC form the master regulation operon for the synthesis of flagella in<em> E. coli. </em> They are responsible for the downstream transcription of the class 2 flagella genes. flhD is directly induced by several separate systems; among these is the two-component quorum sensing response system, qseB and qseC.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842001"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842001</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. FlhD is to be used in conjunction with flhC in order to form the transcriptional operon for the production of flagella. This part can be used either to generate a flagella in an <em>E. coli</em> strain in which they are not already present, or to promote further formation of pre-existing flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Flagellar transcriptional regulator FlhD &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0A8S9</span></td><br />
</tr><br />
</tbody><br />
</table><br />
<p><strong><span style="color:#000000;">New Constructs Being Tested:</span></strong></p><br />
<p><span style="color:#000000;">Because cheY and cheZ have opposing functions, we wanted to test the expression of both in a single E.coli. To ensure equal transcription from this construct, we utilized the commercially availible pCOLA-Duet and pCDF-Duet vectors from Novagen, which have two RBS sites flanking two unique multiple cloning sites, and are all under the control of a single T7 promoter.</span></p><br />
<p><span style="color:#000000;">These new constructs worked perfectly, and we are currently designing and constructing a similar system in the pSBC1C3 backbone as a new BioBrick.</span></p><br />
<p><span style="color:#000000;">Although we have not included these in the registry yet, as they have not been fully tested, we have generated the following constructs:</span></p><br />
<p><span style="color:#000000;">pCOLA-Duet-CheY</span></p><br />
<p><span style="color:#000000;">pCOLA-Duet-CheZ</span></p><br />
<p><span style="color:#000000;">pCDF-Duet-CheY</span></p><br />
<p><span style="color:#000000;">pCDF-Duet-CheZ</span></p><br />
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<center><br />
<groupparts>iGEM012 USC</groupparts><br />
</center></div>
Sgenyk
http://2012.igem.org/Team:USC/Parts
Team:USC/Parts
2012-10-04T01:48:06Z
<p>Sgenyk: </p>
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<meta name="description" content="Below are the BioBrick parts our team created and submitted to the iGEM Registry. Click images to enlarge. Featured BioBricks are highlighted in yellow. BBa_K842006 <em>cheA</em> Description and Function cheA is a&hellip; Read More &rarr;" /><br />
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<a href="https://2012.igem.org/Team:USC" title="E. musici" rel="home">E. musici</a><br />
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<h1><br />
<a href="http://uscigem2012.wordpress.com/">E. musici</a><br />
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University of Southern California iGEM 2012 </div><!-- .desc --><br />
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Our&nbsp;BioBricks </h2><!-- .postitle --><br />
<br />
<p style="text-align:center;"><span style="color:#000000;">Below are the BioBrick parts our team created and submitted to the iGEM Registry.</span></p><br />
<p style="text-align:center;"><span style="color:#000000;">Click images to enlarge. Featured BioBricks are highlighted in yellow.</span></p><br />
<p><span style="color:#000000;"><span style="color:#000000;"><a href="http://uscigem2012.files.wordpress.com/2012/07/biobrick-gene-pathway.gif"><img class="aligncenter" title="BioBrick-Gene-Pathway" src="http://uscigem2012.files.wordpress.com/2012/07/biobrick-gene-pathway.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></span></p><br />
<table width="918" border="0" cellspacing="0" cellpadding="0"><br />
<tbody><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chea1.gif"><img class="aligncenter size-medium wp-image-205" title="cheA" src="http://uscigem2012.files.wordpress.com/2012/07/chea1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842006</strong></span></p><br />
<p><span style="color:#000000;"><em>cheA</em></span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;"><em>cheA</em> is a regulatory protein that becomes activated when the methyl-accepting chemotaxis protein (MCP) is not bound to any attractant. The activated cheA self-phosphorylates itself by hydrolyzing ATP in order to gain a phosphate group. <em>cheA</em> then donates its phosphate group to activate <em>cheB</em> or <em>cheA</em>.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis protein CheA &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P07363</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/cheb1.gif"><img class="aligncenter size-medium wp-image-206" title="cheB" src="http://uscigem2012.files.wordpress.com/2012/07/cheb1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842007</strong></span></p><br />
<p><span style="color:#000000;">cheB</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;"><em>CheB</em> removes the methyl groups that are attached to the gamma-glutamyl methyl ester residues located in the methyl-accepting chemotaxis protein (MCP). This allows for the bacteria to remain alert to the changes in the environment.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842007"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842007</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella. </span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis response regulator protein-glutamate methylesterase &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P04042</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif"><img class="aligncenter size-medium wp-image-208" title="cheY" src="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842009</strong></span></p><br />
<p><span style="color:#000000;">cheY</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">CheY is a regulatory protein that reverses the direction of flagella rotation from counter-clockwise to clockwise. When the methyl-accepting chemotaxis protein (MCP) is not bound to any attractant, cheA is activated and told to self-phosphorylate itself by hydrolyzing ATP. Once the cheA is phosphorylated, it transfers its phosphate group to cheY, therefore activating it. CheY then moves to the cytoplasmic side of the flagella apparatus and binds to it. When flagella is not bound to cheY, it rotates in a counter-clockwise direction, which allows the bacteria to move in a “forward” direction. Flagella that is bound to phosphorylated cheY switches its rotation to clockwise, which induces tumbling.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842009"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842009</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis protein CheY &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0A2D5</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chez1.gif"><img class="aligncenter size-medium wp-image-209" title="cheZ" src="http://uscigem2012.files.wordpress.com/2012/07/chez1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842010</strong></span></p><br />
<p><span style="color:#000000;">cheZ</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">CheZ is a regulatory protein that constantly dephosphorylates cheY that is attached to a flagellum. The incessant dephosphorylation is what causes bacteria to run, stop, and tumble when in an environment that has a concentration gradient and remain responsive to any changes in chemical concentration.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Protein phosphatase CheZ &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P07800</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/mota1.gif"><img class="aligncenter size-medium wp-image-214" title="motA" src="http://uscigem2012.files.wordpress.com/2012/07/mota1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842011</strong></span></p><br />
<p><span style="color:#000000;">motA</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">motA is a structural protein for the stationary element of the flagella motor. The protein works in conjunction with motB to form this structure, which then in turn controls the flagella’s rotation.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. The protein works in conjunction with motB to form the stationary base of the flagella motor, which then in turn controls the flagella’s rotation. This part can be used in the complete recreation of an <em>E. coli’s</em> genetic flagellar pathway.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Motility protein A &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P09348</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/motb1.gif"><img class="aligncenter size-medium wp-image-215" title="motB" src="http://uscigem2012.files.wordpress.com/2012/07/motb1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842012</strong></span></p><br />
<p><span style="color:#000000;">motB</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">motB is a structural protein for the stationary element of the flagella motor. The protein works in conjunction with motA to form this structure, which then in turn controls the flagella’s rotation. It is also believed that motB is responsible for forming the structure that connects the flagella motor to the cell wall.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. The protein works in conjunction with motA to form the stationary base of the flagella motor, which then in turn controls the flagella’s rotation. This part can be used in the complete recreation of an <em>E. coli’s</em> genetic flagellar pathway.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Motility protein B &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0AF06</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/t7_rbs_chez.gif"><img class="aligncenter size-medium wp-image-217" title="T7_RBS_CheZ" src="http://uscigem2012.files.wordpress.com/2012/07/t7_rbs_chez.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842014</strong></span></p><br />
<p><span style="color:#000000;">T7 RBS cheZ</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">T7 serves as a promoter that is stimulated by IPTG. RBS is a sequence in DNA located upstream of the start codon. It affects the rate at which the open reading frame is translated. CheZ is a regulatory protein that constantly dephosphorylates cheY that is attached to a flagellum. The incessant dephosphorylation is what causes bacteria to run, stop, and tumble when in an environment that has a concentration gradient and remain responsive to any changes in chemical concentration. Together, cheZ is induced by the presence of IPTG.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Protein phosphatase CheZ &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P07800</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif"><img class="aligncenter size-medium wp-image-208" title="cheY" src="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842015</strong></span></p><br />
<p><span style="color:#000000;">T7 RBS cheY</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">T7 serves as a promoter that is stimulated by IPTG. RBS is a sequence in DNA located upstream of the start codon. It affects the rate at which the open reading frame is translated. CheY is regulatory protein that reverses the direction of flagella rotation from counter-clockwise to clockwise. When the methyl-accepting chemotaxis protein (MCP) is not bound to any attractant, cheA is activated and told to self-phosphorylate itself by hydrolyzing ATP. Once the cheA is phosphorylated, it transfers its phosphate group to cheY, therefore activating it. CheY then moves to the cytoplasmic side of the flagella apparatus and binds to it. When flagella are not bound to cheY, it rotates in a counter-clockwise direction, which allows the bacteria to move in a “forward” direction. Flagella that are bound to phosphorylated cheY switches its rotation to clockwise, which induces tumbling.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842015"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842015</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis protein CheY &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0A2D5</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flgj.gif"><img class="aligncenter size-medium wp-image-220" title="flgJ" src="http://uscigem2012.files.wordpress.com/2012/07/flgj.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842002</strong></span></p><br />
<p><span style="color:#000000;">flgJ</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">flgJ transcribes a flagellum specific muramidase which hydrolyzes the peptidoglycan layer in the cell membrane. This creates a small hole in the side of the bacterium in which the flagellar rod is formed. It is most commonly believed that flgJ is exported via the flagellum specific exportation system, making it a class 3 flagella gene.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used in conjunction with the entire flagella synthesis genetic pathway to reproduce a complete flagella apparatus. Additionally, if this part is used separate from the flagellar genetic pathway and connected to a gene promoter, it will continually react with the cell wall until the peptidoglycan layer of the cell membrane can no longer sustain the structure of the bacterium.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Nambu, T., Minamino, T., Macnab, R., &amp; Kutsukake, K. (1999). Peptidoglycan-Hydrolyzing Activity of the FlgJ Protein, Essential for Flagellar Rod Formation in Salmonella typhimurium. <em>Journal of Bacteriology</em>, <em>181</em>(5), 1555-1561. Retrieved October 1, 2012, from http://jb.asm.org/content/181/5/1555</span></p><br />
<p><span style="color:#000000;">Peptidoglycan hydrolase flgJ &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P75942</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flia.gif"><img class="aligncenter size-medium wp-image-222" title="fliA" src="http://uscigem2012.files.wordpress.com/2012/07/flia.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842003</strong></span></p><br />
<p><span style="color:#000000;">fliA</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">fliA is an alternate sigma factor for the class 3 flagella operons. When transcribed, it forms a component of RNA polymerase sigma 28.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used in conjunction with the entire flagella synthesis genetic pathway to reproduce a complete flagella apparatus.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">fliA:Quickview &#8211; EcoliWiki. (n.d.). <em>EcoliWiki</em>. Retrieved October 1, 2012, from http://ecoliwiki.net/colipedia/index.php/fliA</span></p><br />
<p><span style="color:#000000;">Ide, N., &amp; Kutsukake, K. (n.d.). Identification of a novel Escherichia coli gene whose e&#8230; [Gene. 1997] &#8211; PubMed &#8211; NCBI. <em>National Center for Biotechnology Information</em>. Retrieved October 1, 2012, from http://www.ncbi.nlm.nih.gov/pubmed/9358034</span></p><br />
<p><span style="color:#000000;">Claret, L., Miquel, S., Vieille, N., Ryjenkov, D., Gomelsky, M., &amp; Darfeuille-Michaud, A. A. (2007). The Flagellar Sigma Factor FliA Regulates Adhesion and Invasion of Crohn Disease-associated Escherichia coli via a Cyclic Dimeric GMP-dependent Pathway. <em>The Journal Of Biological Chemistry</em>, <em>282</em>(46), 33275-33283. Retrieved October 1, 2012, from http://www.jbc.org/content/282/46/33275.full.pdf</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flih1.gif"><img class="aligncenter size-medium wp-image-213" title="fliH" src="http://uscigem2012.files.wordpress.com/2012/07/flih1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842005</strong></span></p><br />
<p><span style="color:#000000;">fliH</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">FliH functions as part of the flagella specific export system and is necessary for flagellar growth and assembly. It is believed to be part of a two-component system consisting of fliH and fliL that hydrolyses ATP until construction of the flagella apparatus is completed.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used in conjunction with the entire flagella synthesis genetic pathway to reproduce a complete flagella apparatus. </span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Minamino, T., &amp; MacNab, R. (n.d.). FliH, a soluble component of the type III flag&#8230; [Mol Microbiol. 2000] &#8211; PubMed &#8211; NCBI. <em>National Center for Biotechnology Information</em>. Retrieved October 1, 2012, from http://www.ncbi.nlm.nih.gov/pubmed/10998179</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flic_gfp.gif"><img class="aligncenter size-medium wp-image-212" title="fliC_GFP" src="http://uscigem2012.files.wordpress.com/2012/07/flic_gfp.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842013</strong></span></p><br />
<p><span style="color:#000000;">fliC-GFP</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">When transcribed, RBS-fliC-GFP generates the flagellum protein bonded with a green fluorescent protein. This structure would then be exported to the growing flagella apparatus and used in the construction of the filament. When strongly expressed, this structure would directly result in the flagella filament's containing GFP, allowing for the flagella to be detectable. Unfortunately we were unable to promote the sequence enough to test whether this construct actually functions in this manner.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">The fliC gene was cloned via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. Due to the fact that fliC has multiple S and P restriction enzyme sites located within its DNA sequence, it was generated with XbaI sites on the forward and reverse primer. This is so as to allow for the placement of a gene promoter in the E-X sites upstream of the construct in an iGEM biobrick vector. It is important to note though that the current structure consists of the following so as to avoid the accidental loss of a gene during the cloning procedure.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;">EcoRI-XbaI-RBS-fliC-XbaI-GFP-SpeI-PstI</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">fliC:Quickview &#8211; EcoliWiki. (n.d.). <em>EcoliWiki</em>. Retrieved October 1, 2012, from http://ecoliwiki.net/colipedia/index.php/fliC</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flhb1.gif"><img class="aligncenter size-medium wp-image-210" title="flhB" src="http://uscigem2012.files.wordpress.com/2012/07/flhb1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842000</strong></span></p><br />
<p><span style="color:#000000;">flhB</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">Class 2 flagella operon. When transcribed, flhB is one of three flagella genes responsible for the generation of proteins that export flagellin to the flagella apparatus. The flagellin is then directly used in the synthesis of the structure's basal body. Additionally, flhB directly transcribes the operon flhBAE.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842000"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842000</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">flhB was use generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is directly downstream of the class 1 flagella operons flhD and flhC. It also works in conjunction with fliL and fliH to make up the flagellin export system. This part can be used either to generate a flagella in an <em>E. coli</em> strain in which they are not already present, or to promote further formation of pre-existing flagella.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Flagellar biosynthetic protein flhB &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P76299</span></p><br />
<p><span style="color:#000000;">flhB:Quickview &#8211; EcoliWiki. (n.d.). <em>EcoliWiki</em>. Retrieved October 1, 2012, from http://ecoliwiki.net/colipedia/index.php/flhB:Quickview</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flhd.gif"><img class="aligncenter size-medium wp-image-221" title="flhD" src="http://uscigem2012.files.wordpress.com/2012/07/flhd.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842001</strong></span></p><br />
<p><span style="color:#000000;">flhD</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">flhD along with flhC form the master regulation operon for the synthesis of flagella in<em> E. coli. </em> They are responsible for the downstream transcription of the class 2 flagella genes. flhD is directly induced by several separate systems; among these is the two-component quorum sensing response system, qseB and qseC.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842001"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842001</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. FlhD is to be used in conjunction with flhC in order to form the transcriptional operon for the production of flagella. This part can be used either to generate a flagella in an <em>E. coli</em> strain in which they are not already present, or to promote further formation of pre-existing flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Flagellar transcriptional regulator FlhD &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0A8S9</span></td><br />
</tr><br />
</tbody><br />
</table><br />
<p><strong><span style="color:#000000;">New Constructs Being Tested:</span></strong></p><br />
<p><span style="color:#000000;">Because cheY and cheZ have opposing functions, we wanted to test the expression of both in a single E.coli. To ensure equal transcription from this construct, we utilized the commercially availible pCOLA-Duet and pCDF-Duet vectors from Novagen, which have two RBS sites flanking two unique multiple cloning sites, and are all under the control of a single T7 promoter.</span></p><br />
<p><span style="color:#000000;">These new constructs worked perfectly, and we are currently designing and constructing a similar system in the pSBC1C3 backbone as a new BioBrick.</span></p><br />
<p><span style="color:#000000;">Although we have not included these in the registry yet, as they have not been fully tested, we have generated the following constructs:</span></p><br />
<p><span style="color:#000000;">pCOLA-Duet-CheY</span></p><br />
<p><span style="color:#000000;">pCOLA-Duet-CheZ</span></p><br />
<p><span style="color:#000000;">pCDF-Duet-CheY</span></p><br />
<p><span style="color:#000000;">pCDF-Duet-CheZ</span></p><br />
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<center><br />
<groupparts>iGEM012 USC</groupparts><br />
</center></div>
Sgenyk
http://2012.igem.org/Team:USC/Parts
Team:USC/Parts
2012-10-04T01:46:42Z
<p>Sgenyk: </p>
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<meta name="application-name" content="E. musici" /><meta name="msapplication-window" content="width=device-width;height=device-height" /><meta name="msapplication-tooltip" content="University of Southern California iGEM 2012" /><meta name="msapplication-task" content="name=Subscribe;action-uri=http://uscigem2012.wordpress.com/feed/;icon-uri=http://s2.wp.com/i/favicon.ico" /><meta name="msapplication-task" content="name=Sign up for a free blog;action-uri=http://wordpress.com/signup/;icon-uri=http://s2.wp.com/i/favicon.ico" /><meta name="msapplication-task" content="name=WordPress.com Support;action-uri=http://support.wordpress.com/;icon-uri=http://s2.wp.com/i/favicon.ico" /><meta name="msapplication-task" content="name=WordPress.com Forums;action-uri=http://forums.wordpress.com/;icon-uri=http://s2.wp.com/i/favicon.ico" /><meta name="title" content="Our&nbsp;BioBricks | E. musici on WordPress.com" /><br />
<meta name="description" content="Below are the BioBrick parts our team created and submitted to the iGEM Registry. Click images to enlarge. Featured BioBricks are highlighted in yellow. BBa_K842006 <em>cheA</em> Description and Function cheA is a&hellip; Read More &rarr;" /><br />
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<h1><br />
<a href="http://uscigem2012.wordpress.com/">E. musici</a><br />
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University of Southern California iGEM 2012 </div><!-- .desc --><br />
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Our&nbsp;BioBricks </h2><!-- .postitle --><br />
<br />
<p style="text-align:center;"><span style="color:#000000;">Below are the BioBrick parts our team created and submitted to the iGEM Registry.</span></p><br />
<p style="text-align:center;"><span style="color:#000000;">Click images to enlarge. Featured BioBricks are highlighted in yellow.</span></p><br />
<p><span style="color:#000000;"><span style="color:#000000;"><a href="http://uscigem2012.files.wordpress.com/2012/07/biobrick-gene-pathway.gif"><img class="aligncenter" title="BioBrick-Gene-Pathway" src="http://uscigem2012.files.wordpress.com/2012/07/biobrick-gene-pathway.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></span></p><br />
<table width="918" border="0" cellspacing="0" cellpadding="0"><br />
<tbody><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chea1.gif"><img class="aligncenter size-medium wp-image-205" title="cheA" src="http://uscigem2012.files.wordpress.com/2012/07/chea1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842006</strong></span></p><br />
<p><span style="color:#000000;"><em>cheA</em></span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;"><em>cheA</em> is a regulatory protein that becomes activated when the methyl-accepting chemotaxis protein (MCP) is not bound to any attractant. The activated cheA self-phosphorylates itself by hydrolyzing ATP in order to gain a phosphate group. <em>cheA</em> then donates its phosphate group to activate <em>cheB</em> or <em>cheA</em>.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis protein CheA &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P07363</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/cheb1.gif"><img class="aligncenter size-medium wp-image-206" title="cheB" src="http://uscigem2012.files.wordpress.com/2012/07/cheb1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842007</strong></span></p><br />
<p><span style="color:#000000;">cheB</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">CheB removes the methyl groups that are attached to the gamma-glutamyl methyl ester residues located in the methyl-accepting chemotaxis protein (MCP). This allows for the bacteria to remain alert to the changes in the environment.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842007"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842007</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella. </span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis response regulator protein-glutamate methylesterase &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P04042</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif"><img class="aligncenter size-medium wp-image-208" title="cheY" src="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842009</strong></span></p><br />
<p><span style="color:#000000;">cheY</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">CheY is a regulatory protein that reverses the direction of flagella rotation from counter-clockwise to clockwise. When the methyl-accepting chemotaxis protein (MCP) is not bound to any attractant, cheA is activated and told to self-phosphorylate itself by hydrolyzing ATP. Once the cheA is phosphorylated, it transfers its phosphate group to cheY, therefore activating it. CheY then moves to the cytoplasmic side of the flagella apparatus and binds to it. When flagella is not bound to cheY, it rotates in a counter-clockwise direction, which allows the bacteria to move in a “forward” direction. Flagella that is bound to phosphorylated cheY switches its rotation to clockwise, which induces tumbling.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842009"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842009</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis protein CheY &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0A2D5</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chez1.gif"><img class="aligncenter size-medium wp-image-209" title="cheZ" src="http://uscigem2012.files.wordpress.com/2012/07/chez1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842010</strong></span></p><br />
<p><span style="color:#000000;">cheZ</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">CheZ is a regulatory protein that constantly dephosphorylates cheY that is attached to a flagellum. The incessant dephosphorylation is what causes bacteria to run, stop, and tumble when in an environment that has a concentration gradient and remain responsive to any changes in chemical concentration.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Protein phosphatase CheZ &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P07800</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/mota1.gif"><img class="aligncenter size-medium wp-image-214" title="motA" src="http://uscigem2012.files.wordpress.com/2012/07/mota1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842011</strong></span></p><br />
<p><span style="color:#000000;">motA</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">motA is a structural protein for the stationary element of the flagella motor. The protein works in conjunction with motB to form this structure, which then in turn controls the flagella’s rotation.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. The protein works in conjunction with motB to form the stationary base of the flagella motor, which then in turn controls the flagella’s rotation. This part can be used in the complete recreation of an <em>E. coli’s</em> genetic flagellar pathway.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Motility protein A &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P09348</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/motb1.gif"><img class="aligncenter size-medium wp-image-215" title="motB" src="http://uscigem2012.files.wordpress.com/2012/07/motb1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842012</strong></span></p><br />
<p><span style="color:#000000;">motB</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">motB is a structural protein for the stationary element of the flagella motor. The protein works in conjunction with motA to form this structure, which then in turn controls the flagella’s rotation. It is also believed that motB is responsible for forming the structure that connects the flagella motor to the cell wall.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842010</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. The protein works in conjunction with motA to form the stationary base of the flagella motor, which then in turn controls the flagella’s rotation. This part can be used in the complete recreation of an <em>E. coli’s</em> genetic flagellar pathway.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Motility protein B &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0AF06</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/t7_rbs_chez.gif"><img class="aligncenter size-medium wp-image-217" title="T7_RBS_CheZ" src="http://uscigem2012.files.wordpress.com/2012/07/t7_rbs_chez.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842014</strong></span></p><br />
<p><span style="color:#000000;">T7 RBS cheZ</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">T7 serves as a promoter that is stimulated by IPTG. RBS is a sequence in DNA located upstream of the start codon. It affects the rate at which the open reading frame is translated. CheZ is a regulatory protein that constantly dephosphorylates cheY that is attached to a flagellum. The incessant dephosphorylation is what causes bacteria to run, stop, and tumble when in an environment that has a concentration gradient and remain responsive to any changes in chemical concentration. Together, cheZ is induced by the presence of IPTG.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Protein phosphatase CheZ &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P07800</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif"><img class="aligncenter size-medium wp-image-208" title="cheY" src="http://uscigem2012.files.wordpress.com/2012/07/chey1.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842015</strong></span></p><br />
<p><span style="color:#000000;">T7 RBS cheY</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">T7 serves as a promoter that is stimulated by IPTG. RBS is a sequence in DNA located upstream of the start codon. It affects the rate at which the open reading frame is translated. CheY is regulatory protein that reverses the direction of flagella rotation from counter-clockwise to clockwise. When the methyl-accepting chemotaxis protein (MCP) is not bound to any attractant, cheA is activated and told to self-phosphorylate itself by hydrolyzing ATP. Once the cheA is phosphorylated, it transfers its phosphate group to cheY, therefore activating it. CheY then moves to the cytoplasmic side of the flagella apparatus and binds to it. When flagella are not bound to cheY, it rotates in a counter-clockwise direction, which allows the bacteria to move in a “forward” direction. Flagella that are bound to phosphorylated cheY switches its rotation to clockwise, which induces tumbling.</span></p><br />
<p><span style="color:#000000;">Used for researcher control of signaling mechanisms that influence flagella function.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842015"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842015</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used with other chemotaxis genes to form a complete regulatory pathway that controls the response system in MCP and the direction of rotation in flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Chemotaxis protein CheY &#8211; Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0A2D5</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flgj.gif"><img class="aligncenter size-medium wp-image-220" title="flgJ" src="http://uscigem2012.files.wordpress.com/2012/07/flgj.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842002</strong></span></p><br />
<p><span style="color:#000000;">flgJ</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">flgJ transcribes a flagellum specific muramidase which hydrolyzes the peptidoglycan layer in the cell membrane. This creates a small hole in the side of the bacterium in which the flagellar rod is formed. It is most commonly believed that flgJ is exported via the flagellum specific exportation system, making it a class 3 flagella gene.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used in conjunction with the entire flagella synthesis genetic pathway to reproduce a complete flagella apparatus. Additionally, if this part is used separate from the flagellar genetic pathway and connected to a gene promoter, it will continually react with the cell wall until the peptidoglycan layer of the cell membrane can no longer sustain the structure of the bacterium.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Nambu, T., Minamino, T., Macnab, R., &amp; Kutsukake, K. (1999). Peptidoglycan-Hydrolyzing Activity of the FlgJ Protein, Essential for Flagellar Rod Formation in Salmonella typhimurium. <em>Journal of Bacteriology</em>, <em>181</em>(5), 1555-1561. Retrieved October 1, 2012, from http://jb.asm.org/content/181/5/1555</span></p><br />
<p><span style="color:#000000;">Peptidoglycan hydrolase flgJ &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P75942</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flia.gif"><img class="aligncenter size-medium wp-image-222" title="fliA" src="http://uscigem2012.files.wordpress.com/2012/07/flia.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842003</strong></span></p><br />
<p><span style="color:#000000;">fliA</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">fliA is an alternate sigma factor for the class 3 flagella operons. When transcribed, it forms a component of RNA polymerase sigma 28.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842002</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used in conjunction with the entire flagella synthesis genetic pathway to reproduce a complete flagella apparatus.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">fliA:Quickview &#8211; EcoliWiki. (n.d.). <em>EcoliWiki</em>. Retrieved October 1, 2012, from http://ecoliwiki.net/colipedia/index.php/fliA</span></p><br />
<p><span style="color:#000000;">Ide, N., &amp; Kutsukake, K. (n.d.). Identification of a novel Escherichia coli gene whose e&#8230; [Gene. 1997] &#8211; PubMed &#8211; NCBI. <em>National Center for Biotechnology Information</em>. Retrieved October 1, 2012, from http://www.ncbi.nlm.nih.gov/pubmed/9358034</span></p><br />
<p><span style="color:#000000;">Claret, L., Miquel, S., Vieille, N., Ryjenkov, D., Gomelsky, M., &amp; Darfeuille-Michaud, A. A. (2007). The Flagellar Sigma Factor FliA Regulates Adhesion and Invasion of Crohn Disease-associated Escherichia coli via a Cyclic Dimeric GMP-dependent Pathway. <em>The Journal Of Biological Chemistry</em>, <em>282</em>(46), 33275-33283. Retrieved October 1, 2012, from http://www.jbc.org/content/282/46/33275.full.pdf</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flih1.gif"><img class="aligncenter size-medium wp-image-213" title="fliH" src="http://uscigem2012.files.wordpress.com/2012/07/flih1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842005</strong></span></p><br />
<p><span style="color:#000000;">fliH</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">FliH functions as part of the flagella specific export system and is necessary for flagellar growth and assembly. It is believed to be part of a two-component system consisting of fliH and fliL that hydrolyses ATP until construction of the flagella apparatus is completed.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842005</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is to be used in conjunction with the entire flagella synthesis genetic pathway to reproduce a complete flagella apparatus. </span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Minamino, T., &amp; MacNab, R. (n.d.). FliH, a soluble component of the type III flag&#8230; [Mol Microbiol. 2000] &#8211; PubMed &#8211; NCBI. <em>National Center for Biotechnology Information</em>. Retrieved October 1, 2012, from http://www.ncbi.nlm.nih.gov/pubmed/10998179</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flic_gfp.gif"><img class="aligncenter size-medium wp-image-212" title="fliC_GFP" src="http://uscigem2012.files.wordpress.com/2012/07/flic_gfp.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842013</strong></span></p><br />
<p><span style="color:#000000;">fliC-GFP</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">When transcribed, RBS-fliC-GFP generates the flagellum protein bonded with a green fluorescent protein. This structure would then be exported to the growing flagella apparatus and used in the construction of the filament. When strongly expressed, this structure would directly result in the flagella filament's containing GFP, allowing for the flagella to be detectable. Unfortunately we were unable to promote the sequence enough to test whether this construct actually functions in this manner.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842013</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">The fliC gene was cloned via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. Due to the fact that fliC has multiple S and P restriction enzyme sites located within its DNA sequence, it was generated with XbaI sites on the forward and reverse primer. This is so as to allow for the placement of a gene promoter in the E-X sites upstream of the construct in an iGEM biobrick vector. It is important to note though that the current structure consists of the following so as to avoid the accidental loss of a gene during the cloning procedure.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;">EcoRI-XbaI-RBS-fliC-XbaI-GFP-SpeI-PstI</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">fliC:Quickview &#8211; EcoliWiki. (n.d.). <em>EcoliWiki</em>. Retrieved October 1, 2012, from http://ecoliwiki.net/colipedia/index.php/fliC</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flhb1.gif"><img class="aligncenter size-medium wp-image-210" title="flhB" src="http://uscigem2012.files.wordpress.com/2012/07/flhb1.gif?w=214&h=300" alt="" width="214" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842000</strong></span></p><br />
<p><span style="color:#000000;">flhB</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">Class 2 flagella operon. When transcribed, flhB is one of three flagella genes responsible for the generation of proteins that export flagellin to the flagella apparatus. The flagellin is then directly used in the synthesis of the structure's basal body. Additionally, flhB directly transcribes the operon flhBAE.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842000"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842000</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">flhB was use generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. It is directly downstream of the class 1 flagella operons flhD and flhC. It also works in conjunction with fliL and fliH to make up the flagellin export system. This part can be used either to generate a flagella in an <em>E. coli</em> strain in which they are not already present, or to promote further formation of pre-existing flagella.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Flagellar biosynthetic protein flhB &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P76299</span></p><br />
<p><span style="color:#000000;">flhB:Quickview &#8211; EcoliWiki. (n.d.). <em>EcoliWiki</em>. Retrieved October 1, 2012, from http://ecoliwiki.net/colipedia/index.php/flhB:Quickview</span></td><br />
</tr><br />
<tr><br />
<td rowspan="3" valign="top" width="377"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/flhd.gif"><img class="aligncenter size-medium wp-image-221" title="flhD" src="http://uscigem2012.files.wordpress.com/2012/07/flhd.gif?w=300&h=300" alt="" width="300" height="300" /></a></span></td><br />
<td rowspan="3" valign="top" width="120"><span style="color:#000000;"><strong>BBa_K842001</strong></span></p><br />
<p><span style="color:#000000;">flhD</span></td><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Description and Function</strong></span></p><br />
<p><span style="color:#000000;">flhD along with flhC form the master regulation operon for the synthesis of flagella in<em> E. coli. </em> They are responsible for the downstream transcription of the class 2 flagella genes. flhD is directly induced by several separate systems; among these is the two-component quorum sensing response system, qseB and qseC.</span></p><br />
<p><span style="color:#000000;">Used for reconstitution of flagella apparatus in <em>E. coli</em> B strains.</span></p><br />
<p><span style="color:#000000;"><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K842001"><span style="color:#000000;">http://partsregistry.org/wiki/index.php?title=Part:BBa_K842001</span></a></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Cloning and Uses</strong></span></p><br />
<p><span style="color:#000000;">This part was generated via PCR from the <em>E. coli</em> strain DH5α and cloned into the vector pSB1C3. FlhD is to be used in conjunction with flhC in order to form the transcriptional operon for the production of flagella. This part can be used either to generate a flagella in an <em>E. coli</em> strain in which they are not already present, or to promote further formation of pre-existing flagella.</span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="420"><span style="color:#000000;"><strong>Sources</strong></span></p><br />
<p><span style="color:#000000;">Flagellar transcriptional regulator FlhD &#8211; Escherichia coli (strain K12). (n.d.). <em>UniProt</em>. Retrieved October 1, 2012, from http://www.uniprot.org/uniprot/P0A8S9</span></td><br />
</tr><br />
</tbody><br />
</table><br />
<p><strong><span style="color:#000000;">New Constructs Being Tested:</span></strong></p><br />
<p><span style="color:#000000;">Because cheY and cheZ have opposing functions, we wanted to test the expression of both in a single E.coli. To ensure equal transcription from this construct, we utilized the commercially availible pCOLA-Duet and pCDF-Duet vectors from Novagen, which have two RBS sites flanking two unique multiple cloning sites, and are all under the control of a single T7 promoter.</span></p><br />
<p><span style="color:#000000;">These new constructs worked perfectly, and we are currently designing and constructing a similar system in the pSBC1C3 backbone as a new BioBrick.</span></p><br />
<p><span style="color:#000000;">Although we have not included these in the registry yet, as they have not been fully tested, we have generated the following constructs:</span></p><br />
<p><span style="color:#000000;">pCOLA-Duet-CheY</span></p><br />
<p><span style="color:#000000;">pCOLA-Duet-CheZ</span></p><br />
<p><span style="color:#000000;">pCDF-Duet-CheY</span></p><br />
<p><span style="color:#000000;">pCDF-Duet-CheZ</span></p><br />
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<groupparts>iGEM012 USC</groupparts><br />
</center></div>
Sgenyk
http://2012.igem.org/Team:USC/Team
Team:USC/Team
2012-10-04T00:50:01Z
<p>Sgenyk: </p>
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Team </h2><!-- .postitle --><br />
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<td valign="top" width="168"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/curran.jpeg"><span style="color:#000000;"><img class="aligncenter size-thumbnail wp-image-93" title="curran" src="http://uscigem2012.files.wordpress.com/2012/07/curran.jpeg?w=150&h=150" alt="" width="150" height="150" /></span></a></span></td><br />
<td valign="top" width="527"><span style="color:#000000;"><strong>Advisor: </strong></span><span style="color:#000000;"><strong>Sean Curran, Ph.D.</strong></span><br><span> <em>Sean Curran is an assistant professor in Gerontology at the USC Davis School of Gerontology with joint appointments in Molecular and Computational Biology (USC Dana and David Dornsife College of Letters, Arts, and Sciences) and Biochemistry and Molecular Biology (Keck School of Medicine of USC). He studies molecular genetics of exceptional longevity. In his free time, Sean loves to watch NCAA sports, experiment in the kitchen, swim, and watch movies.</em></span></td><br />
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<td valign="top" width="168"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/percy.png"><span style="color:#000000;"><img class="aligncenter size-thumbnail wp-image-94" title="Percy Genyk" src="http://uscigem2012.files.wordpress.com/2012/07/percy.png?w=109&h=150" alt="" width="109" height="150" /></span></a></span></td><br />
<td valign="top" width="527"><span style="color:#000000;"><strong>Graduate Student Advisor: </strong></span><span style="color:#000000;"><strong>Percy Genyk</strong></span><br><span><em> Percy Genyk is a recent graduate of USC Viterbi School of Engineering as a biomedical engineer. In his free time, he enjoys reading about synthetic biology and taking out his frustrations in the lab at the golf course. </em></span></td><br />
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<td valign="top" width="168"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/megan-photo-igem-copy.png"><span style="color:#000000;"><img class="aligncenter size-thumbnail wp-image-88" title="Megan Bernstein" src="http://uscigem2012.files.wordpress.com/2012/07/megan-photo-igem-copy.png?w=98&h=150" alt="" width="98" height="150" /></span></a></span></td><br />
<td valign="top" width="527"><span style="color:#000000;"><strong>Megan Bernstein </strong></span><br><span style="color:#000000;"><em>Megan is a junior majoring in Biological Sciences with a minor in News Media. In addition to iGEM, she directs USC&#8217;s Xpressions Dance Company and is a board member for USC Global Medical Brigades. In her free time, she enjoys volunteering, teaching science to 3rd graders, making crafts and baking!</em></span></td><br />
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<td valign="top" width="168"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/rachel.png"><span style="color:#000000;"><img class="aligncenter size-thumbnail wp-image-85" title="Rachel Kohan" src="http://uscigem2012.files.wordpress.com/2012/07/rachel.png?w=96&h=150" alt="" width="96" height="150" /></span></a></span></td><br />
<td valign="top" width="527"><span style="color:#000000;"><strong>Rachel Kohan </strong></span><br><span style="color:#000000;"><em>Rachel is a senior from Salt Lake City, UT studying biomedical engineering. In addition to iGEM, she coordinates an on-campus discussion group. In her free time she likes to draw, hike, and ski.</em></span></td><br />
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<td valign="top" width="168"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/63187_1499451847350_7277539_n.jpeg"><span style="color:#000000;"><img class="aligncenter size-thumbnail wp-image-89" title="Ellen Park" src="http://uscigem2012.files.wordpress.com/2012/07/63187_1499451847350_7277539_n.jpeg?w=102&h=150" alt="" width="102" height="150" /></span></a></span></td><br />
<td valign="top" width="527"><span style="color:#000000;"><strong>Ellen Park </strong></span><br><span style="color:#000000;"><em>Ellen Park is a senior Biology major at USC. Although she is a science major, she loves writing, singing, and art. Ellen is not only part of iGEM but is also the President of Overflow acappella, a small group leader in Korean Campus Missions, a member of USC pre-pharmacy society, and a research volunteer in the Curran lab.</em></span></td><br />
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<td valign="top" width="168"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/stephan.png"><span style="color:#000000;"><img class="aligncenter size-thumbnail wp-image-90" title="Stephan Genyk" src="http://uscigem2012.files.wordpress.com/2012/07/stephan.png?w=127&h=150" alt="" width="127" height="150" /></span></a></span></td><br />
<td valign="top" width="527"><span style="color:#000000;"><strong>Stephan Genyk </strong></span><br><span style="color:#000000;"><em>Stephan Genyk is senior at USC studying kinesiology and psychology. During his spare time, he enjoys swimming, cycling, playing chess, or just listening to his favorite genre of music, classical and electronic. Stephan knew that he wanted to join iGEM after writing an essay on synthetic biology sparked his passion.</em></span></td><br />
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<td valign="top" width="168"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/eric-siryj.jpg"><span style="color:#000000;"><img class="aligncenter size-thumbnail wp-image-87" title="Eric Siryj" src="http://uscigem2012.files.wordpress.com/2012/07/eric-siryj.jpg?w=123&h=150" alt="" width="123" height="150" /></span></a></span></td><br />
<td valign="top" width="527"><span style="color:#000000;"><strong>Eric Siryj </strong></span><br><span style="color:#000000;"><em>Eric Siryj is junior from Los Angeles studying biomedical engineering with an emphasis in mechanical engineering. In addition to iGEM, Eric researches crack propagation in USC&#8217;s &#8220;shock wave lab.&#8221; In his free time, he loves playing guitar and leads a Christian worship band on campus. He also loves all sports, especially basketball!</em></span></td><br />
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<td valign="top" width="168"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/396204_2293402709292_114923926_n.png"><span style="color:#000000;"><img class="aligncenter size-full wp-image-91" title="Luke Quinto" src="http://uscigem2012.files.wordpress.com/2012/07/396204_2293402709292_114923926_n.png?w=960" alt="" /></span></a></span></td><br />
<td valign="top" width="527"><span style="color:#000000;"><strong>Luke Quinto </strong></span><br><span style="color:#000000;"><em>Luke is a freshmen (Bio)Chemical Engineering major as well as a brother of the Theta Xi social fraternity. He works for Marshall Technology Support over at the school of business and when he&#8217;s not working or studying he&#8217;s usually either sleeping or eating. Aside from these, he also enjoys ultimate frisbee, swimming, skiing, orienteering, and just about anything else that gets him outside or off campus for a while.</em></span></td><br />
</tr><br />
<tr><br />
<td valign="top" width="168"><span style="color:#000000;"> <a href="http://uscigem2012.files.wordpress.com/2012/07/subuwoli.jpg"><span style="color:#000000;"><img class="aligncenter size-thumbnail wp-image-86" title="Rebecca Gao" src="http://uscigem2012.files.wordpress.com/2012/07/subuwoli.jpg?w=103&h=150" alt="" width="103" height="150" /></span></a></span></td><br />
<td valign="top" width="527"><span style="color:#000000;"><strong>Rebecca Gao </strong></span><br><span style="color:#000000;"><em>Rebecca Gao is a junior from Fremont, CA majoring in Global Health and Biological Sciences. She is the co-Editor-in-Chief of Trojan Health Connection and works as a research intern for Drs. Tower, Samet, and Charuzi. Rebecca enjoys sleeping and running in her free time.</em></span></td><br />
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Sgenyk
http://2012.igem.org/Team:USC
Team:USC
2012-10-04T00:43:36Z
<p>Sgenyk: </p>
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E.&nbsp;Musici </h2><!-- .postitle --><br />
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<p style="text-align:center;"><span style="color:#000000;"><strong>Abstract</strong></span></p><br />
<p style="text-align:justify;"><span style="color:#000000;">Our project aims to create a new system to quantify the environmental conditions and their effect on the bacteria <em>E.coli</em> by engineering a system that causes a predictable response to a controlled environment. Many strains of <em>E.coli</em> possess flagella, a whip-like appendage that can be found protruding from both eukaryotic and prokaryotic single-celled organisms. The assembly and motor control of <em>E.coli</em> flagella is under the control of a key group of genetic factors primarily for flagella assembly and chemotactic control. Control of these genes can be regulated by creating various combinations of genetic promoters which control expression and activity of individual components of the flagella mechanism. By promoting the <em>E.coli</em> flagella genes in various conditions, such as salt concentration, nitrate concentration, pH and temperature, we can prove a significant change in flagella rotation and frequency. Ultimately, this frequency can be translated into an audible range which can act as an indicator of the bacteria’s distress based on varied environmental conditions. Thus, by specifying the bacteria’s response to an environment on a genetic level, the resulting frequency can be used as a convenient assay for future researchers to determine the response of their test subjects to a controlled environment.</span></p><br />
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