| - | 9/10/14</h2><p>Objective: assemble decoy binding site arrays</p><ul><li>Setting up two PCRs with same cycling program as last time: </li></ul><ul><li>one with 4x 50uL reactions using the same concs of everything as the 0.5 uL rx from 9/9/14 (blue labels)</li><li>one with equimolar oligo1 and oligo2 and varying amounts of reaction 0.5 from 9/9/14 (green labels)</li></ul><ul><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 10, 2014 6:28:14 PM.jpg" src="images/image56.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>So something’s happening. PCR cleanup’d reactions 1-4 (green) and 5-8 (blue) in to one tube each labeled Decoy array (1-4 or 5-8) PCR1. Tomorrow will PCR with the end oligos and try gibson, dig/lig in to pSB1C3 and/or pSB1A2</li></ul><h2><a name="h.dmhnzg83ygzw"></a>9/11/14</h2><p>Objective: assemble decoy binding site arrays. Step 2: add prefix & suffix to arrays</p><ul><li>Faw setting up two series of PCRs with 1c337prfGfp1Space and 1c337sufGfp1Space as oligos on 0.5-2.0 ng of PCR cleanup’d green and blue rx from 9/10/14 and running with the same cycling conditions.</li><li>CBC did not take a picture and doesn’t remember why. PCR looked bad for some reason.</li></ul><h2><a name="h.knhldp48br8h"></a>9/12/14</h2><p>Objective: assemble decoy binding site arrays. Step 2: add prefix & suffix to arrays</p><ul><li>Garima setting up two series of PCRs with 1c337prfGfp1Space and 1c337sufGfp1Space as oligos on higher concs than yesterday ofPCR cleanup’d green and blue rx from 9/10/14 and running with the same cycling conditions.</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 16, 2014 11:21:26 AM.jpg" src="images/image06.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>PCR cleanup to pool tubes 2-4 for both series and labeled Decoy Array PCR2</li></ul><h2><a name="h.qg2pv53zrivc"></a>9/13/14</h2><p>Objective: assemble decoy binding site arrays. Step 3: insert putative arrays in to pSB backbone</p><ul><li>CBC attempts a SLIC with 0.5 uL of the “Super Clean” XbaI/PstI pSB1C3 backbone. Note that puts 15 bp of nonhomology in the way of successful SLIC. I did this because sometimes it works despite mismatches and there was no EcoRI/PstI dig’d pSB1C3 for me to work with. Green series looks like there is a bigger difference between no-template and +template tubes, so I will try that</li></ul><ul><li>set up tubes for backbone-only, backbone + 0.5, 1, 2, 3uL of insert from Green tube at 88.8 ng/uL, and 3uL insert-only.</li></ul><ul><li>Didn’t work.</li></ul><p>Objective: observe fluorescence changes with antitracr time course over time</p><ul><li>Last night antitracr strains started in LB+AmpKanCm and let shake overnight. Diluted this morning in to +/- aTc and began collection at time indicated. Replaced LB with PBS before flowing</li><li>Isolated “singlets” which are probably not singlets (smalls) or left alone (bugs) and got mean GFPA or gfpa/fsca or /ssca to normalize roughly to size.</li><li></li></ul><a href="#" name="504882174bdbc3a066b549e955cdc3b34f759d86"></a><a href="#" name="7"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p>time point</p></td><td><p>n/a</p></td><td><p>n/a</p></td><td><p>1</p></td><td><p>2</p></td><td><p>3</p></td><td><p>4</p></td><td><p>5</p></td></tr><tr><td><p>time (min)</p></td><td><p>n/a</p></td><td><p>n/a</p></td><td><p>120</p></td><td><p>180</p></td><td><p>240</p></td><td><p>300</p></td><td><p>360</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.52</p></td><td><p>2.74</p></td><td><p>3.17</p></td><td><p>3.16</p></td><td><p>2.81</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.39</p></td><td><p>2.8</p></td><td><p>3.18</p></td><td><p>3.42</p></td><td><p>2.88</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.45</p></td><td><p>2.92</p></td><td><p>3.31</p></td><td><p>3.53</p></td><td><p>2.88</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.53</p></td><td><p>3.13</p></td><td><p>3.72</p></td><td><p>3.6</p></td><td><p>2.89</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>45.6</p></td><td><p>56</p></td><td><p>68.4</p></td><td><p>76.8</p></td><td><p>74.9</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>46.9</p></td><td><p>54</p></td><td><p>67.8</p></td><td><p>73.8</p></td><td><p>74.7</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>48.4</p></td><td><p>53.1</p></td><td><p>65.8</p></td><td><p>73</p></td><td><p>76.2</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>45.6</p></td><td><p>54.2</p></td><td><p>68.2</p></td><td><p>74.3</p></td><td><p>78.4</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>0.109</p></td><td><p>0.16</p></td><td><p>0.185</p></td><td><p>0.139</p></td><td><p>0.143</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.43</p></td><td><p>3.08</p></td><td><p>3.65</p></td><td><p>3.67</p></td><td><p>3.07</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.32</p></td><td><p>3.17</p></td><td><p>3.67</p></td><td><p>3.88</p></td><td><p>3.07</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.73</p></td><td><p>3.3</p></td><td><p>4.15</p></td><td><p>3.99</p></td><td><p>3.18</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>1.57</p></td><td><p>3.47</p></td><td><p>4.16</p></td><td><p>3.83</p></td><td><p>3.29</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>41.7</p></td><td><p>53.4</p></td><td><p>67</p></td><td><p>75.8</p></td><td><p>75.6</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>41.4</p></td><td><p>51.3</p></td><td><p>67.4</p></td><td><p>74.3</p></td><td><p>71.7</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>39.4</p></td><td><p>52.4</p></td><td><p>66</p></td><td><p>73.9</p></td><td><p>60.8</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>40</p></td><td><p>52.7</p></td><td><p>67</p></td><td><p>76</p></td><td><p>73.2</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa (smalls)</p></td><td><p>0.0949</p></td><td><p>0.156</p></td><td><p>0.162</p></td><td><p>0.129</p></td><td><p>0.122</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>83.9</p></td><td><p>157</p></td><td><p>188</p></td><td><p>189</p></td><td><p>172</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>70.6</p></td><td><p>159</p></td><td><p>186</p></td><td><p>205</p></td><td><p>177</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>72.8</p></td><td><p>169</p></td><td><p>193</p></td><td><p>208</p></td><td><p>175</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>80.1</p></td><td><p>172</p></td><td><p>206</p></td><td><p>199</p></td><td><p>166</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2526</p></td><td><p>3227</p></td><td><p>3986</p></td><td><p>4592</p></td><td><p>4520</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2700</p></td><td><p>3078</p></td><td><p>4044</p></td><td><p>4486</p></td><td><p>4543</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2892</p></td><td><p>3028</p></td><td><p>3795</p></td><td><p>4291</p></td><td><p>4553</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2641</p></td><td><p>3131</p></td><td><p>4038</p></td><td><p>4531</p></td><td><p>4614</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>6.26</p></td><td><p>9.36</p></td><td><p>11.1</p></td><td><p>8.92</p></td><td><p>9.11</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>72.8</p></td><td><p>177</p></td><td><p>221</p></td><td><p>228</p></td><td><p>194</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>60.5</p></td><td><p>187</p></td><td><p>220</p></td><td><p>241</p></td><td><p>193</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>94.8</p></td><td><p>192</p></td><td><p>245</p></td><td><p>245</p></td><td><p>199</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>79.6</p></td><td><p>193</p></td><td><p>235</p></td><td><p>222</p></td><td><p>193</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2184</p></td><td><p>3100</p></td><td><p>4012</p></td><td><p>4663</p></td><td><p>4666</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2188</p></td><td><p>2957</p></td><td><p>4160</p></td><td><p>4554</p></td><td><p>4386</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2004</p></td><td><p>3047</p></td><td><p>4035</p></td><td><p>4565</p></td><td><p>3842</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>2039</p></td><td><p>3051</p></td><td><p>4107</p></td><td><p>4630</p></td><td><p>4512</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (smalls)</p></td><td><p>5.64</p></td><td><p>9.13</p></td><td><p>9.66</p></td><td><p>8.24</p></td><td><p>7.79</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>47.7</p></td><td><p>75.1</p></td><td><p>78.8</p></td><td><p>75.1</p></td><td><p>64.8</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>46.3</p></td><td><p>76.2</p></td><td><p>79.1</p></td><td><p>81</p></td><td><p>66</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>48</p></td><td><p>78.7</p></td><td><p>82.6</p></td><td><p>83.3</p></td><td><p>66.4</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>51.5</p></td><td><p>85.9</p></td><td><p>96.7</p></td><td><p>93.4</p></td><td><p>71.1</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1273</p></td><td><p>1469</p></td><td><p>1666</p></td><td><p>1779</p></td><td><p>1718</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1226</p></td><td><p>1413</p></td><td><p>1636</p></td><td><p>1719</p></td><td><p>1688</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1182</p></td><td><p>1382</p></td><td><p>1605</p></td><td><p>1704</p></td><td><p>1739</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1182</p></td><td><p>1391</p></td><td><p>1645</p></td><td><p>1714</p></td><td><p>1790</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>3.43</p></td><td><p>4.38</p></td><td><p>4.82</p></td><td><p>3.89</p></td><td><p>3.69</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>46.3</p></td><td><p>80.9</p></td><td><p>89.5</p></td><td><p>87.5</p></td><td><p>71</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>45.6</p></td><td><p>80.6</p></td><td><p>89.6</p></td><td><p>91.3</p></td><td><p>71.4</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>50.4</p></td><td><p>84.4</p></td><td><p>101</p></td><td><p>95</p></td><td><p>74</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>55.3</p></td><td><p>93.8</p></td><td><p>108</p></td><td><p>100</p></td><td><p>83.8</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1250</p></td><td><p>1375</p></td><td><p>1606</p></td><td><p>1758</p></td><td><p>1715</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1215</p></td><td><p>1320</p></td><td><p>1606</p></td><td><p>1734</p></td><td><p>1635</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1203</p></td><td><p>1319</p></td><td><p>1563</p></td><td><p>1718</p></td><td><p>1425</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>1230</p></td><td><p>1338</p></td><td><p>1595</p></td><td><p>1754</p></td><td><p>1661</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (smalls)</p></td><td><p>3.05</p></td><td><p>4.55</p></td><td><p>4.63</p></td><td><p>3.67</p></td><td><p>3.35</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.17</p></td><td><p>3.7</p></td><td><p>5.12</p></td><td><p>5.27</p></td><td><p>5.68</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.1</p></td><td><p>3.82</p></td><td><p>5.06</p></td><td><p>5.77</p></td><td><p>5.85</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.17</p></td><td><p>3.92</p></td><td><p>5.48</p></td><td><p>6.04</p></td><td><p>5.89</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.07</p></td><td><p>4.2</p></td><td><p>5.49</p></td><td><p>5.95</p></td><td><p>5.85</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>70.3</p></td><td><p>82.2</p></td><td><p>105</p></td><td><p>120</p></td><td><p>133</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>74.4</p></td><td><p>78.2</p></td><td><p>102</p></td><td><p>122</p></td><td><p>137</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>75.6</p></td><td><p>76.9</p></td><td><p>102</p></td><td><p>114</p></td><td><p>130</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>75.3</p></td><td><p>79.9</p></td><td><p>103</p></td><td><p>120</p></td><td><p>136</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>0.169</p></td><td><p>0.26</p></td><td><p>0.396</p></td><td><p>0.319</p></td><td><p>0.34</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.26</p></td><td><p>4.28</p></td><td><p>6.12</p></td><td><p>6.62</p></td><td><p>6.7</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.01</p></td><td><p>4.53</p></td><td><p>6.07</p></td><td><p>7.13</p></td><td><p>6.9</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.72</p></td><td><p>4.59</p></td><td><p>6.58</p></td><td><p>7.36</p></td><td><p>7.43</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>2.16</p></td><td><p>4.63</p></td><td><p>6.39</p></td><td><p>7.15</p></td><td><p>7.22</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>63.4</p></td><td><p>76.5</p></td><td><p>107</p></td><td><p>127</p></td><td><p>141</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>64.1</p></td><td><p>74.2</p></td><td><p>112</p></td><td><p>127</p></td><td><p>134</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>61.2</p></td><td><p>77.2</p></td><td><p>110</p></td><td><p>127</p></td><td><p>126</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>63.3</p></td><td><p>78.4</p></td><td><p>110</p></td><td><p>128</p></td><td><p>138</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa (bugs)</p></td><td><p>0.157</p></td><td><p>0.26</p></td><td><p>0.357</p></td><td><p>0.331</p></td><td><p>0.287</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>88.5</p></td><td><p>159</p></td><td><p>205</p></td><td><p>206</p></td><td><p>216</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>75.6</p></td><td><p>163</p></td><td><p>197</p></td><td><p>228</p></td><td><p>220</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>77.3</p></td><td><p>172</p></td><td><p>210</p></td><td><p>232</p></td><td><p>217</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>83.4</p></td><td><p>174</p></td><td><p>215</p></td><td><p>227</p></td><td><p>223</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2559</p></td><td><p>3229</p></td><td><p>4012</p></td><td><p>4818</p></td><td><p>5003</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2751</p></td><td><p>3078</p></td><td><p>4107</p></td><td><p>4768</p></td><td><p>5130</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>3006</p></td><td><p>3014</p></td><td><p>3787</p></td><td><p>4456</p></td><td><p>4927</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2695</p></td><td><p>3129</p></td><td><p>4076</p></td><td><p>4738</p></td><td><p>5032</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>6.33</p></td><td><p>10.1</p></td><td><p>15.8</p></td><td><p>12.9</p></td><td><p>14.1</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>78.4</p></td><td><p>182</p></td><td><p>242</p></td><td><p>263</p></td><td><p>257</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>64.6</p></td><td><p>194</p></td><td><p>236</p></td><td><p>278</p></td><td><p>259</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>101</p></td><td><p>195</p></td><td><p>263</p></td><td><p>287</p></td><td><p>277</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>82.5</p></td><td><p>195</p></td><td><p>250</p></td><td><p>270</p></td><td><p>274</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2213</p></td><td><p>3106</p></td><td><p>4099</p></td><td><p>5045</p></td><td><p>5411</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2212</p></td><td><p>2976</p></td><td><p>4289</p></td><td><p>5022</p></td><td><p>5084</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2022</p></td><td><p>3060</p></td><td><p>4164</p></td><td><p>4964</p></td><td><p>4574</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>2061</p></td><td><p>3053</p></td><td><p>4158</p></td><td><p>4990</p></td><td><p>5205</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa/fsca*1000 (bugs)</p></td><td><p>5.59</p></td><td><p>10</p></td><td><p>14.4</p></td><td><p>13.3</p></td><td><p>12.3</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>49</p></td><td><p>73.4</p></td><td><p>74.8</p></td><td><p>71.9</p></td><td><p>69</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>48.4</p></td><td><p>74.5</p></td><td><p>74.4</p></td><td><p>77.2</p></td><td><p>68.9</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>49.8</p></td><td><p>77.1</p></td><td><p>79.2</p></td><td><p>80.5</p></td><td><p>69.6</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>53</p></td><td><p>84.8</p></td><td><p>90.9</p></td><td><p>88.6</p></td><td><p>74.1</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1267</p></td><td><p>1433</p></td><td><p>1538</p></td><td><p>1591</p></td><td><p>1583</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1210</p></td><td><p>1380</p></td><td><p>1489</p></td><td><p>1535</p></td><td><p>1541</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1170</p></td><td><p>1359</p></td><td><p>1497</p></td><td><p>1514</p></td><td><p>1583</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1174</p></td><td><p>1356</p></td><td><p>1512</p></td><td><p>1531</p></td><td><p>1634</p></td></tr><tr><td><p>pdCas9</p></td><td><p>+ aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>3.25</p></td><td><p>4.27</p></td><td><p>5.57</p></td><td><p>4.17</p></td><td><p>4.13</p></td></tr><tr><td><p>GFP1-9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>48.5</p></td><td><p>77.5</p></td><td><p>84</p></td><td><p>84.1</p></td><td><p>77.8</p></td></tr><tr><td><p>GFP1-10</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>47.8</p></td><td><p>77.4</p></td><td><p>83.9</p></td><td><p>88.1</p></td><td><p>76.9</p></td></tr><tr><td><p>GFP1-11</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>51.9</p></td><td><p>80.9</p></td><td><p>94.2</p></td><td><p>92.9</p></td><td><p>82.8</p></td></tr><tr><td><p>GFP1-12</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>56.8</p></td><td><p>90.5</p></td><td><p>101</p></td><td><p>94.2</p></td><td><p>90.5</p></td></tr><tr><td><p>blankpdCas9-9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1246</p></td><td><p>1323</p></td><td><p>1446</p></td><td><p>1575</p></td><td><p>1613</p></td></tr><tr><td><p>blankpdCas9-10</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1208</p></td><td><p>1269</p></td><td><p>1413</p></td><td><p>1553</p></td><td><p>1529</p></td></tr><tr><td><p>blankpdCas9-11</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1196</p></td><td><p>1255</p></td><td><p>1395</p></td><td><p>1529</p></td><td><p>1366</p></td></tr><tr><td><p>blankpdCas9-12</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>1231</p></td><td><p>1282</p></td><td><p>1420</p></td><td><p>1561</p></td><td><p>1553</p></td></tr><tr><td><p>pdCas9</p></td><td><p>- aTc</p></td><td><p>gfpa/ssca*1000 (bugs)</p></td><td><p>2.82</p></td><td><p>4.23</p></td><td><p>4.92</p></td><td><p>4.26</p></td><td><p>3.62</p></td></tr></tbody></table><ul><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 585.00px; height: 925.00px;"><img alt="" src="images/image49.jpg" style="width: 585.00px; height: 925.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>It looks like the decrease in fluor for pdCas9-GFP1 coli manifests fairly early and persists for 6 hours. Lutz & Bujard’s paper use slightly less aTc for full induction of pTet, but if the decrease we see here were due to aTc inhibiting translation then we would expect to see a decrease in +aTc cultures with pdCas9. If pTet expression of aTc was removing trasncription/translation resources away from GFP production then you would also expect that to manifest in pdCas9. However it might be possible that the pdCas9 strains are experiencing the same decrease from aTc or growht burden but the strong GFP expression is masking the effect.</li><li>The other possibility is that something scientifically interesting, but not useful for iGEM purposes, is going on.</li></ul><h2><a name="h.oz7brdgn00cg"></a>9/15/14</h2><p>Objective: assemble decoy binding site arrays. Step 2: add prefix & suffix to arrays (retry)</p><ul><li>CBC sets up PCR with 0.5 ng/uL green or blue PCR1 template with 0, 0.4, 0.7, 1.0 uL in 50uL reactions with same cycling conditions and oligo concs etc as before. Maybe with less template the dominant species in the tubes will be that with prefix & suffix added. Maybe</li><li>No difference between + template and notemplate tubes. No picture taken.</li><li></li></ul><h2><a name="h.bvksvj6lqq8i"></a>9/16/14</h2><p>Objective: assemble decoy binding site arrays. Step 3: insert putative arrays in to pSB backbone</p><ul><li>CBC prays, puts PCR2 products used on 9/13/14 in a digestion reaction with EcoRI/PstI and incubates 37C for 4h. PCR cleanup and CIP (buffer 2) for 20m then cleanup again. Both tubes have ~35 ng/uL and Dusseldorf ligation calculator calls for 12 ng of 100 (ish) bp insert for 86 ng 2000 bp bb. Assuming that the large majority of the inserts lack the prefix and suffix, I will try 1 and 2 uL of insert for 1 of the bb. Also bb only and insert only, of course. Ligating 90m at RT</li><li>Shocking in to Z1; 30m on ice with 10uL of the ligation mix, 80m recovery before plating on LB+Cm</li><li>next morning: no colonies on any plates. Cm works</li></ul><p>9/17/14</p><p>Objective: PCR to assemble decoy binding site array</p><ul><li>MZ does PCR on 4 tubes: 0 um, 0.5 um, 1 um, and 2 um of ~200 ng/uL prefix oligo respectively + 50 uL standard mix (Q5 buffer and polymerase, repeat-spacer-repeat and spacer-repeat-spacer oligos, dNTPS, H2O) </li><li>PCR for 75 min</li><li>Gel to confirm (picture)</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 18, 2014 2:40:25 PM.jpg" src="images/image03.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>From left to right, 0, 0.5, 1, 2 uL of prefix oligo. Shortening of schmear is consistent with intended effect but not conclusive evidence thereof.</li><li>CBC takes 0.3 uL out of wells 0.5, 1, 2 to serve as template for new PCR2 with prefix & suffix oligos. MCF runs the gel</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 18, 2014 2:40:52 PM.jpg" src="images/image15.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li></ul><p>9/18/14</p><p>Objective: PCR to assemble decoy binding site array</p><ul><li>CBC sets up 3 new PCR series, each with 0.3, 0.6, 0.9, 1.2 uL of tubes 2, 3, 4 from previous reaction as template. </li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 18, 2014 2:44:02 PM.jpg" src="images/image14.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Left to right is series from reactions templated with tube 2, 3, and 4. Gels look more like small pieces are dominating (primer dimer??) but we will try gibson anyway</li></ul><p>Objective: PCR to assemble decoy binding site array</p><ul><li></li><li>MZ with CBC get Mert’s homemade 1.33x gibson assembly mix and make reactions with backbone only, then backbone + insert</li><li>1uL of (conc) backbone (=EcoRI/PstI/CIP pSB1C3-R0040 made by CBC on [date] conc = [conc]) and 0.3uL of PCR products from series 2, 3, 4 at concs [conc conc conc] respectively.</li><li>Next morning: 100s of colonies.</li></ul><p>Objective: PCR to assemble decoy binding site array</p><ul><li>Master mix X13: (of a standard 50 uL reaction)</li></ul><ul><li>494 uL dH2O</li><li>130 uL buffer (Q5)</li><li>13 uL dNTPs</li><li>3.25 uL each oligo (prefix & suffix oligos)</li><li>6.25 uL enzyme (Q5)</li><li>0.3 uL of each template (0.5, 1, 2)</li></ul><ul><li>Ran PCR with iGEM protocol (previously saved)</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 19, 2014 9:48:04 AM.jpg" src="images/image48.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Some uneven loading in gel or at addition of template to tube step; either way it’s fine. Pool with PCR cleanup protocol and then can use this for GA. 0.5 and 1.0 series look the most promising for this. </li></ul><p>9/19/14</p><p>Objective: confirm insert presence & size of decoy array inserts from gibson assembly yesterday</p><ul><li>100s of colonies on bb+insert plates 2, 3, 4. None on insert-only plates or backbone-only. </li><li>Colony PCR with pSB1C3-up and -dn with 62C annealing temp (see 7/25/2014 entry, Farnitano figured this temp out for these oligos)</li><li>1ng pSB1C3-R0040, no template control, then 4 colonies from plate 2, 4 from 3, 6 from 4. </li><li>No amplification on anything. No picture</li></ul><p>Objective: assemble decoy arrays. Insert putative arrays in to pSB1C3</p><ul><li>Delta pools reactions from Garima’s PCR/gel produced yesterday in to three tubes. CBC sets up gibson assembly. 1 uL backbone and 0.3 insert as appropriate, H2O to 5 uL, vortexed briefly then added 2.5uL of this to 7.5 uL of master mix. Only enough master mix was in the freezer for 6 of these half-sized reactions so there is no insert-only control for reactions labeled 2 in Garima’s gel.</li><li>Transforming all 10 uL in to 80uL of competent Z1s. </li><li>Next morning, lots of colonies on all the places there should be colonies. Huzzah</li></ul><p>9/21/14</p><p>Objective: confirm insert presence & size of decoy array inserts from gibson assembly </p><ul><li>Picked 18 colonies from gibson/transformation performed on 9/19/14 yesterday and miniprepped. EcoRI/PstI digest for 3h then run 15m on 0.8% gel</li></ul><ul><li>Unadorned pSB1C3 is 41bp between EcoRI and PstI, 107 for minimal intended length of the four assembly oligos. It looks like there might be some incomplete digestion happening on these maybe, or just overloaded. </li><li>taaggatgatttctgg aattcgcggccgcttctagagCCATCTAATTCAACAAGAATTGGtgatgttaatCCATCTAATTCAACAAGAATTGGtgatgttaattactagtagcggccgctgca gtccggcaaaaaagggc</li></ul><ul><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 21, 2014 4:12:27 PM.jpg" src="images/image51.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Left to right is 1-18. Two probably aberrant clones but they’re getting sequenced anyway. Sent to bio sequencing core seqd from pSB1C3-up-1 and -dn-1. Clones 1-4, 15-18. </li></ul><p>9/22/14</p><p>Objective: confirm insert presence & size of decoy array inserts from gibson assembly </p><ul><li>Mike picks 12 more colonies for miniprepping tomorrow</li></ul><p>9/23/14</p><p>Objective: confirm insert presence & size of decoy array inserts from gibson assembly </p><ul><li>Mike starts miniprep for 12 new colonies, charlie takes over after N3 addition & spin. Mike sets up EcoRI-HF/PstI-HF digest (cutsmart) for each clone. </li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 23, 2014 4:41:21 PM.jpg" src="images/image38.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Garima loaded this 1.3% TAE gel. Lane 7 (ie clone 25) has one big band at ~600 but also looks to have a small band down at the 1x size. Contamination? Unstable repeats? Another gel 0.8% has the same thing, but smearier. </li><li>Sequencing results come back!! As expected and consistent with the gel above, almost all colonies have a single insert, but clone 17 has 5! It works, kind of!!! The chromatograms taper off in quality around the repeats which makes it difficult for seqman to put it assemble properly, but manually getting called bases and finding repeats makes it clear. Clone 2 from yesterday was trash. </li></ul><p><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 806.50px; height: 48.18px;"><img alt="" src="images/image12.png" style="width: 806.50px; height: 48.18px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></p><ul><li>ctrl-F in text editor highlights each decoy site. To the left and right are prefix & suffixes</li></ul><p>Objective: transfer decoy arrays to pSB1A2</p><ul><li>CBC sets up prep scale digest of EcoRI/PstI of clones 17 & 18 & pSB1A2-R0040-antitracr. PCR cleanup and CIP treatment of clones 17 & 18 followed by a second PCR cleanup. Gel extraction of pSB1A2 backbone leaves only 22 ng/uL of dirty DNA by nanodrop. Tomorrow will try ligating, and also will set up another EcoRI/PstI digest of more pSB1A2-R0040-antitracr</li></ul><p>Objective: optimize colony PCR conditions for testing pSB1C3-decoy arrays</p><ul><li>Garima sets up master mix with 9x50uL rx. pSB1C3-up-1 and -dn-1 included at 0.25*9uL from 100uM stock. </li></ul><ul><li>45 uL Taq buffer</li><li>2.25 uL Taq polymerase</li><li>9 uL dNTPs</li><li>9 uL of each pSB1C3-up-1 and -dn-1</li><li>375.75 uL dH2O</li><li>9 uL template in positive tube/ 0 uL template in negative tubes</li></ul><ul><li>Ran colony pcr using saved protocol:</li></ul><ul><li>edited annealing step of protocol from 62 degrees to a gradient : 58-65 degrees</li><li>extension time of 15s (was 30)</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 24, 2014 1:13:46 PM.jpg" src="images/image35.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Fairly crappy snapshot (crapshot?) but you can see that the primer dimers on the - gel are all at ~100bp and the product on +template are at 2-300 which is consistent with R0040-antitracr size. the best amplification took place at higher temps. New protocol: 65C with 15s extension for short pieces, next try with some actual colonies.</li></ul><p>9/24/14</p><p>Objective: transfer decoy arrays to pSB1A2</p><ul><li>Setting up ligation reaction with EcoRI/PstI/CIP treated clone 17 and 18 from yesterday (5x and 1x decoys respectively) and EcoRI/PstI/gel-purified pSB1A2 bb. Also including controls below</li><li>used ligation calculator from dusseldorf </li><li></li></ul><a href="#" name="b2d32455b7b3438d6a59a365920ec8095fe8bb0e"></a><a href="#" name="8"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td></td><td><p>17 (5x)</p></td><td><p>18 (1x)</p></td><td><p>1A2 bb</p></td></tr><tr><td><p>length</p></td><td><p>~500</p></td><td><p>~100</p></td><td><p>2252</p></td></tr><tr><td><p>conc</p></td><td><p>84.1</p></td><td><p>63.8</p></td><td><p>22.8</p></td></tr><tr><td><p>ng needed</p></td><td><p>66</p></td><td><p>13</p></td><td><p>100</p></td></tr><tr><td><p>using uL</p></td><td><p>1</p></td><td><p>0.5</p></td><td><p>5</p></td></tr></tbody></table><a href="#" name="5465b9bebaea090720acd450041ece042a9665b5"></a><a href="#" name="9"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p>thing</p></td><td><p>bb+ins 17</p></td><td><p>insert only 17</p></td><td><p>bb+ins 17</p></td><td><p>insert only 17</p></td><td><p>bb only</p></td></tr><tr><td><p>backbone</p></td><td><p>5uL</p></td><td><p>0</p></td><td><p>5</p></td><td><p>0</p></td><td><p>5</p></td></tr><tr><td><p>insert</p></td><td><p>1uL</p></td><td><p>1</p></td><td><p>0.5</p></td><td><p>0.5</p></td><td><p>0</p></td></tr><tr><td><p>T4 buffer</p></td><td><p>2uL</p></td><td><p>2</p></td><td><p>2</p></td><td><p>2</p></td><td><p>2</p></td></tr><tr><td><p>enzyme</p></td><td><p>1uL</p></td><td><p>1</p></td><td><p>1</p></td><td><p>1</p></td><td><p>1</p></td></tr><tr><td><p>h2o</p></td><td><p>11uL</p></td><td><p>16</p></td><td><p>11.5</p></td><td><p>16.5</p></td><td><p>12</p></td></tr></tbody></table><ul><li>Mixed andl et sit for 3h at room temp on bench. Took 10uL and transformed with 80uL of frozen competent Z1s</li><li>next day: lots of colonies on bb+insert plates, none on bb-only or insert-only. huzzah</li></ul><p>Objective: find more long decoy arrays</p><ul><li>miniprepping 12 more colonies from plate 1.0, middle one from 9/18/14</li><li>EcoRI/PstI digest for 3h gives all small bands at 1x size. No picture taken, all tubes discarded. </li></ul><p>9/25/14</p><p>Objective: find more long decoy arrays</p><ul><li>colony PCR on 4 colonies from plate 1.0 next to no-template, 1ng of pSB1C3-R0040-antitracr, pSB1C3-5xGFP1 decoy, and pSB1C3-1xGFP1 decoy. 10s extension at 64C</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 25, 2014 5:39:52 PM.jpg" src="images/image39.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""> No amplification on any of the colonies?</li></ul><ul><li>no template, pSB1C3-R0040-antitracr, 5x, 1x, then 4 colonies. At first I thought this was no amplification but actually I think it worked but all of the colonies are 1x or maybe a 2x for colony 4. Threw them all away in any case and before I had the realization that they amplified I already set up the next PCR with 8 colonies on a 58-65C gradient. </li></ul><ul><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 25, 2014 6:04:08 PM.jpg" src="images/image22.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li></ul><ul><li>left to right is 65 to 58. My strong suspicion is that the long band in lane 1 is from a long template rather than 65. Picking colonies 1 and 3 for further investigation. Labeled 43 and 44 on the tubes, because that’s where we are in the screening.</li></ul><p>Objective: confirm insert presence & size of decoy array inserts from gibson assembly </p><ul><li>sequencing clone 25 with pSB1C3-up and -dn, dugsim job 1827</li></ul><p>It might be better to take the 5x repeat and then expand it with some other scheme. </p><ul><li>It is possible to do this by stepwise dig/lig/confirm cycles with XbaI/PstI dig’d insert and SpeI/PstI dig’d backbone (gel purified). </li><li>Another possible trick to speed up the expansion would be to cycle from 16C to 37C a couple of times with XbaI/SpeI dig’d pSB1C3-5xGFP1 and also add XbaI/SpeI & T4 ligase and a third enzyme that cuts somewhere else in the backbone. </li></ul><ul><li>When XbaI and SpeI ligate it will destroy the recognition site. Any sites re-ligating to self will be cleaved in the next 37C cycle. The last step in the cycling reaction will be a long digest.</li><li>Follow that with a ligation to XbaI/SpeI/CIP treated backbone. </li><li>Circularization is possible with this scheme</li></ul><ul><li>Third possibility: cycle XbaI/PstI/CIPd insert with backbone, SpeI, PstI, ligase and cycle</li></ul><ul><li>XbaI ligs to SpeI and destroys site. XbaI ligs to XbaI and gets re-cut, same with PstI. On re-cutting this would restore the phosphate removed by CIP so that doesn’t help much. Hmm</li></ul><ul><li>…..or just the stepwise dig/lig/shock will work. I think the problems of circularization will be there no matter what I do with CIP & including restriction enzymes the ligase.</li></ul><p>9/26/14</p><p>Objective: Confirm pSB1A2-GFP1 decoy arrays</p><ul><li>Yesterday picked 4 colonies from transformation of 5x and 1x repeat arrays and set up with EcoRI/PstI digest, alongside colonies 43 and 44 from yesterday’s colony PCR and pSB1C3 5x and 1x parents. </li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 26, 2014 2:19:47 PM.jpg" src="images/image34.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Slightly hard to read on the picture, but they’re loaded in the order mentioned above. 43 might be longer than 5x, 44 looks like 1x so discarding. </li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 26, 2014 2:21:25 PM.jpg" src="images/image30.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Zoom in on the 5x on 1A2 and Colony 1 might be slightly shorter than colony 4? Making bacteria with both of these just in case. Should probably sequence as well. pSB1C3-up-1 binds to 1A2, but not pSB1C3-dn</li><li>Shocking Z1 to make a couple of strains:</li></ul><ul><li>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-1</li><li>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-4</li><li>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-1xGFPdecoy-1</li><li>pdCas9, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-1</li><li>pdCas9, pSB4K5-K(gfp), pSB1A2-5xGFPdecoy-4</li><li>pdCas9, pSB4K5-K(gfp), pSB1A2-1xGFPdecoy-1</li></ul><p>9/27/14</p><p>Objective: extend decoy arrays</p><ul><li>XbaI/PstI digest of 1C3-5xGFP1decoy followed by CIP, then SpeI/PstI of 1C3-5xGFP1 (same plasmid) with NO GEL PURIFICATION makes the backbone. I am hoping that since SpeI to PstI is only 18bp that the PCR cleanup kit gets rid of the fragment. Ligating with 3-fold molar excess of CIP-treated insert for 20m then shocking 10uL in to 70uL Z1. This is rushing the protocol; the other 10uL of the ligation mix will sit at RT for 90m in case the rushed version fails. </li></ul><ul><li>Update: it looks like it did not fail; there are some colonies on bb-only and ins-only but ~10x as many on b+i.</li></ul><p>9/28/14</p><p>Objective: test 1x and 5x decoy arrays</p><ul><li>4 colonies each picked from plates from</li></ul><ul><li>pdCas9-GFP1, pSB4K5-K608012, pSB1A2-5xGFPdecoy-1 G51</li><li>pdCas9-GFP1, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 G54</li><li>pdCas9-GFP1, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 G11</li><li>pdCas9, pSB4K5-K608012, pSB1A2-5xGFPdecoy-1 C51</li><li>pdCas9, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 C54</li><li>pdCas9, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 C11</li></ul><ul><li>….and grown overnight (started 9/27/14). 9:15am today diluted 1/200 in 3 mL fresh LB+AmpKanCm. 3h45m in 37C shaker. 1mL collected, spun down, and resuspended in PBS. Flowed 75000 events and got something that looks super promising. Means here are written down from stats view on MQ during acquisition and might change after full processing. There is a lot of variability in G51</li></ul><a href="#" name="225acd26b42536ecbf50f90518b34a9be550eb1e"></a><a href="#" name="10"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p>2014-09-28 flow data, not fully processed</p></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><p>(constitutive GFP reporter)</p></td><td><p>pdCas9 with or without GFP-targeting crRNA</p></td><td><p>decoy binding site count & colony</p></td><td><p>expected GFP if working</p></td><td><p>tube label</p></td><td><p>unprocessed mean gfp</p></td><td><p>avg</p></td><td><p>std</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>derepressed</p></td><td><p>G51-1</p></td><td><p>42</p></td><td><p>69.25</p></td><td><p>28.14</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>derepressed</p></td><td><p>G51-2</p></td><td><p>50</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>derepressed</p></td><td><p>G51-3</p></td><td><p>83</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>derepressed</p></td><td><p>G51-4</p></td><td><p>102</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>derepressed</p></td><td><p>G54-1</p></td><td><p>64</p></td><td><p>68.33</p></td><td><p>5.13</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>derepressed</p></td><td><p>G54-2</p></td><td><p>67</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>derepressed</p></td><td><p>G54-3</p></td><td><p>nan</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>derepressed</p></td><td><p>G54-4</p></td><td><p>74</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>near repressed levels</p></td><td><p>G11-1</p></td><td><p>16</p></td><td><p>16.00</p></td><td><p>0.82</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>near repressed levels</p></td><td><p>G11-2</p></td><td><p>15</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>near repressed levels</p></td><td><p>G11-3</p></td><td><p>17</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9-GFP1</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>near repressed levels</p></td><td><p>G11-4</p></td><td><p>16</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C51-1</p></td><td><p>131</p></td><td><p>141.75</p></td><td><p>7.46</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C51-2</p></td><td><p>143</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C51-3</p></td><td><p>145</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C51-4</p></td><td><p>148</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>unrepressed</p></td><td><p>C54-1</p></td><td><p>153</p></td><td><p>149.00</p></td><td><p>3.16</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>unrepressed</p></td><td><p>C54-2</p></td><td><p>150</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>unrepressed</p></td><td><p>C54-3</p></td><td><p>146</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-5xGFP1decoy-4</p></td><td><p>unrepressed</p></td><td><p>C54-4</p></td><td><p>147</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C11-1</p></td><td><p>150</p></td><td><p>153.25</p></td><td><p>3.95</p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C11-2</p></td><td><p>152</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C11-3</p></td><td><p>159</p></td><td><p> </p></td><td><p> </p></td></tr><tr><td><p>pSB4K5-K608012</p></td><td><p>pdCas9</p></td><td><p>pSB1A2-1xGFP1decoy-1</p></td><td><p>unrepressed</p></td><td><p>C11-4</p></td><td><p>152</p></td><td><p> </p></td><td><p> </p></td></tr></tbody></table><ul><li>Saved G51-1 through -4, then colony 1 for one of each different strain as glycerol stocks. Labeled very minimally with the bolded labels in first sub-bullet list. </li><li>Next steps:</li></ul><ul><li>repeat</li><li>try with more decoy site numbers: 10x is on the way, need to also make 0x</li></ul>
| |
| | + | <p>10/1/14</p><p>Objective: sequence confirm 1C3-2x5xGFP</p><ul><li>Sequenced with pSB1C3-up-1 and -dn-1, and both clones look like they have a duplication of 6x, rather than 5x. That means that clone 43 from yesterday is not useful so it will be thrown away.</li></ul><p>Objective: test RT-PCR primers</p><ul><li>MZ setting up two test PCRs</li><li>4 samples of tracr amplification on pdcas9 - 0, 0.3, 0.6, 0.9 ng/uL tubes, 4 samples of anti-tracr amplification on pdcas9</li><li>results: no good</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Oct 2, 2014 4:29:22 PM.jpg" src="images/image05.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>We can guess at what’s happening here, but probably the first thing to do is just to try it again.</li></ul><p>10/2/14</p><p>Objective: confirm 1A2-2x5x</p><ul><li>Picked 6 colonies and subjecting to EcoRI/PstI digestion</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Oct 2, 2014 4:19:54 PM.jpg" src="images/image32.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li></ul><ul><li>Left to right are clones 1-6 of 2x5x, rightmost = 1A2-5x. Looks perfect, freezing clones 1 and 2 and will sequence this weekend. </li></ul><p>Objective: extract RNA and test for expression & aTc response of tracr, antitracr</p><ul><li><a href="http://www.google.com/url?q=http%3A%2F%2Fopenwetware.org%2Fwiki%2FSauer%3ARNA_Purification_from_E._coli&sa=D&sntz=1&usg=AFQjCNEUMG4dvztCAaiAneGoVZIW0PKINA">http://openwetware.org/wiki/Sauer:RNA_Purification_from_E._coli</a></li><li>TYler of Gersbach lab recommends RNAzap, lab coat, holding breath, filter tips. Random hexamers are better if you want to RT everything which can be more generically useful than specific primers for this step. He uses hexamers for RT step then does specific amplification following that. </li></ul><ul><li>Last night started from frozen some Z1 with pdCas9 and 1A2-R0040-antitracr in LB + antibioic. This morning measured 1/10 diluted OD600 and calculated amount to add to 1) balance cultures ODs and 2) dilute back to a sufficient degree that after several generations in +/- aTc the cultures would not be at too high a density to easily harvest the max number of cells called for by the agilent RNA purification kit. This means diluting to the equivalent of 1/1000 from a culture of OD600=3.0</li></ul><ul><li><a href="http://www.google.com/url?q=http%3A%2F%2Fwww.genomics.agilent.com%2Fbiocalculators%2FcalcODBacterial.jsp&sa=D&sntz=1&usg=AFQjCNHJj1DKIVUO326ebS70jsxbDBDHNw">http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp</a> for OD -> cell count calculations</li><li><a href="http://www.google.com/url?q=http%3A%2F%2Fwww.chem-agilent.com%2Fpdf%2Fstrata%2F400800.pdf&sa=D&sntz=1&usg=AFQjCNGRsizNL201WP4khmvIUEEuqwIKKA">http://www.chem-agilent.com/pdf/strata/400800.pdf</a></li></ul><ul><li>manual</li></ul><ul><li><a href="http://www.google.com/url?q=http%3A%2F%2Fcp.literature.agilent.com%2Flitweb%2Fpdf%2F5990-5152EN.pdf&sa=D&sntz=1&usg=AFQjCNGvbTOiNbW_-5dNRECt1JbqQC9CQA">http://cp.literature.agilent.com/litweb/pdf/5990-5152EN.pdf</a></li></ul><ul><li>Has details for lysozyme treatment of coli before following the rest of the lysis/extraction protocol. This one might be just for the microprep kit, rather than the miniprep kit which we have.</li></ul><ul><li>1 mg/mL lysozyme, ethanol dilutions were prepared with filtered EtOH and/or water. Not DEPC treated or certified RNAse free. </li></ul><ul><li>Kind of a big fucking mess. Centrifuge at CBC’s bench is broken so rna back and forth between Mert’s bench to do centrifugations. Lids broke off so the real ID of some samples isn’t actually known, and accidentally made too little lysis buffer so one sample is just gone. Ended up with low 20s to 50s of some nucleic acid as measured by ND but all was pretty contaminated with ethanol, or whatever it is that gives a low 260/230 ratio. Will try this again. </li><li>Also the pdCas9 dilutions grew less quickly than did the 1A2-R0040antitracr ones</li></ul><ul><li>~.065 after 3h in new medium pdCas9</li><li>~.112 after 3h in new medium 1A2-R0040antitracr</li></ul><ul><li>Tomorrow AM: retry test PCR on DNA with the anti/tracr oligos.</li></ul><p>Objective: make more decoy strains for testing</p><ul><li>Shocking Z1 with</li></ul><ul><li>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-2x5xGFPdecoy-1</li><li>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-B0011 (0x)</li><li>pdCas9, pSB4K5-K(gfp), pSB1A2-2x5xGFPdecoy-1</li><li>pdCas9, pSB4K5-K(gfp), pSB1A2-B0011 (0x)</li></ul><p>10/3/14</p><p>Objective: test PCR of tracr & antitracr oligos on pdCas9</p><ul><li>tracr oligos were designed wrong and will not work. Also the antitracr ones should amplify both tracr and antitracr as long as they’re part of a one-mix RTPCR kit. And also also it might not be possible to make one that amplifies tracr without also amplifying antitracr for the same reason. </li><li>Ran 5s extension and 62C annealing. </li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Oct 3, 2014 12:57:14 PM.jpg" src="images/image07.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>left to right: antitracr-up and -dn with 1ng/uL pdCas9 at 0.3, 0.6, 0.9 uL, then the same with tracr-up and -dn. The bigger bands are partial amplification of the entire backbone. There is more of a small product in the +_template lanes for antitracr than there is in the no-template. It might be possible to improve this with different cycling conditions.</li></ul><p>10/4/14</p><p>Objective: test more decoy arrays for repression & cooperativity</p><ul><li>Last night started from plates</li></ul><ul><li>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-2x5xGFPdecoy-1 G251</li><li>pdCas9-GFP1, pSB4K5-K(gfp), pSB1A2-B0011 (0x) C251</li><li>pdCas9, pSB4K5-K(gfp), pSB1A2-2x5xGFPdecoy-1 G01</li><li>pdCas9, pSB4K5-K(gfp), pSB1A2-B0011 (0x) C01</li></ul><ul><li>...and also from frozen, three cultures started from same frozen stock</li></ul><ul><li>pdCas9-GFP1, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 G54</li><li>pdCas9-GFP1, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 G11</li><li>pdCas9, pSB4K5-K608012, pSB1A2-5xGFPdecoy-4 C54</li><li>pdCas9, pSB4K5-K608012, pSB1A2-1xGFPdecoy-1 C11</li></ul><ul><li>….then diluted 1/200 in fresh LB + AmpKanCm. Let roll for 3h30m before spinning and resuspending in PBS for flow. Collected 75000 events, 350 350 350V on fsc ssc b1</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 362.00px; height: 218.00px;"><img alt="" src="images/image13.png" style="width: 362.00px; height: 218.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li></ul><ul><li>This is after guessing on some of the sample IDs. This flow should be redone since I somehow came up one sample short during acquisition (possibly lost a 0x?)<hr style="page-break-before:always;display:none;"></li><li></li></ul><a href="#" name="1480a8636207f9da790124f8da1e79de0ae77bab"></a><a href="#" name="11"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p>Name</p></td><td><p>Count/ml</p></td><td><p>GFP/FITC-A Mean</p></td><td><p>putative decoy count</p></td></tr><tr><td><p>if142014-10-04.001</p></td><td><p>7032348.63</p></td><td><p>65.24</p></td><td><p>6</p></td></tr><tr><td><p>if142014-10-04.002</p></td><td><p>7693097.17</p></td><td><p>58.46</p></td><td><p>6</p></td></tr><tr><td><p>if142014-10-04.003</p></td><td><p>13638844.73</p></td><td><p>60.12</p></td><td><p>6</p></td></tr><tr><td><p>if142014-10-04.004</p></td><td><p>6406423.83</p></td><td><p>4.64</p></td><td><p>1</p></td></tr><tr><td><p>if142014-10-04.005</p></td><td><p>6806425.29</p></td><td><p>9.83</p></td><td><p>1</p></td></tr><tr><td><p>if142014-10-04.006</p></td><td><p>8183306.15</p></td><td><p>9.88</p></td><td><p>1</p></td></tr><tr><td><p>if142014-10-04.007</p></td><td><p>30012003.91</p></td><td><p>7.32</p></td><td><p>0</p></td></tr><tr><td><p>if142014-10-04.008</p></td><td><p>7628153.32</p></td><td><p>7.33</p></td><td><p>0</p></td></tr><tr><td><p>if142014-10-04.009</p></td><td><p>4245683.59</p></td><td><p>77.01</p></td><td><p>12</p></td></tr><tr><td><p>if142014-10-04.010</p></td><td><p>32425421.88</p></td><td><p>88.33</p></td><td><p>12</p></td></tr><tr><td><p>if142014-10-04.011</p></td><td><p>19679875</p></td><td><p>81.53</p></td><td><p>12</p></td></tr><tr><td><p>if142014-10-04.012</p></td><td><p>9475678.71</p></td><td><p>155.04</p></td><td><p>6</p></td></tr><tr><td><p>if142014-10-04.013</p></td><td><p>8393017.58</p></td><td><p>145.65</p></td><td><p>6</p></td></tr><tr><td><p>if142014-10-04.014</p></td><td><p>8781172.85</p></td><td><p>148.24</p></td><td><p>6</p></td></tr><tr><td><p>if142014-10-04.015</p></td><td><p>9550490.23</p></td><td><p>150.54</p></td><td><p>1</p></td></tr><tr><td><p>if142014-10-04.016</p></td><td><p>10143360.35</p></td><td><p>148.22</p></td><td><p>1</p></td></tr><tr><td><p>if142014-10-04.017</p></td><td><p>9304056.64</p></td><td><p>142.56</p></td><td><p>1</p></td></tr><tr><td><p>if142014-10-04.018</p></td><td><p>7726383.3</p></td><td><p>150.92</p></td><td><p>0</p></td></tr><tr><td><p>if142014-10-04.019</p></td><td><p>8277232.42</p></td><td><p>148.32</p></td><td><p>0</p></td></tr><tr><td><p>if142014-10-04.020</p></td><td><p>8675535.16</p></td><td><p>150.66</p></td><td><p>0</p></td></tr><tr><td><p>if142014-10-04.021</p></td><td><p>8471705.08</p></td><td><p>148.45</p></td><td><p>12</p></td></tr><tr><td><p>if142014-10-04.022</p></td><td><p>7073470.21</p></td><td><p>145.62</p></td><td><p>12</p></td></tr><tr><td><p>live (if142014-10-04.023)</p></td><td><p>6844938.96</p></td><td><p>148.57</p></td><td><p>12</p></td></tr></tbody></table><ul><li>Derepression happens with even a single copy? </li><li>There is more derepression with 12 than with 6, but not tremendously more. Still not derepressing all the way to pdCas9-only levels. Eyeballing, it doesn’t look frightfully cooperative but we probably need more than 4 points to be sure. </li><li>Conclusions and next steps</li></ul><ul><li>We need more arrays between 1 and 6x</li><li>We need more arrays longer than 12x</li></ul><ul><li>Possible explanations for mild increase in derepression</li></ul><ul><li>Plasmid copy number makes the difference between 0x and 1x decoys on the plasmid translate to no copies in the cell to 100s. The difference between 1x and 6x is 6-fold, 6x to 12x is just 2-fold. Bigger decoy arrays are needed to test this. Copies of decoys vs copies of site of repression might be important</li><li>Instability: there is published work where long arrays of TF binding sites can lead to DNA breakage, and CBC has seen this in his own work with LacI arrays. It might be that 12x repeats of the decoy get broken, recombined, and shrunk down to something smaller. This is testable by extracting plasmid and looking at size of EcoRI/PstI fragments, but keep in mind that there are other plasmids in these bugs. </li></ul><p>10/7/14</p><p>Objective: optimize tracr/antitracr PCR</p><ul><li>Set up test PCR with 5s extension and a gradient from 58 - 68C for annealing temp. 30s at annealing, 0.5 uL/rx for + template, 0uL/rx for -template</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Oct 7, 2014 5:09:57 PM.jpg" src="images/image24.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Left to right is (+ template) 58 -> 68 and then (-template) 58-68 again there is more product with template, but right around the same size as primer dimer</li></ul><p>Objective: try again RNA extraction, aim for more RNA</p><ul><li>Last night started cultures in LB+amp or +Cm of Z1 + pdCas9 or 1A2-R0040-antitracr. Diluted 1/400 in new fresh medium + or 100nM aTc/50% EtOH. Let shake for 5h and collected 1 mL</li><li>Final ODs………...RNA concs (nanodropped)</li></ul><ul><li>1A2-R0040-antitracr + aTc 0.000 ………..</li><li>1A2-R0040-antitracr - aTc 0.000 ………..</li><li>pdCas9 + aTc 0.000 ………..</li><li>pdCas9 - aTc 0.000 ………..</li></ul><ul><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Oct 7, 2014 5:09:01 PM.jpg" src="images/image18.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Ran ~100ng out on a gel for 16m. They all look like 2 bands of something smeary. Maybe not plasmid? pdCas9 is ~10000 bp. tRNA is less than 100bp. Do this again but with DH5alphaZ1 in LB+Spec </li></ul><p>10/8/14</p><p>Objective: another RNA isolation</p><ul><li>This time with empty Z1 as well as 1A2-R0040-antitracr and pdCas9 strains alls +/- aTc</li><li>One of the pdCas9 culture’s pre-filter spin filtered very, very slowly and incompletely so it was discarded. </li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Oct 9, 2014 1:23:47 PM.jpg" src="images/image45.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li></ul><ul><li>Left to right, empty Z1 +aTc, -aTc, then Z1+R0040-antitracr +aTc -aTc</li></ul><ul><li>Asked Tony Burnetti of the Buchler lab what RNA should look like and he said it would be mainly two bands which correspond to the big and small ribosomal subunits. RNA extraction successful!</li><li></li></ul><p>10/9/14</p><p>Objective: send parts for iGEM competition</p><ul><li>Submitted 1x, 6x, and 12x GFP1 decoy arrays in pSB1C3 and sent 10uL 25ng/uL plasmids in dH2O via FedEx priority overnight in accordance with the iGEM instructions. </li></ul><ul><li>Tracking number 771440542338</li><li>Shipment number 02826 (written on tube holding PCR tube strip)</li><li>iGEM website lists as received on 10/10/14. Huzzah!</li><li></li></ul><p>10/14/14</p><p>Objective: Final reflow of decoy array strains</p><ul><li>Started last night from frozen 3 replicate cultures each of pdCas9-GFP1, reporter, and 1A2-0x 1x 6x and 12x, and the same but with pdCas9 sans GFP1 crRNA. Diluted 1/400 in new LB+AmpKanCm and let shake for 4h before collecting 900uL and replacing medium with PBS. Vortex and flow</li><li>Gated for “singlets,” which are probably just smaller bacteria but did it for consistency with other plots and normalized by FSCA for the same area. Made ugly box plots with matlab: bam<span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 560.00px; height: 420.00px;"><img alt="" src="images/image09.jpg" style="width: 560.00px; height: 420.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>legend indicates pdCas9 variant. All strians have reporter and some number of pSB1A2-decoy</li><li>If you plot it on a log scale it 1) wonks up the box widths but 2) shows that the derepression looks pretty smooth. The kind of ultrasensitivity we are looking for manifests on the log scale so this might be something to be concerned about. One way to improve this is to make the binding to the decoy sites stronger than it is to the actual site of repression. You could do this by intentionally having a mismatch between crRNA and the site of repression, but having the decoys be a perfect match. </li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 560.00px; height: 420.00px;"><img alt="" src="images/image02.jpg" style="width: 560.00px; height: 420.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li></ul><p>Objective: RTPCR of antitracr strains to check for aTc-responsive antitracr RNA levels</p><ul><li>RNA extracted DH5alphaZ1 and Z1+R0040, and Z1+pdCas9 in +/- aTc used as a template for one-step RT-PCR kit with antitracr-up and -dn according to bioline’s instructions. 1.5uL of 1 ng/uL dilution of RNA used as template, also ran no RT controls for each strain as well as no template and 1.5ng pdCas9 (DNA)</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Oct 14, 2014 2:02:31 PM.jpg" src="images/image29.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Left to right: </li></ul><ul><li>0 ladder</li><li>1 no template + RT</li><li>2 ng pdCas9 template (DNA) + RT</li><li>Z1 +aTc + RT</li><li>Z1 -aTc + RT</li><li>Z1+1A2-R0040-antitracr +aTc + RT</li><li>Z1+1A2-R0040-antitracr -aTc + RT</li><li>Z1+pdCas9 +aTc, Z1+pdCas9 -aTc + RT</li><li>Z1 +aTc - RT</li><li>Z1 -aTc - RT</li><li>Z1+1A2-R0040-antitracr +aTc - RT</li><li>Z1+1A2-R0040-antitracr -aTc - RT</li><li>Z1+pdCas9 +aTc, Z1+pdCas9 -aTc - RT</li></ul><ul><li></li><li>Quantifying with heavy caveats by drawing boxes in fiji.</li></ul><ul><li>Draw little boxes outside of the bands but near to get background </li><li>Draw little boxes inside each band</li><li>Measure gives mean pixel intensity. Subtract band mean intensity - bg mean intensity and you get this:</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 552.00px; height: 332.00px;"><img alt="" src="images/image23.png" style="width: 552.00px; height: 332.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>HOWEVER you have to take these with a fairly big grain of salt because this gel did not have a loading control and this is end-point RT-PCR so we don’t know whether the PCR was stopped in the linear range. </li></ul><ul><li>Product on -RT indicates some DNA contamination. +RT bands are stronger where they’re supposed to be stronger, though, and Z1 with RT having no bands might mean that there is less than the bands would indicate. The test of this is to take some, treat with RNAse, and then PCR the product of that</li><li>On +RT lanes we have no bands as expected for Z1, stronger on 1A2-R0040-antitracr +aTc than -aTc as expected, and levels unresponsive to aTc for pdCas9 lanes also as expected. Everything is as expected, except maybe the leakiness of pTet. That could be either DNA contamination or genuine leak. </li><li>The question motivating this RT PCR was whether pTet is actually expressing antitracr, and this gel indicates that it is. Possibilities for why the fluorescence changes aren’t what you’d expect when doing flow are</li></ul><ul><li>Insufficient expression of antitracr</li></ul><ul><li>In vivo binding might not be as strong as you’d expect from RNAfold etc predictions. If this is the case then you’d need a lot of antitracr to see derepression</li><li>tracr is co-cistronic with dCas9 on pdCas9 - one could imagine that maybe each tracrRNA that gets made is in close proximity to dCas9 proteins and is quickly bound to dCas9 before antitracr has access. More antitracr may also overcome this</li></ul><ul><li>maybe in vivo hairpinning prevents antitracr from grabbing tracr. </li></ul><ul><li>It’s worth noting that antitracr has a little extra sequence on 5’ from PCR/oligo design realities during construction and also the terminators on 3’. antitracr is probably not getting processed in the same way that tracr is so everything on the plasmid after TSS and including terminators are probably present.</li><li>If this is the case then expressing a full-on gRNA with non-targeting targeting sequence (20bp determining binding specificity) might be a better choice for universal dCas9 derepression.</li><li>You could also think about doing anti-crRNAs for specific derepression. </li></ul><p>10/15/14</p><p>Objective: look for (in)stability of decoy arrays</p><ul><li>Is the mediocre increase in derepression of 12x decoy GFP1 relative to 6x due to the long repeats getting broken down from repeated collisions of dCas9 with DNAP? Charlie has seen this before with the Lac repressor</li><li>DNA was miniprepped from the overnight cultures that made yesterdays’ flow data and EcoRI/PstI digested.</li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Oct 15, 2014 4:33:14 PM.jpg" src="images/image20.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>Left ot right</li></ul><ul><li>ladder</li><li>G0</li><li>G1</li><li>G6</li><li>G12</li><li>C0</li><li>C1</li><li>C6</li><li>C12</li></ul><ul><li>C6’s band is faint and smeary, but C12 looks nice? Reason for that is unclear, but all of the G bands’ clear bandiness and C12’s suggests that, for this round at least, the arrays are stable.</li></ul> |
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