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| <div class="hero-image"> | | <div class="hero-image"> |
| <div class="hero-text"> | | <div class="hero-text"> |
− | <h1>Notebook</h1> | + | <h1> Notebook </h1> |
− | <p>A detailed description of what we did in the lab everyday!</p>
| + | |
| </div> | | </div> |
| </div> | | </div> |
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| <h3>Friday (06/02/17)</h3> | | <h3>Friday (06/02/17)</h3> |
| <p>On the second day, the students began learning and overviewing basic lab | | <p>On the second day, the students began learning and overviewing basic lab |
− | techniques. These techniques include: conversion factors, serial dilutions, | + | techniques. These techniques include conversion factors, serial dilutions, |
| micro pipetting, and creating and pouring plates of LB agar. Under the | | micro pipetting, and creating and pouring plates of LB agar. Under the |
| tender supervision of knowledgable mentors, the students learned to accurately | | tender supervision of knowledgable mentors, the students learned to accurately |
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| <h2>Week 2: June 5-9</h2> | | <h2>Week 2: June 5-9</h2> |
| <h3>Monday (06/05/17) and Tuesday (06/06/17)</h3> | | <h3>Monday (06/05/17) and Tuesday (06/06/17)</h3> |
− | <p>The first two days of this week were spent on Wright Patt Air Force Base | + | <p>The first two days of this week were spent on Wright Patterson Air Force Base |
| (WPAFB). The team members toured the two labs that they would be | | (WPAFB). The team members toured the two labs that they would be |
| working in and received safety training in both. The student researchers | | working in and received safety training in both. The student researchers |
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| probability that the E. coli absorbed the plasmid. The tired but excited young | | probability that the E. coli absorbed the plasmid. The tired but excited young |
| researchers went home with fingers crossed.</p> | | researchers went home with fingers crossed.</p> |
− | <p><b>Packaging: </b>The lab members attended a review meeting for the Materials Lab. This is where people gave mini presentations on how their projects were going and what problems they have run into. The Packaging members and Chia considered the advantages of using different surface proteins in order to package E. coli and decided on curli. The four Packaging students searched the iGEM site for parts (cellulose binding domains, Csg A, etc) was discussed. | + | <p><b>Packaging: </b>The lab members attended a review meeting for the Materials Lab. This is where people gave mini presentations on how their projects were going and what problems they have run into. The Packaging members and Chia considered the advantages of using different surface proteins in order to package E. coli and decided on curli. The four Packaging students searched the iGEM site for parts (cellulose binding domains, CsgA, etc) was discussed. |
| </p> | | </p> |
| | | |
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| to confirm that E.coli JM109 had absorbed the | | to confirm that E.coli JM109 had absorbed the |
| plasmid PRhl_GFPa1</p> | | plasmid PRhl_GFPa1</p> |
− | <p><b>Packaging: </b> The Packaging team clarified the goals of their project by deciding to attach a cellulose binding domain to the Csg A portion of a curli protein on E. coli bacteria. In the lab, the team worked with producing agarose gel, diluting a buffer, and using these components to run gel electrophoresis on DNA samples. Additionally, the team poured LB agar plates. | + | <p><b>Packaging: </b> The Packaging team clarified the goals of their project by deciding to attach a cellulose binding domain to the CsgA portion of a curli protein on E. coli bacteria. In the lab, the team worked with producing agarose gel, diluting a buffer, and using these components to run gel electrophoresis on DNA samples. Additionally, the team poured LB agar plates. |
| | | |
| </p> | | </p> |
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| | | |
| <tr> | | <tr> |
− | <td>Tina</td> | + | <th>Tina</th> |
| <td>1.099</td> | | <td>1.099</td> |
| <td>0.620</td> | | <td>0.620</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Hayley</td> | + | <th>Hayley</th> |
| <td>1.594</td> | | <td>1.594</td> |
| <td>0.867</td> | | <td>0.867</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Annie</td> | + | <th>Annie</th> |
| <td>1.366</td> | | <td>1.366</td> |
| <td>0.776</td> | | <td>0.776</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Jason</td> | + | <th>Jason</th> |
| <td>1.599</td> | | <td>1.599</td> |
| <td>0.886</td> | | <td>0.886</td> |
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| </tr> | | </tr> |
| </table> | | </table> |
− | | + | <p class="caption2">Table 1: Nanodrop results</p> |
| + | <img src="https://static.igem.org/mediawiki/2017/8/82/US_AFRL_CarrollHS_Labnotes1.png" style="width: 80%; margin-left: 10%; margin-right: 10%"> |
| <p><b>Packaging: </b>The Packaging team prepared DNA samples and analyzed the samples with the Nanodrop device. Later, the team used restriction enzymes to slice open the DNA and then ran the samples through gel electrophoresis. The project idea continued to progress with the team finding parts form the iGEM inventory and researching DNA sequences of the various parts. | | <p><b>Packaging: </b>The Packaging team prepared DNA samples and analyzed the samples with the Nanodrop device. Later, the team used restriction enzymes to slice open the DNA and then ran the samples through gel electrophoresis. The project idea continued to progress with the team finding parts form the iGEM inventory and researching DNA sequences of the various parts. |
| | | |
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| confirming that the Colony + 3OC12 glowed green. The ecstatic students celebrated | | confirming that the Colony + 3OC12 glowed green. The ecstatic students celebrated |
| as anticipated results showed through the light.</p> | | as anticipated results showed through the light.</p> |
| + | |
| + | <table> |
| + | <tr> |
| + | <th></th> |
| + | <th>Colony+3OC12</th> |
| + | <th>Colony+DMSO</th> |
| + | <th>LB+3OC12</th> |
| + | <th>LB+DMSO</th> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Dallas</th> |
| + | <td>0.929</td> |
| + | <td>1.048</td> |
| + | <td>0.039</td> |
| + | <td>0.037</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Tina</th> |
| + | <td>1.075</td> |
| + | <td>1.084</td> |
| + | <td>0.048</td> |
| + | <td>524.53</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Hayley</th> |
| + | <td>1.094</td> |
| + | <td>1.079</td> |
| + | <td>0.042</td> |
| + | <td>0.041</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Annie</th> |
| + | <td>1.096</td> |
| + | <td>1.079</td> |
| + | <td>0.044</td> |
| + | <td>1.227</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Jason</th> |
| + | <td>1.229</td> |
| + | <td>1.213</td> |
| + | <td>0.055</td> |
| + | <td>1.227</td> |
| + | </tr> |
| + | </table> |
| + | <p class="caption2">Table 2: Optical Density of PLCD-GFPa1</p> |
| + | <br> |
| + | <table> |
| + | <tr> |
| + | <th></th> |
| + | <th>Colony+3OC12</th> |
| + | <th>Colony+DMSO</th> |
| + | <th>LB+3OC12</th> |
| + | <th>LB+DMSO</th> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Dallas</th> |
| + | <td>2359.7</td> |
| + | <td>889.14</td> |
| + | <td>594.84</td> |
| + | <td>645.10</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Tina</th> |
| + | <td>35454</td> |
| + | <td>841.14</td> |
| + | <td>524.53</td> |
| + | <td>1074.5</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Hayley</th> |
| + | <td>27733</td> |
| + | <td>844.70</td> |
| + | <td>508.87</td> |
| + | <td>513.41</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Annie</th> |
| + | <td>28791</td> |
| + | <td>837.42</td> |
| + | <td>906.98</td> |
| + | <td>950.71</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Jason</th> |
| + | <td>35088</td> |
| + | <td>958.79</td> |
| + | <td>673.31</td> |
| + | <td>950.71</td> |
| + | </tr> |
| + | </table> |
| + | <p class="caption2">Table 3: Fluorescence of LCD-GFPA1+3OC12</p> |
| + | <img src="https://static.igem.org/mediawiki/2017/e/e2/US_AFRL_CarrollHS_Labnotes5.png" style="width: 80%; margin-left: 10%; margin-right: 10%"> |
| + | |
| <p>In the afternoon, the students were back at work again preparing a mini-prep on the | | <p>In the afternoon, the students were back at work again preparing a mini-prep on the |
| PLCD-GFPa1 (the plate streaked on Monday). The remaining DNA containing the | | PLCD-GFPa1 (the plate streaked on Monday). The remaining DNA containing the |
| GFP was then placed in the -20o C freezer for storage. Having much more | | GFP was then placed in the -20o C freezer for storage. Having much more |
− | experience the second time doing the mini-prep, the trailblazing students had a | + | experience the second time doing the mini-prep, the students had a |
| much better grasp and spent less time with more accuracy.</p> | | much better grasp and spent less time with more accuracy.</p> |
| <table> | | <table> |
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| | | |
| <tr> | | <tr> |
− | <td>Dallas</td> | + | <th>Dallas</th> |
| <td>2.106</td> | | <td>2.106</td> |
| <td>1.273</td> | | <td>1.273</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Tina</td> | + | <th>Tina</th> |
| <td>0.927</td> | | <td>0.927</td> |
| <td>0.456</td> | | <td>0.456</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Hayley</td> | + | <th>Hayley</th> |
| <td>0.723</td> | | <td>0.723</td> |
| <td>0.766</td> | | <td>0.766</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Annie</td> | + | <th>Annie</th> |
| <td>0.705</td> | | <td>0.705</td> |
| <td>0.357</td> | | <td>0.357</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Jason</td> | + | <th>Jason</th> |
| <td>0.561</td> | | <td>0.561</td> |
| <td>0.276</td> | | <td>0.276</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Blank</td> | + | <th>Blank</th> |
| <td>-0.50</td> | | <td>-0.50</td> |
| <td>-0.048</td> | | <td>-0.048</td> |
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| </tr> | | </tr> |
| </table> | | </table> |
| + | <p class="caption2">Table 4: Nanodrop results</p> |
| | | |
| | | |
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| | | |
| <tr> | | <tr> |
− | <td>Dallas- PLas1s</td> | + | <th>Dallas- PLas1s</th> |
| <td>2.429</td> | | <td>2.429</td> |
| <td>1.275</td> | | <td>1.275</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Dallas- PLas2</td> | + | <th>Dallas- PLas2</th> |
| <td>1.363</td> | | <td>1.363</td> |
| <td>0.701</td> | | <td>0.701</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Tina- PLas1</td> | + | <th>Tina- PLas1</th> |
| <td>2.059</td> | | <td>2.059</td> |
| <td>1.065</td> | | <td>1.065</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Tina- PLas2</td> | + | <th>Tina- PLas2</th> |
| <td>1.883</td> | | <td>1.883</td> |
| <td>0.990</td> | | <td>0.990</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Hayley- PLas1</td> | + | <th>Hayley- PLas1</th> |
| <td>0.994</td> | | <td>0.994</td> |
| <td>0.516</td> | | <td>0.516</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Hayley- PLas2</td> | + | <th>Hayley- PLas2</th> |
| <td>1.691</td> | | <td>1.691</td> |
| <td>0.883</td> | | <td>0.883</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Annie- PLas1</td> | + | <th>Annie- PLas1</th> |
| <td>2.186</td> | | <td>2.186</td> |
| <td>1.149</td> | | <td>1.149</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Annie- PLas2</td> | + | <th>Annie- PLas2</th> |
| <td>1.488</td> | | <td>1.488</td> |
| <td>0.796</td> | | <td>0.796</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Jason- PLas1</td> | + | <th>Jason- PLas1</th> |
| <td>1.3494</td> | | <td>1.3494</td> |
| <td>0.723</td> | | <td>0.723</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Jason- PLas2</td> | + | <th>Jason- PLas2</th> |
| <td>2.160</td> | | <td>2.160</td> |
| <td>1.104</td> | | <td>1.104</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Blank</td> | + | <th>Blank</th> |
| <td>0.024</td> | | <td>0.024</td> |
| <td>0.026</td> | | <td>0.026</td> |
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| | | |
| </table> | | </table> |
| + | <p class="caption2">Table 5: Nanodrop results</p> |
| | | |
| | | |
− | <p><b>Packaging: </b> The RX team performed a Restriction Enzyme Digest on the pBAD containing Csg A DNA and then purified the cut pBAD using a clean-up kit. The team then analyzed the results of the clean-up using the Nanodrop. When the Nanodrop confirmed the presence of DNA, the team created a Master Mix and ran PCR on the Csg A DNA. Upon the completion of the PCR, the products were purified using a purification protocol. | + | <p><b>Packaging: </b> The RX team performed a Restriction Enzyme Digest on the pBAD containing CsgA DNA and then purified the cut pBAD using a clean-up kit. The team then analyzed the results of the clean-up using the Nanodrop. When the Nanodrop confirmed the presence of DNA, the team created a Master Mix and ran PCR on the CsgA DNA. Upon the completion of the PCR, the products were purified using a purification protocol. |
| </p> | | </p> |
| | | |
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| treat. All 12 days of hard work deserved a celebration and the students and mentors | | treat. All 12 days of hard work deserved a celebration and the students and mentors |
| bonded over frozen custard and ice cream.</p> | | bonded over frozen custard and ice cream.</p> |
| + | <img src="https://static.igem.org/mediawiki/2017/4/40/US_AFRL_CarrollHS_Labnotes2.png" style="width: 80%; margin-left: 10%; margin-right: 10%"> |
| + | |
| </div> | | </div> |
− | <p><b>Packaging: </b>The RX team prepared agarose gel and 1X TAE Buffer in order to run the Csg A DNA PCR products on a gel. Images were taken of the results of the gel electrophoresis in order to confirm the presence of the correct DNA. Colonies began to appear from the transformation using the dCBD DNA. | + | <p><b>Packaging: </b>The RX team prepared agarose gel and 1X TAE Buffer in order to run the CsgA DNA PCR products on a gel. Images were taken of the results of the gel electrophoresis in order to confirm the presence of the correct DNA. Colonies began to appear from the transformation using the dCBD DNA. |
| | | |
| </p> | | </p> |
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| | | |
| <tr> | | <tr> |
− | <td>Dallas</td> | + | <th>Dallas</th> |
| <td>3.099</td> | | <td>3.099</td> |
| <td>1.700</td> | | <td>1.700</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Tina</td> | + | <th>Tina</th> |
| <td>2.355</td> | | <td>2.355</td> |
| <td>1.357</td> | | <td>1.357</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Jason</td> | + | <th>Jason</th> |
| <td>1.756</td> | | <td>1.756</td> |
| <td>1.005</td> | | <td>1.005</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Hayley</td> | + | <th>Hayley</th> |
| <td>2.3</td> | | <td>2.3</td> |
| <td>1.3</td> | | <td>1.3</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Annie</td> | + | <th>Annie</th> |
| <td>3.793</td> | | <td>3.793</td> |
| <td>2.050</td> | | <td>2.050</td> |
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| | | |
| <tr> | | <tr> |
− | <td>Blank</td> | + | <th>Blank</th> |
| <td>0.013</td> | | <td>0.013</td> |
| <td>0.058</td> | | <td>0.058</td> |
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| </tr> | | </tr> |
| </table> | | </table> |
| + | <p class="caption2">Table 6: Nanodrop results</p> |
| + | <img src="https://static.igem.org/mediawiki/2017/8/8e/US_AFRL_CarrollHS_Labnotes6.png" style="width: 80%; margin-left: 10%; margin-right: 10%"> |
| | | |
| | | |
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| | | |
| | | |
− | <h3>Thursday (6/22/17)</h3> | + | <h3>Thursday (06/22/17)</h3> |
| <p><b>Sense/Respond: </b>The day began with another PCR. Today, the students were ready to | | <p><b>Sense/Respond: </b>The day began with another PCR. Today, the students were ready to |
| completely do the PCR by themselves with little guidance from the mentors. | | completely do the PCR by themselves with little guidance from the mentors. |
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| | | |
| | | |
− | <h3>Friday (6/23/17)</h3> | + | <h3>Friday (06/23/17)</h3> |
| <p><b>Sense/Respond: </b>The day began bright and early with two PCR digests. This time a PCR | | <p><b>Sense/Respond: </b>The day began bright and early with two PCR digests. This time a PCR |
| Vector Digest was conducted on the same from yesterday and a PCR GFP | | Vector Digest was conducted on the same from yesterday and a PCR GFP |
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| connect again. During the students' lunch break, another meeting was held with teacher | | connect again. During the students' lunch break, another meeting was held with teacher |
| leaders who wanted to know information about progress and future proceedings.</p> | | leaders who wanted to know information about progress and future proceedings.</p> |
| + | <table> |
| + | |
| + | <tr> |
| + | <th></th> |
| + | <th>260</th> |
| + | <th>280</th> |
| + | <th>Ratio 260/280</th> |
| + | <th>ng/μl</th> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Dallas</th> |
| + | <td>2.106</td> |
| + | <td>1.273</td> |
| + | <td>1.65</td> |
| + | <td>105.3</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Tina</th> |
| + | <td>0.927</td> |
| + | <td>0.456</td> |
| + | <td>2.03</td> |
| + | <td>46.4</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Jason</th> |
| + | <td>1.188</td> |
| + | <td>0.719</td> |
| + | <td>1.65</td> |
| + | <td>59.6</td> |
| + | </tr> |
| + | </table> |
| + | <p class="caption2">Table 7: Nanodrop results</p> |
| <br> | | <br> |
| + | <table> |
| + | <tr> |
| + | <th>DNA</th> |
| + | <td>10μl</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>10x Buffer</th> |
| + | <td>1.5μl</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Kpn1-HF</th> |
| + | <td>1μl</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>BsrG1-HF</th> |
| + | <td>1μl</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>H2O</th> |
| + | <td>1.5μl</td> |
| + | </tr> |
| + | </table> |
| + | <p class="caption2">Table 8: GFP Digest (15μl)</p> |
| + | <br> |
| + | <table> |
| + | <tr> |
| + | <th>DNA</th> |
| + | <td>8.5μl</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>10x Buffer</th> |
| + | <td>1.5μl</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Kpn1-HF</th> |
| + | <td>1μl</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>BsrG1-HF</th> |
| + | <td>1μl</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>H2O</th> |
| + | <td>3μl</td> |
| + | </tr> |
| + | </table> |
| + | <p class="caption2">Table 9: Vector Digest (15μl)</p> |
| + | |
| <p><b>Packaging: </b>The RXers extracted the TOPO plasmids from the E. coli and ran a restriction digest. To ensure the correct dCBD length, both cut and uncut DNA was run on a gel. The results of the gel determined the possibility of sending in plasmids with dCBD for sequencing on Monday. | | <p><b>Packaging: </b>The RXers extracted the TOPO plasmids from the E. coli and ran a restriction digest. To ensure the correct dCBD length, both cut and uncut DNA was run on a gel. The results of the gel determined the possibility of sending in plasmids with dCBD for sequencing on Monday. |
| </p> | | </p> |
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| | | |
| <h2>Week 5: June 26-30</h2> | | <h2>Week 5: June 26-30</h2> |
− | <h3>Monday (6/26/17)</h3> | + | <h3>Monday (06/26/17)</h3> |
| <p><b>Sense/Respond: </b>The students began another week of hard work. They made a gel to run | | <p><b>Sense/Respond: </b>The students began another week of hard work. They made a gel to run |
| their previous Plasmid Digest. Next, they looked at the gel under the blue | | their previous Plasmid Digest. Next, they looked at the gel under the blue |
Line 553: |
Line 754: |
| very uneven graph, but with the desired numerals. Afterwards, the mentors discussed | | very uneven graph, but with the desired numerals. Afterwards, the mentors discussed |
| and determined to still do a legation the next day and see the results.</p> | | and determined to still do a legation the next day and see the results.</p> |
| + | |
| + | <table> |
| + | |
| + | <tr> |
| + | <th></th> |
| + | <th>260</th> |
| + | <th>280</th> |
| + | <th>Ratio 260/280</th> |
| + | <th>ng/μl</th> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>TD+AB+JD</th> |
| + | <td>0.046</td> |
| + | <td>0.027</td> |
| + | <td>1.71</td> |
| + | <td>2.3</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>DM+HJ</th> |
| + | <td>0.227</td> |
| + | <td>0.183</td> |
| + | <td>1.24</td> |
| + | <td>11.3</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Blank</th> |
| + | <td>0.015</td> |
| + | <td>-0.005</td> |
| + | <td>-2.86</td> |
| + | <td>0.7</td> |
| + | </tr> |
| + | </table> |
| + | <p class="caption2">Table 10: Nanodrop results</p> |
| + | |
| <p><b>Packaging: </b>The RX side of the team did a nanodrop on the uncut dCBD TOPO plasmid and found the concentration of each sample. Water was added to each samples to create an equal concentration at 50 nanograms per microliter and 500 ng in all. We then sent our samples to be sequenced. | | <p><b>Packaging: </b>The RX side of the team did a nanodrop on the uncut dCBD TOPO plasmid and found the concentration of each sample. Water was added to each samples to create an equal concentration at 50 nanograms per microliter and 500 ng in all. We then sent our samples to be sequenced. |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Tuesday (6/27/17)</h3> | + | <h3>Tuesday (06/27/17)</h3> |
| <p><b>Sense/Respond: </b>Today was the student's first ligation. The goal was to put the sticky ends | | <p><b>Sense/Respond: </b>Today was the student's first ligation. The goal was to put the sticky ends |
| back together to form a complete plasmid. A lot of math was done to | | back together to form a complete plasmid. A lot of math was done to |
Line 573: |
Line 811: |
| | | |
| | | |
− | <h3>Wednesday (6/28/17)</h3> | + | <h3>Wednesday (06/28/17)</h3> |
| <p><b>Sense/Respond: </b>The students began the day with a new PCR. After examining the plates from | | <p><b>Sense/Respond: </b>The students began the day with a new PCR. After examining the plates from |
| yesterday, it was determined that there indeed was green fluorescence on the | | yesterday, it was determined that there indeed was green fluorescence on the |
Line 599: |
Line 837: |
| | | |
| | | |
− | <h3>Thursday (6/29/17)</h3> | + | <h3>Thursday (06/29/17)</h3> |
| <p><b>Sense/Respond: </b>Today the students began to mini-prep the samples from the previous day. | | <p><b>Sense/Respond: </b>Today the students began to mini-prep the samples from the previous day. |
| Right from the start, the students noticed fluorescent green coming from | | Right from the start, the students noticed fluorescent green coming from |
Line 609: |
Line 847: |
| of DNA. Everyone cheered as they realized that the "response" component of the | | of DNA. Everyone cheered as they realized that the "response" component of the |
| project was successful.</p> | | project was successful.</p> |
− | <p><b>Packaging: </b> Sequencing results for dCBD and Csg A DNA were reviewed using the program Blast. Each of the six dCBD in TOPO plasmid samples contained no errors on either the SP6 or the T7 primer sequences; however, some samples contained the gene in the reverse orientation. Chia's Csg A in TOPO plasmid samples were also entirely correct. | + | <p><b>Packaging: </b> Sequencing results for dCBD and CsgA DNA were reviewed using the program Blast. Each of the six dCBD in TOPO plasmid samples contained no errors on either the SP6 or the T7 primer sequences; however, some samples contained the gene in the reverse orientation. Chia's CsgA in TOPO plasmid samples were also entirely correct. |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Friday (6/30/17)</h3> | + | <h3>Friday (06/30/17)</h3> |
| <p><b>Sense/Respond: </b>Since all the lab work is currently done until the DNA sample is sent to Ohio State | | <p><b>Sense/Respond: </b>Since all the lab work is currently done until the DNA sample is sent to Ohio State |
| University for DNA sequencing, the students had the whole day to make website | | University for DNA sequencing, the students had the whole day to make website |
| improvements and wiki renovations. New logos were also created as well as | | improvements and wiki renovations. New logos were also created as well as |
| graphics. The students will continue to add content to the wiki weekly.</p> | | graphics. The students will continue to add content to the wiki weekly.</p> |
− | <p><b>Packaging: </b>The RXers learned how to design a restriction digest and created the digests for Csg A, dCBD, and pBAD samples. Later, the team performed the three digests, each with one sample, and left the samples to incubate in a heat block for several hours. In the afternoon, the whole iGEM team met with mentors to review the projects. | + | |
| + | <table> |
| + | <tr> |
| + | <th></th> |
| + | <th>260</th> |
| + | <th>280</th> |
| + | <th>Ratio 260/280</th> |
| + | <th>ng/μl</th> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>1</th> |
| + | <td>2.899</td> |
| + | <td>1.657</td> |
| + | <td>1.75</td> |
| + | <td>145.0</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>2</th> |
| + | <td>2.364</td> |
| + | <td>1.268</td> |
| + | <td>1.86</td> |
| + | <td>118.2</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>3</th> |
| + | <td>1.948</td> |
| + | <td>1.030</td> |
| + | <td>1.89</td> |
| + | <td>97.4</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>4</th> |
| + | <td>2.152</td> |
| + | <td>1.143</td> |
| + | <td>1.88</td> |
| + | <td>107.6</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>5</th> |
| + | <td>2.071</td> |
| + | <td>1.123</td> |
| + | <td>1.84</td> |
| + | <td>103.5</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>6</th> |
| + | <td>2.421</td> |
| + | <td>1.318</td> |
| + | <td>1.84</td> |
| + | <td>121.1</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>7</th> |
| + | <td>2.072</td> |
| + | <td>1.103</td> |
| + | <td>1.88</td> |
| + | <td>103.6</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>8</th> |
| + | <td>1.764</td> |
| + | <td>0.963</td> |
| + | <td>1.83</td> |
| + | <td>88.3</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>9</th> |
| + | <td>2.287</td> |
| + | <td>1.228</td> |
| + | <td>1.86</td> |
| + | <td>114.4</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>11</th> |
| + | <td>6.402</td> |
| + | <td>3.492</td> |
| + | <td>1.83</td> |
| + | <td>320.1</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <td>Blank</td> |
| + | <td>0.0003</td> |
| + | <td>0.029</td> |
| + | <td>0.11</td> |
| + | <td>0.2</td> |
| + | </tr> |
| + | </table> |
| + | <p class="caption2">Table 11: Nanodrop results</p> |
| + | |
| + | <p><b>Packaging: </b>The RXers learned how to design a restriction digest and created the digests for CsgA, dCBD, and pBAD samples. Later, the team performed the three digests, each with one sample, and left the samples to incubate in a heat block for several hours. In the afternoon, the whole iGEM team met with mentors to review the projects. |
| </p> | | </p> |
| | | |
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| <div class="text2"> | | <div class="text2"> |
| <h2>Week 6: July 5-6</h2> | | <h2>Week 6: July 5-6</h2> |
− | <h3>Wednesday (7/5/17)</h3> | + | <h3>Wednesday (07/05/17)</h3> |
| <p>“Ring, ring, ring!” came from the laptop as the students huddled around | | <p>“Ring, ring, ring!” came from the laptop as the students huddled around |
| to receive a Skype call from the Singapore iGem team. They were greeted | | to receive a Skype call from the Singapore iGem team. They were greeted |
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| | | |
| | | |
− | <h3>Thursday (7/6/16)</h3> | + | <h3>Thursday (07/06/16)</h3> |
| <p><b>Sense/Respond: </b>The next day, the group decided to do the InterLab in order to be | | <p><b>Sense/Respond: </b>The next day, the group decided to do the InterLab in order to be |
| considered for medal criteria. They sifted through the kit and found many | | considered for medal criteria. They sifted through the kit and found many |
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| students had the wait since the lab was going under routine inspection. The students | | students had the wait since the lab was going under routine inspection. The students |
| spent the rest of the day designing more graphics and made wiki edits. <br> | | spent the rest of the day designing more graphics and made wiki edits. <br> |
− | <p><b>Packaging: </b>The morning began with running a gel on the results of the gel extraction. While the gel was solidifying, the team determined the concentrations of the samples with the Nanodrop. After the gel confirmed the pBAD, csgA fragment, and dCBD fragment, the RXers performed a ligation in order to clone the csgA gene into the pBAD plasmid.</p> | + | <p><b>Packaging: </b>The morning began with running a gel on the results of the gel extraction. While the gel was solidifying, the team determined the concentrations of the samples with the Nanodrop. After the gel confirmed the pBAD, <i>csgA</i> fragment, and dCBD fragment, the RXers performed a ligation in order to clone the <i>csgA</i> gene into the pBAD plasmid.</p> |
| + | <table> |
| + | <tr> |
| + | <th>Gel Extraction Results</th> |
| + | <th>Concentration (ng/uL)</th> |
| + | <th>260/280</th> |
| + | <th>260/230</th> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>pBAD 1 (w/ Ncol and Kpnl)</th> |
| + | <td>6.2</td> |
| + | <td>1.64</td> |
| + | <td>0.02</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>pBAD 2 (w/ Ncol and Kpnl)</th> |
| + | <td>7.4</td> |
| + | <td>2.11</td> |
| + | <td>0.01</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>CsgA 1 (w/ Ncol and Kpnl)</th> |
| + | <td>17.0</td> |
| + | <td>1.88</td> |
| + | <td>0.03</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>CsgA 2 (w/ Ncol and Kpnl)</th> |
| + | <td>21.5</td> |
| + | <td>1.79</td> |
| + | <td>0.05</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>dCBD 1 (w/ Ncol and HindIII)</th> |
| + | <td>8.0</td> |
| + | <td>1.75</td> |
| + | <td>0.02</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>dCBD 2 (w/ Ncol and HindIII)</th> |
| + | <td>8.8</td> |
| + | <td>1.98</td> |
| + | <td>0.02</td> |
| + | </tr> |
| + | </table> |
| + | <p class="caption2">Table 12: Nanodrop results</p> |
| + | |
| <p>In the | | <p>In the |
| afternoon, the whole team got together with mentors to meet at the Air Force | | afternoon, the whole team got together with mentors to meet at the Air Force |
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| | | |
| | | |
− | <h3>Friday (7/7/16)</h3> | + | <h3>Friday (07/07/16)</h3> |
| <p><b>Packaging: </b>To finish the week, the RXers transformed pBAD-CsgA plasmids into DH5-alpha competent cells and patched the samples onto LB agar plates with ampicillin. Additionally, the team made and poured a multitude of LB agar plates with ampicillin. The entire team met at UES in the afternoon to discuss the Wiki.</p> | | <p><b>Packaging: </b>To finish the week, the RXers transformed pBAD-CsgA plasmids into DH5-alpha competent cells and patched the samples onto LB agar plates with ampicillin. Additionally, the team made and poured a multitude of LB agar plates with ampicillin. The entire team met at UES in the afternoon to discuss the Wiki.</p> |
| <br> | | <br> |
Line 666: |
Line 1,056: |
| <h2>Week 7: July 10-14</h2> | | <h2>Week 7: July 10-14</h2> |
| | | |
− | <h3>Monday (7/10/17)</h3> | + | <h3>Monday (07/10/17)</h3> |
| <p><b>Sense/Respond: </b>At 9 AM, the students went to their weekly lab meeting. Interns from their | | <p><b>Sense/Respond: </b>At 9 AM, the students went to their weekly lab meeting. Interns from their |
| department always have a weekly meeting where they share ideas and summer | | department always have a weekly meeting where they share ideas and summer |
Line 679: |
Line 1,069: |
| | | |
| | | |
− | <h3>Tuesday (7/11/17)</h3> | + | <h3>Tuesday (07/11/17)</h3> |
| <p><b>Sense/Respond: </b>The student researchers began the InterLab the next morning. They split up in 2 | | <p><b>Sense/Respond: </b>The student researchers began the InterLab the next morning. They split up in 2 |
| groups while one group worked with the competent cells kit and completed Day | | groups while one group worked with the competent cells kit and completed Day |
Line 688: |
Line 1,078: |
| slideshow for the website.</p> | | slideshow for the website.</p> |
| <p><b>Packaging: </b>The RXers spent the morning miniprepping twenty-one samples of DH5 alpha competent cells with pBAD-CsgA. Around five milliliters of each sample from the culture tubes of the previous day were pelleted for miniprepping and one milliliter was reserved for storage. | | <p><b>Packaging: </b>The RXers spent the morning miniprepping twenty-one samples of DH5 alpha competent cells with pBAD-CsgA. Around five milliliters of each sample from the culture tubes of the previous day were pelleted for miniprepping and one milliliter was reserved for storage. |
− | Wednesday</p>
| |
− | <p>The Nanodrop was used to check the concentration of several samples of the miniprep results. Then, a restriction digest was performed on the miniprepped pBAD-CsgA plasmids with NcoI and KpnI. Digested and undigested pBAD-CsgA samples were run on a gel in order to confirm the lengths of the fragments. The samples with the clearest CsgA bands were chosen to have a second restriction digest with KpnI and HindIII performed on them to prepare for cloning the dCBD gene into the plasmid.
| |
| | | |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Wednesday (7/12/17)</h3> | + | <h3>Wednesday (07/12/17)</h3> |
| <p><b>Sense/Respond: </b>The next morning, the young students attended a lecture taught by biologists | | <p><b>Sense/Respond: </b>The next morning, the young students attended a lecture taught by biologists |
| from the Purdue University. It was very interesting and higher level, and the | | from the Purdue University. It was very interesting and higher level, and the |
Line 704: |
Line 1,092: |
| amount of colonies in order to proceed. | | amount of colonies in order to proceed. |
| </p> | | </p> |
| + | <p><b>Packaging: </b>The Nanodrop was used to check the concentration of several samples of the miniprep results. Then, a restriction digest was performed on the miniprepped pBAD-CsgA plasmids with NcoI and KpnI. Digested and undigested pBAD-CsgA samples were run on a gel in order to confirm the lengths of the fragments. The samples with the clearest CsgA bands were chosen to have a second restriction digest with KpnI and HindIII performed on them to prepare for cloning the dCBD gene into the plasmid. |
| + | </p> |
| + | <table> |
| + | <tr> |
| + | <th>Spot Samples of |
| + | pBAD-CsgA</th> |
| + | <th>Concentration (ng/uL)</th> |
| + | <th>260/280</th> |
| + | <th>260/230</th> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>AS2 pBAD-CsgA</th> |
| + | <td>100.0</td> |
| + | <td>1.90</td> |
| + | <td>1.91</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>AS6 pBAD-CsgA</th> |
| + | <td>105.2</td> |
| + | <td>1.89</td> |
| + | <td>1.93</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>AP1 pBAD-CsgA</th> |
| + | <td>102.1</td> |
| + | <td>1.94</td> |
| + | <td>2.00</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>AP2 pBAD-CsgA</th> |
| + | <td>106.0</td> |
| + | <td>1.88</td> |
| + | <td>1.71</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>MH1 pBAD-CsgA</th> |
| + | <td>109.7</td> |
| + | <td>1.94</td> |
| + | <td>1.92</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>MH7 pBAD CsgA</th> |
| + | <td>64.8</td> |
| + | <td>1.92</td> |
| + | <td>2.23</td> |
| + | </tr> |
| + | |
| + | |
| + | </table> |
| + | <p class="caption2">Table 13: Nanodrop results</p> |
| + | |
| + | <br> |
| + | <table> |
| + | <tr> |
| + | <th>Chosen Samples of |
| + | pBAD-CsgA</th> |
| + | <th>Concentration (ng/uL)</th> |
| + | <th>260/280</th> |
| + | <th>260/230</th> |
| + | </tr> |
| + | |
| + | |
| + | <tr> |
| + | <th>AS3 pBAD-CsgA</th> |
| + | <td>129.2</td> |
| + | <td>1.96</td> |
| + | <td>1.98</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>MH2 pBAD-CsgA</th> |
| + | <td>83.1</td> |
| + | <td>1.94</td> |
| + | <td>1.89</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>MH3 pBAD-CsgA</th> |
| + | <td>114.3</td> |
| + | <td>1.96</td> |
| + | <td>1.98</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>MH5 pBAD-CsgA</th> |
| + | <td>93.4</td> |
| + | <td>1.94</td> |
| + | <td>1.97</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>MH6 pBAD-CsgA</th> |
| + | <td>108.0</td> |
| + | <td>1.96</td> |
| + | <td>2.06</td> |
| + | </tr> |
| + | </table> |
| + | <p class="caption2">Table 14: Nanodrop results</p> |
| | | |
| | | |
− | <h3>Thursday (7/13/17)</h3> | + | <h3>Thursday (07/13/17)</h3> |
| <p><b>Sense/Respond: </b>Continuing the next morning, the students immediately continued the Interlab. | | <p><b>Sense/Respond: </b>Continuing the next morning, the students immediately continued the Interlab. |
| Today they focused on completing the OD600 and Serial Dilution part of the | | Today they focused on completing the OD600 and Serial Dilution part of the |
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| | | |
| | | |
− | <h3>Friday (7/14/17)</h3> | + | <h3>Friday (07/14/17)</h3> |
| <p>-“I brought doughnuts!” exclaimed Andrea as she walked in the door in the | | <p>-“I brought doughnuts!” exclaimed Andrea as she walked in the door in the |
| morning. The group had planned a Skype meeting with the U.S. Navy and U.S. | | morning. The group had planned a Skype meeting with the U.S. Navy and U.S. |
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| <div class="text2"> | | <div class="text2"> |
| <h2>Week 8: June 17-21</h2> | | <h2>Week 8: June 17-21</h2> |
− | <h3>Monday (7/17/17)</h3> | + | <h3>Monday (07/17/17)</h3> |
| <p><b>Sense/Respond: </b>The student sat around nervously waiting for | | <p><b>Sense/Respond: </b>The student sat around nervously waiting for |
| their presentation to begin. In a few minutes | | their presentation to begin. In a few minutes |
Line 749: |
Line 1,241: |
| up to date so the young researchers dedicated the rest of | | up to date so the young researchers dedicated the rest of |
| the day for wiki organization and miscellaneous tasks.</p> | | the day for wiki organization and miscellaneous tasks.</p> |
− | <p><b>Packaging: </b>GoTaq Flexi PCR protocol was performed on 28 colonies of the transformants. After the PCR was completed, a gel was run in order to verify which of the chosen colonies contained both the csgA and the dCBD gene fragments. Based off of the PCR products, 6 of the 28 chosen colonies were grown on LB with ampicillin plates and broth in an incubator overnight. | + | <p><b>Packaging: </b>GoTaq Flexi PCR protocol was performed on 28 colonies of the transformants. After the PCR was completed, a gel was run in order to verify which of the chosen colonies contained both the <i>csgA</i> and the dCBD gene fragments. Based off of the PCR products, 6 of the 28 chosen colonies were grown on LB with ampicillin plates and broth in an incubator overnight. |
− | Tuesday</p>
| + | |
− | <p>The six liquid culture samples were taken out of the incubator and 3ml from each tube was miniprepped for four restriction digests. The Nanodrop was used to check the concentration of DNA in each sample. After the digests were performed using Nco1, Kpn1, HindII, and Nco1 and HindIII, a gel was run to analyze the results. The results obtained were unexpected, but several samples, 14 and 32, were chosen as likely candidates.
| + | |
| | | |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Tuesday (7/18/17)</h3> | + | |
| + | <h3>Tuesday (07/18/17)</h3> |
| <p><b>Sense/Respond: </b>Mentor Mike was finally back from vacation and the students were very excited | | <p><b>Sense/Respond: </b>Mentor Mike was finally back from vacation and the students were very excited |
| to see him and update him on current progress. They discussed the plan moving | | to see him and update him on current progress. They discussed the plan moving |
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| described. Afterwards the students continued working on | | described. Afterwards the students continued working on |
| collaboration and organizing wiki pages.</p> | | collaboration and organizing wiki pages.</p> |
− | <p><b>Packaging: </b> | + | <p><b>Packaging: </b>The six liquid culture samples were taken out of the incubator and 3ml from each tube was miniprepped for four restriction digests. The Nanodrop was used to check the concentration of DNA in each sample. After the digests were performed using Nco1, Kpn1, HindII, and Nco1 and HindIII, a gel was run to analyze the results. The results obtained were unexpected, but several samples, 14 and 32, were chosen as likely candidates. |
| </p> | | </p> |
| + | <table> |
| + | <tr> |
| + | <th>Chosen Samples of |
| + | Prepped DNA</th> |
| + | <th>Concentration (ng/uL)</th> |
| + | <th>260/280</th> |
| + | <th>260/230</th> |
| + | </tr> |
| | | |
− | <h3>Wednesday (7/19/17)</h3> | + | <tr> |
| + | <th>Sample 14</th> |
| + | <td>8.3</td> |
| + | <td>1.61</td> |
| + | <td>1.50</td></tr> |
| + | |
| + | <tr> |
| + | <th>Sample 15</th> |
| + | <td>33.0</td> |
| + | <td>1.49</td> |
| + | <td>0.71</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Sample 110</th> |
| + | <td>21.0</td> |
| + | <td>1.67</td> |
| + | <td>0.82</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Sample 22</th> |
| + | <td>17.6</td> |
| + | <td>1.69</td> |
| + | <td>1.30</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Sample 32</th> |
| + | <td>43.6</td> |
| + | <td>1.75</td> |
| + | <td>1.85</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Sample 34</th> |
| + | <td>23.7</td> |
| + | <td>1.06</td> |
| + | <td>1.59</td> |
| + | </tr> |
| + | </table> |
| + | <p class="caption2">Table 15: Nanodrop results</p> |
| + | |
| + | <h3>Wednesday (07/19/17)</h3> |
| <p><b>Packaging: </b> | | <p><b>Packaging: </b> |
| In the morning, the entire iGEM team met with mentors at RH to discuss the projects and Wiki. At the lab, the RXers miniprepped samples 14 and 32 of NEB 5-alpha/ pJAAM1. Later, restriction digests with Nco1, Kpn1, HindII, and Nco1 and HindII were performed on the miniprepped samples. Lastly, cultures of NEB 5-alpha/pJAAM 1 were diluted and spread on LB agar with ampicillin plates. | | In the morning, the entire iGEM team met with mentors at RH to discuss the projects and Wiki. At the lab, the RXers miniprepped samples 14 and 32 of NEB 5-alpha/ pJAAM1. Later, restriction digests with Nco1, Kpn1, HindII, and Nco1 and HindII were performed on the miniprepped samples. Lastly, cultures of NEB 5-alpha/pJAAM 1 were diluted and spread on LB agar with ampicillin plates. |
| + | |
| </p> | | </p> |
| | | |
− | <h3>Thursday (7/20/17)</h3> | + | <h3>Thursday (07/20/17)</h3> |
| <p><b>Sense/Respond: </b>The day started at UES and the students | | <p><b>Sense/Respond: </b>The day started at UES and the students |
| began to look up literature that would help | | began to look up literature that would help |
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| | | |
| | | |
− | <h3>Friday (7/21/17)</h3> | + | <h3>Friday (07/21/17)</h3> |
| <p><b>Sense/Respond: </b>The students finally began the lab | | <p><b>Sense/Respond: </b>The students finally began the lab |
| work today for "sensing". The | | work today for "sensing". The |
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| order the primers. Afterwards, the RH lab held | | order the primers. Afterwards, the RH lab held |
| their weekly lab meeting with the RX lab and | | their weekly lab meeting with the RX lab and |
− | shared the exciting news of their new discovery.</p><br> | + | shared the exciting news of their new discovery.</p> |
| + | <img src="https://static.igem.org/mediawiki/2017/0/06/US_AFRL_CarrollHS_Labnotes3.png" style="width: 80%; margin-left: 10%; margin-right: 10%"> |
| + | |
| <p><b>Packaging: </b>The week ended with the RX team miniprepping three milliliters of the cultures from the previous day and digesting the results. However, when a gel was run on the digest, the results still appeared incorrect. Consequently, the RXers decided to return to a previous step in order to retry the ligation between pBAD-CsgA and dCBD. Previously digested samples of pBAD-CsgA with KpnI and HindIII were further cut with the same enzymes to achieve a more thorough digestion. | | <p><b>Packaging: </b>The week ended with the RX team miniprepping three milliliters of the cultures from the previous day and digesting the results. However, when a gel was run on the digest, the results still appeared incorrect. Consequently, the RXers decided to return to a previous step in order to retry the ligation between pBAD-CsgA and dCBD. Previously digested samples of pBAD-CsgA with KpnI and HindIII were further cut with the same enzymes to achieve a more thorough digestion. |
| </p> | | </p> |
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| <div class="text"> | | <div class="text"> |
| <h2>Week 9: July 24-28</h2> | | <h2>Week 9: July 24-28</h2> |
− | <h3>Monday (7/24/17)</h3> | + | <h3>Monday (07/24/17)</h3> |
| <p><b>Sense/Respond: </b>The day was a bit more relaxed for the Monday morning. The weekly lab | | <p><b>Sense/Respond: </b>The day was a bit more relaxed for the Monday morning. The weekly lab |
| meeting was scheduled and the students were able to learn a lot about other | | meeting was scheduled and the students were able to learn a lot about other |
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| | | |
| | | |
− | <h3>Tuesday (7/25/17)</h3> | + | <h3>Tuesday (07/25/17)</h3> |
| <p><b>Sense/Respond: </b>The adventures continue the next morning as the team went back into the lab to | | <p><b>Sense/Respond: </b>The adventures continue the next morning as the team went back into the lab to |
| look at the overnight cultures. Tina and Hayley were extremely pleased to find | | look at the overnight cultures. Tina and Hayley were extremely pleased to find |
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| </p> | | </p> |
| | | |
− | <h3>Wednesday (7/25/17)</h3> | + | <h3>Wednesday (07/25/17)</h3> |
− | <p><b>Packaging: </b>The entire iGEM team met in the morning at RH to practice the Michigan State presentation and to discuss the trip. Later the RXers and ran a gel of the gel extractions, as well as checking the concentrations with the Nanodrop. The results were not entirely desirable, likely due to the number of protocols performed with the samples, but a ligation was set up and performed. Vanessa helped the team gather pictures and begin work on their Friday presentation. </b> | + | <p><b>Packaging: </b>The entire iGEM team met in the morning at RH to practice the Michigan State presentation and to discuss the trip. Later the RXers ran a gel of the gel extractions, as well as checking the concentrations with the Nanodrop. The results were not entirely desirable, likely due to the number of protocols performed with the samples, but a ligation was set up and performed. Vanessa helped the team gather pictures and begin work on their Friday presentation. </b> |
| | | |
| | | |
| | | |
− | <h3>Thursday (7/27/17)</h3> | + | <h3>Thursday (07/27/17)</h3> |
| <p>Another meeting was held to discuss the presentation and viability of the | | <p>Another meeting was held to discuss the presentation and viability of the |
| Michigan State Trip. Students from both labs went over their combined | | Michigan State Trip. Students from both labs went over their combined |
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| and proceed again tomorrow. Later in the day, the young researchers finalized their plan for | | and proceed again tomorrow. Later in the day, the young researchers finalized their plan for |
| "flat Stanley" and the outreach survey they hope to do in the future.</p> | | "flat Stanley" and the outreach survey they hope to do in the future.</p> |
| + | <img src="https://static.igem.org/mediawiki/2017/8/84/US_AFRL_CarrollHS_Labnotes4.png" style="width: 80%; margin-left: 10%; margin-right: 10%"> |
| + | |
| <p><b>Packaging: </b>To start the morning, the RXers spent a portion of time discussing the Friday presentation with Vanessa. Later the team performed a transformation of the ligation from the previous day and continued preparing for their presentation. | | <p><b>Packaging: </b>To start the morning, the RXers spent a portion of time discussing the Friday presentation with Vanessa. Later the team performed a transformation of the ligation from the previous day and continued preparing for their presentation. |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Friday (7/28/17)</h3> | + | <h3>Friday (07/28/17)</h3> |
| <p>The morning began bright and early with | | <p>The morning began bright and early with |
| a tour of a C-17 cargo plane on base. It | | a tour of a C-17 cargo plane on base. It |
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| protocol with ease and completed the protocol | | protocol with ease and completed the protocol |
| accurately and quickly.</p> | | accurately and quickly.</p> |
− | <p><b>Packaging: </b>Both the RH and the RX teams participated in a tour of a C-17. Afterwards, the RXers presented their project at a Journal Talk. Vanessa suggested troubleshooting by sending samples of pBAD-csgA off for sequencing. Additionally, the University of of Michigan mutant strains of E. coli arrived. Once the preparation for sequencing was completed, the team met at UES for a bit before leaving for the weekend (Michigan State!) | + | <p><b>Packaging: </b>Both the RH and the RX teams participated in a tour of a C-17. Afterwards, the RXers presented their project at a Journal Talk. Vanessa suggested troubleshooting by sending samples of pBAD-CsgA off for sequencing. Additionally, the University of of Michigan mutant strains of E. coli arrived. Once the preparation for sequencing was completed, the team met at UES for a bit before leaving for the weekend (Michigan State!) |
| </p> | | </p> |
| | | |
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| <div class="text2"> | | <div class="text2"> |
| <h2>Week 10: July 31-August 4</h2> | | <h2>Week 10: July 31-August 4</h2> |
− | <h3>Monday (7/31/17)</h3> | + | <h3>Monday (07/31/17)</h3> |
| <p><b>Sense/Respond: </b>The morning started with the students performing a digest over the mini prep | | <p><b>Sense/Respond: </b>The morning started with the students performing a digest over the mini prep |
| from several days pervious. Two digests were preformed using different sets of | | from several days pervious. Two digests were preformed using different sets of |
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| cut enzymes PacI and BmrI. The Digest was then confirmed using a gel, which | | cut enzymes PacI and BmrI. The Digest was then confirmed using a gel, which |
| showed the accurate number of base pairs.</p> | | showed the accurate number of base pairs.</p> |
− | <p><b>Packaging: </b>The week started with the RXers streaking and growing cultures of certain colonies of the transformants from Friday. The samples of pBAD-csgA were sent off for sequencing and a plan was drawn up to further troubleshoot and proceed with the project. | + | <p><b>Packaging: </b>The week started with the RXers streaking and growing cultures of certain colonies of the transformants from Friday. The samples of pBAD-CsgA were sent off for sequencing and a plan was drawn up to further troubleshoot and proceed with the project. |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Tuesday (8/1/17)</h3> | + | <h3>Tuesday (08/01/17)</h3> |
| <p><b>Sense/Respond: </b>The students set up another run of their GFP Project. The students used a | | <p><b>Sense/Respond: </b>The students set up another run of their GFP Project. The students used a |
| Kinetics reading of the Plate Reader. The kinetics were taken over 24 hours, every | | Kinetics reading of the Plate Reader. The kinetics were taken over 24 hours, every |
| 20 minutes. The students then worked on the wiki, trying to create a template.</p> | | 20 minutes. The students then worked on the wiki, trying to create a template.</p> |
− | <p><b>Packaging: </b>The day began with developing a plan for troubleshooting the project throughout the week. Later, the RXers made up a stock of LB agar and LB agar with Kanamycin and miniprepped three milliliters of the liquid cultures from Monday. Restriction digests were performed on each miniprep and a gel was run, and one sample's results seemed hopeful. New restriction digests were performed on the sample, but the outcomes of the sequencing arrived and suggested that success with the new restriction digests would be unlikely. The sequencing results showed a fragment of the TOPO plasmid had cloned in before csgA in each pBAD-csgA sample. Solutions of magnesium chloride, calcium chloride, and glycerol were made in order to perform a competent cell protocol on the mutant strains of E. coli, which were left in an incubator overnight to grow. | + | <p><b>Packaging: </b>The day began with developing a plan for troubleshooting the project throughout the week. Later, the RXers made up a stock of LB agar and LB agar with Kanamycin and miniprepped three milliliters of the liquid cultures from Monday. Restriction digests were performed on each miniprep and a gel was run, and one sample's results seemed hopeful. New restriction digests were performed on the sample, but the outcomes of the sequencing arrived and suggested that success with the new restriction digests would be unlikely. The sequencing results showed a fragment of the TOPO plasmid had cloned in before <i>csgA</i> in each pBAD-CsgA sample. Solutions of magnesium chloride, calcium chloride, and glycerol were made in order to perform a competent cell protocol on the mutant strains of E. coli, which were left in an incubator overnight to grow. |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Wednesday (8/2/17)</h3> | + | <h3>Wednesday (08/02/17)</h3> |
| <p>Today was an exciting day for the students as they got to perform their survey | | <p>Today was an exciting day for the students as they got to perform their survey |
| at their high school. The iGEM students volunteered to help the high school on | | at their high school. The iGEM students volunteered to help the high school on |
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| | | |
| huge success for the students.</p> | | huge success for the students.</p> |
− | <p><b>Packaging: </b>The RXers chose samples of the pBAD-csgA containing TOPO plasmid to digest with NcoI in the hopes that the TOPO fragment would separate from the rest. A gel showed the digests to be successful, and a gel extraction was performed. The concentrations of the results from the gel extraction were checked using a Nanodrop. Lastly, the mutant strains with csgA and without csgA were each inoculated onto plates with LB and Kanamycin and with just LB respectively, and these plates were stored in an incubator overnight to grow. | + | <p><b>Packaging: </b>The RXers chose samples of the pBAD-CsgA containing TOPO plasmid to digest with NcoI in the hopes that the TOPO fragment would separate from the rest. A gel showed the digests to be successful, and a gel extraction was performed. The concentrations of the results from the gel extraction were checked using a Nanodrop. Lastly, the mutant strains with <i>csgA</i> and without <i>csgA</i> were each inoculated onto plates with LB and Kanamycin and with just LB respectively, and these plates were stored in an incubator overnight to grow. |
| </p> | | </p> |
| | | |
− | <h3>Thursday (8/3/17)</h3> | + | <h3>Thursday (08/03/17)</h3> |
| <p><b>Sense/Respond: </b>Today the students got back to work on the sense plasmid. The students | | <p><b>Sense/Respond: </b>Today the students got back to work on the sense plasmid. The students |
| performed a PCR, because finally their primers came in. The PCR product was | | performed a PCR, because finally their primers came in. The PCR product was |
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| | | |
| performed another digest using the PCR they had just purified.</p> | | performed another digest using the PCR they had just purified.</p> |
| + | |
| + | |
| + | <table> |
| + | <tr> |
| + | <th></th> |
| + | <th>260</th> |
| + | <th>280</th> |
| + | <th>Ratio</th> |
| + | <th>ng/ul</th> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>F1</th> |
| + | <td>7.408</td> |
| + | <td>3.974</td> |
| + | <td>1.86</td> |
| + | <td>370.4</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>F2</th> |
| + | <td>8.412</td> |
| + | <td>4.554</td> |
| + | <td>1.85</td> |
| + | <td>420.6</td> |
| + | </tr> |
| + | |
| + | <tr> |
| + | <th>Blank</th> |
| + | <td>0.089</td> |
| + | <td>0.002</td> |
| + | <td>5.59</td> |
| + | <td>0.4</td> |
| + | </tr> |
| + | </table> |
| + | <p class="caption2">Table 16: Nanodrop results</p> |
| + | |
| + | </p> |
| + | |
| + | |
| <p><b>Packaging: </b>Using the gel extractions from Wednesday, the RXers performed four ligations. After two hours, part of the ligations were removed in order to complete several transformations, while the remaining ligations were left to incubate overnight. Colonies from the plates containing the University of Michigan strains were transferred to flasks containing LB Broth and LB Broth with Kanamycin. | | <p><b>Packaging: </b>Using the gel extractions from Wednesday, the RXers performed four ligations. After two hours, part of the ligations were removed in order to complete several transformations, while the remaining ligations were left to incubate overnight. Colonies from the plates containing the University of Michigan strains were transferred to flasks containing LB Broth and LB Broth with Kanamycin. |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Friday (8/4/17)</h3> | + | <h3>Friday (08/04/17)</h3> |
| <p>The big day to present to the Chief Scientist of the lab had finally come. Early in | | <p>The big day to present to the Chief Scientist of the lab had finally come. Early in |
| the day, the students had a dry presentation with their teachers and mentors. | | the day, the students had a dry presentation with their teachers and mentors. |
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| <div class="text"> | | <div class="text"> |
| <h2>Week 11: August 7-11</h2> | | <h2>Week 11: August 7-11</h2> |
− | <h3>Monday (8/7/17)</h3> | + | <h3>Monday (08/07/17)</h3> |
| <p><b>Sense/Respond: </b>Unfortunately the students were disheartened to learn that none of the plates | | <p><b>Sense/Respond: </b>Unfortunately the students were disheartened to learn that none of the plates |
| grew colonies. The students were at first shocked, but immediately started | | grew colonies. The students were at first shocked, but immediately started |
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| Therefore Mentor Mike recommended that they re-do the ligation from Monday. The ligation | | Therefore Mentor Mike recommended that they re-do the ligation from Monday. The ligation |
| went very smoothly and the transformation will be done tomorrow.</p> | | went very smoothly and the transformation will be done tomorrow.</p> |
− | <p><b>Packaging: </b>Samples of pJAAM01 from early August were digested using Nco1 in the hope that the TOPO fragment would separate from the pBAD-csgA-dCBD plasmid. A gel showed the digests to be successful, and a gel extraction was performed. The concentrations of the results from the gel extraction were checked using a Nanodrop. The RXers then set up ligations using the gel extractions and a gel was run to confirm the gel extractions. Colonies from the transformants of pBAD-csgA from last week were patched on an agar plate and inoculated in culture tubes. Finally, plates were streaked using the freezer stocks of the University of Michigan strains and left to grow overnight. | + | <p><b>Packaging: </b>Samples of pJAAM01 from early August were digested using Nco1 in the hope that the TOPO fragment would separate from the pBAD-CsgA-dCBD plasmid. A gel showed the digests to be successful, and a gel extraction was performed. The concentrations of the results from the gel extraction were checked using a Nanodrop. The RXers then set up ligations using the gel extractions and a gel was run to confirm the gel extractions. Colonies from the transformants of pBAD-CsgA from last week were patched on an agar plate and inoculated in culture tubes. Finally, plates were streaked using the freezer stocks of the University of Michigan strains and left to grow overnight. |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Tuesday (8/8/17)</h3> | + | <h3>Tuesday (08/08/17)</h3> |
| <p><b>Sense/Respond: </b>Today the LabPats were finally able to transform the plasmid from the ligation | | <p><b>Sense/Respond: </b>Today the LabPats were finally able to transform the plasmid from the ligation |
| yesterday. They followed the standard protocol and transformed the cells into E. | | yesterday. They followed the standard protocol and transformed the cells into E. |
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| completion of their "sense" component. The students left the lab in high spirits and | | completion of their "sense" component. The students left the lab in high spirits and |
| headed out for wifi and collaboration space to discuss more wiki work and future work.</p> | | headed out for wifi and collaboration space to discuss more wiki work and future work.</p> |
− | <p><b>Packaging: </b> | + | <p><b>Packaging: </b> We weren't in the lab! |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Wednesday (8/9/17)</h3> | + | <h3>Wednesday (08/09/17)</h3> |
| <p><b>Sense/Respond: </b>The students eagerly went into the lab to check the plates from the | | <p><b>Sense/Respond: </b>The students eagerly went into the lab to check the plates from the |
| transformation. To the students dismay all the plates contained growth even the | | transformation. To the students dismay all the plates contained growth even the |
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| the colonies were already glowing green. With already putting in a long day of work the | | the colonies were already glowing green. With already putting in a long day of work the |
| students made overnights for the next day.</p> | | students made overnights for the next day.</p> |
− | <p><b>Packaging: </b> | + | <p><b>Packaging: </b> We weren't in the lab! |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Thursday (8/10/17)</h3> | + | <h3>Thursday (08/10/17)</h3> |
| <p><b>Sense/Respond: </b>Bright and early the students got their overnights out of the incubator. They then | | <p><b>Sense/Respond: </b>Bright and early the students got their overnights out of the incubator. They then |
| made glycerol stocks and performed a mini prep, so the bacteria could be sent | | made glycerol stocks and performed a mini prep, so the bacteria could be sent |
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| presentations. This was especially helpful for the students, and gave them many | | presentations. This was especially helpful for the students, and gave them many |
| ideas on how to set up their poster.</p> | | ideas on how to set up their poster.</p> |
− | <p><b>Packaging: </b> | + | <p><b>Packaging: </b> We weren't in the lab! |
| </p> | | </p> |
| | | |
| | | |
− | <h3>Friday (8/11/17)</h3> | + | <h3>Friday (08/11/17)</h3> |
| <p><b>Sense/Respond: </b>Today was a sad day in the lab, as the summer was ending and the students | | <p><b>Sense/Respond: </b>Today was a sad day in the lab, as the summer was ending and the students |
| had to return to school. The team spent the morning taking their mentors to | | had to return to school. The team spent the morning taking their mentors to |
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| spent at the lab. The students cleaned up their work benches, and made sure | | spent at the lab. The students cleaned up their work benches, and made sure |
| everything was in order before they left the lab for the last time. The students said | | everything was in order before they left the lab for the last time. The students said |
− |
| |
| their good byes to their coworkers, whom they had enjoyed many lunch breaks with.</p> | | their good byes to their coworkers, whom they had enjoyed many lunch breaks with.</p> |
| </div> | | </div> |
− | <p><b>Packaging: </b> | + | <p><b>Packaging: </b> We weren't in the lab! |
| </p> | | </p> |
| <br> | | <br> |
| | | |
| + | </div> |
| | | |
− | | + | <div class="text2"> |
− | | + | <h2>Parts Submission</h2> |
− | | + | <h3>(10/19-20/17)</h3> |
| + | <p>The parts were submitted according to the protocol provided by iGEM. Minipreped DNA was placed into their respective wells before being dried in a hood overnight. The next day, they were then packaged and shipped.</p> |
| + | <br> |
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