Team:The Agency Escondido/notebook

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                 <a href="http://agency-stem.org"><img src="http://agency-stem.org/static/img/2013-heading-web.png" alt="Agency Logo"/></a>
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<ul>
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<li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido">About</a></li>
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    <li>
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<li id="selectedsection">Notebook</li>
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        <a href="https://2013hs.igem.org/Team:The_Agency_Escondido">About</a>
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<li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/safety">Safety</a></li>
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        <ul>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido">General</a></li>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/sandiego">San Diego</a></li>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/ourteam">Our Team</a></li>
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        </ul>
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        <a href="https://2013hs.igem.org/Team:The_Agency_Escondido/project"><u>Project</u></a>
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        <ul>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/project/rock">Rock</a></li>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/project/paper">Paper</a></li>
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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/project/scissors">Scissors</a></li>
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        </ul>
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    </li>
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    <li>
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        <a href="https://2013hs.igem.org/Team:The_Agency_Escondido/notebook">Notebook</a>
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        <ul>
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            <li></li>
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        </ul>
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    </li>
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    <li>
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        <a href="https://2013hs.igem.org/Team:The_Agency_Escondido/safety">Safety</a>
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        <ul>
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            <li></li>
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<h2>April 9</h2>
<h2>April 9</h2>
<p>Andrew Buss prepared 120 mL of LB broth</p>
<p>Andrew Buss prepared 120 mL of LB broth</p>
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           <td><solid>Vansh Singh Transformation Control</solid></td>
           <td><solid>Vansh Singh Transformation Control</solid></td>
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           <td><solid>Andrew Buss Ligation Control</solid></td>
           <td><solid>Andrew Buss Ligation Control</solid></td>
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           <td><solid>Andrew Buss Transformation Conrol</solid></td>
           <td><solid>Andrew Buss Transformation Conrol</solid></td>
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<p>Vansh Singh and Evan Santos searched for and found the promoters, inducers, and reporters for the project.<p>
<p>Vansh Singh and Evan Santos searched for and found the promoters, inducers, and reporters for the project.<p>
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<p> Grant Hassinger started miniprep on his inoculated E. coli with parts A & B </p>
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<p> Grant Hassinger started miniprep on his inoculated E. coli with parts A & B and attempted to pause the process through freezing at -20C</p>
<h2>April 30</h2>
<h2>April 30</h2>
<p>Andrew Buss obtained a 100/50 volt power supply designed specifically for electrophoresis. A gel loading dye was run alongside a DNA ladder for 50 minutes at 100 volts. The gel was then removed from the tray and soaked in 0.002% methylene blue at room temperature for 3 hours. Before the lab closed, the gel was left to wash in 1X TAE. </p>
<p>Andrew Buss obtained a 100/50 volt power supply designed specifically for electrophoresis. A gel loading dye was run alongside a DNA ladder for 50 minutes at 100 volts. The gel was then removed from the tray and soaked in 0.002% methylene blue at room temperature for 3 hours. Before the lab closed, the gel was left to wash in 1X TAE. </p>
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<p>After Vansh Singh completed plasmid design, Andrew Buss used the Parts Registry to identify existing parts that would eliminate later time-consuming steps for the "rock" plasmid. First, <a href="http://partsregistry.org/Part:BBa_S01022">an intermediate part</a> by Randy Rettberg eliminates the assembly of an RBS and a cyan fluorescent protein. <a href="http://partsregistry.org/Part:BBa_J37033">Another intermediate part</a> eliminates the assembly of an RBS and a LuxR coding region.</p>  
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<p>After Vansh Singh completed plasmid design, Andrew Buss used the Parts Registry to identify existing parts that would eliminate later time-consuming steps for the "rock" plasmid. First, <a href="http://partsregistry.org/Part:BBa_S01022">an intermediate part</a> by Randy Rettberg eliminates the assembly of an RBS and a cyan fluorescent protein. <a href="http://partsregistry.org/Part:BBa_J37033">Another intermediate part</a> eliminates the assembly of an RBS and a LuxR coding region. These reduce the number of required assemblies to three, which cannot be parallelized because of the requirement below that we produce the independent AraC sensor.</p>  
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<p>Andrew Buss documented the final and intermediate steps of assembly of the "rock" plasmid. First, we will construct <a href="http://partsregistry.org/Part:BBa_K1001750">a simple AraC sensor</a>. Then we will assemble this sensor with <a href="http://partsregistry.org/Part:BBa_J37033">a LuxR coding region</a> to form <a href="http://partsregistry.org/Part:BBa_K1001751">our final plasmid</a>. The intermediate AraC sensor will serve as a useful diagnostic tool, and will be submitted to the Registry as well. </p>
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<p>Andrew Buss documented the final and intermediate steps of assembly of the "rock" plasmid. First, we will construct <a href="http://partsregistry.org/Part:BBa_K1001750">a simple AraC sensor</a>. Then we will assemble this sensor with <a href="http://partsregistry.org/Part:BBa_J37033">a LuxR coding region</a> to form <a href="http://partsregistry.org/Part:BBa_K1001751">our final plasmid</a>. The intermediate AraC sensor will serve as a useful diagnostic tool, and will be submitted to the Registry as well.</p>
<p>Andrew Buss began to write a high-level protocol for assembly, detailing the restriction and ligation steps required to complete the plasmid.</p>
<p>Andrew Buss began to write a high-level protocol for assembly, detailing the restriction and ligation steps required to complete the plasmid.</p>
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<p> Grant Hassinger resumed his miniprep but as the next step did not result in the desired effect, he concluded the cells should not be frozen </p>
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 +
<h2>May 1</h2>
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 +
<p>Andrew Buss's agarose gel was a uniform blue color, even after rinsing with TAE overnight. No bands were visible.</p>
 +
<p>Andrew Buss transformed competent cells with the ligation from 4/29.</p>
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<p>Andrew Buss resuspended DNA samples of BBa_R0080, BBa_B0010, BBa_J373033, and pSB1C3 from the Spring 2012 DNA distribution in 50 uL of distilled water each. Transformation is postponed until ampicillin media becomes available (ampicillin and other supplies are on order).</p>
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 +
<h2>May 8</h2>
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<p>Andrew Buss's transformation plate from 5/1 showed no colonies. However, there was large black colony that appeared densest by the edge of the plate. Evidently, contamination was introduced during the transformation process, although the absence of other colonies suggests that the transformation process or a previous step did not work correctly.</p>
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<h2>May 9</h2>
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<p> Grant Jose inoculated lb with parts a and neb 10 </p>
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<p>Andrew Buss inoculated 5 mL of LB media with 100 uL of media containing NEB 10 cells. After inoculation, the spectrophotometer registered an OD 600 of .029. This did not vary over the next hour and the next three spectrophotometer readings. Andrew Segina hypothesized that this was a result of the (previously refrigerated) cells' slow return to a growth stage. The cells were left to incubate overnight.</p>
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<h2>May 10</h2>
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<p>Andrew Buss continued his spectrophotometry work from the previous night. Noticeable increase in OD 600 was observed - a range from .45 to .5. </p>
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<p>Andrew Buss tested a <a href="http://www.med.nyu.edu/medicine/labs/blaserlab/Protocols/E-coli_competent_cells_protocol_&_transformation.pdf">calcium chloride competent cell protocol</a>. Cell clumping presented obstacles to resuspension, so the test was simply to verify that the protocol did not entirely kill NEB 10 cells.</p>
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<p>Ben Jones ran a miniprep on Evan Santos's 3A Construction Protocol Part A</p>
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<p> Evan Santos ran a miniprep on Grant Jose's 5/9/13 culture of 3A Construction Protocol Part B</p>
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<h2>May 15</h2>
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<p>Evan Santos inoculated 600 mL of LB media with 25 uL of media containing NEB-10 cells.</p>
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<h2>May 21</h2>
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<p>Andrew Segina made 1mL 1000x stock solutions of Ampicillin, Kanamycin Sulfate, and Chloramphenicol, as per the protocol found in CASP.</p>
 +
<p>Grant Hassinger and Blaise DeValasco did a run of transforming Cells with I13500 and E0040 but the label wiped off and they became indistinguishable so the work was bleached and disposed of.</p>
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<h2>May 30</h2>
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<p>Evan Santos transformed competent cells for parts BBa_R0062 and BBa_I13500.</p>
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Latest revision as of 23:15, 21 June 2013