Team:The Agency Escondido/project

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                 <a href="http://agency-stem.org"><img width=500px src="http://agency-stem.org/static/img/2013-heading-web.png" alt="Agency Logo"/></a>
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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><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/notebook">Notebook</a></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 id="selectedsection">Project</a></li>
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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/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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            <li><a href="https://2013hs.igem.org/Team:The_Agency_Escondido/ourteam">Our Team</a></li>
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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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</h2>
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<p>
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Our project seeks to create two identical triads of transparent bacteria strains with complementary hydrophobic inducer production to emulate the game of "rock, paper, scissors" (Hydrophobic proteins can move through plasma membranes). Each triad will correspond to a group: group A and group B. Each type of bacteria will produce a specific protein that will act as an inducer to one and only one other bacteria strain(rock strain, paper strain, and scissors strain). Each triad will have different reporter genes designating its champion color. When two bacteria strains are combined in an agar plate, each protein secreted by the victorious strain, when acting as an inducer, changes the color of the cells on the plate identifying the champion.
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Our project seeks to create two identical triads of transparent bacteria strains with complementary hydrophobic inducer production to emulate the game of "rock, paper, scissors" (Hydrophobic proteins can move through plasma membranes). Each triad will correspond to a group: group A and group B. Each type of bacteria will produce a specific protein that will act as an inducer to one and only one other bacteria strain(rock strain, paper strain, and scissors strain). Each triad will have different reporter genes designating its champion color. When two bacteria strains are combined in an agar plate, each protein secreted by the victorious strain, when acting as an inducer, changes the color of the cells on the plate identifying the champion. Through this game, we will learn of the inducers' solubility in water. Only hydrophobic inducers can permeate the cell and induce an operon in a foreign cell. We developed our <a href="http://casp.agency-stem.org/media/G13-LP0013-40.pdf">paper protocol</a>, <a href="http://casp.agency-stem.org/media/G13-LP0009-21.pdf">rock protocol</a>, and <a href="http://casp.agency-stem.org/media/G13-LP0010-57.pdf">scissors protocol</a> before we began our project.
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<p>At the time of writing we are about 75% done with our project. We still need to complete the final assembly of the "scissors" strain and need to complete the second and third assembly of the "rock" and "paper" strains. We anticipate completion by the end of the summer.</p>
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<img height="650px" width="700px" src=https://static.igem.org/mediawiki/2013hs/a/af/The_agency_escondido_plasmid_map_2013_2.gif id="plasmidmap"  usemap ="#plasmidmap"></img>
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<area shape ="rect" coords ="0,0,233,650" href ="https://2013hs.igem.org/Team:The_Agency_Escondido/project/rock" alt="Rock" />
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<area shape ="rect" coords ="233,0,466,650" href ="https://2013hs.igem.org/Team:The_Agency_Escondido/project/paper" alt="Paper" />
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<area shape ="rect" coords ="466,0,700,650" href ="https://2013hs.igem.org/Team:The_Agency_Escondido/project/scissors" alt="Scissors" />
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      <td><a href="#rock"> Rock </a> </td>
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      <td><a href="#paper"> Paper </a> </td>
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      <td><a href="#scissors" > Scissors </a> </td> 
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<a id="rock"></a>
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<h2> Rock </h2>
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<p>The "rock" bacteria strain will have a CFP (<a href = "http://partsregistry.org/Part:BBa_E0020">part E0020</a>) and promoter (<a href = "http://partsregistry.org/Part:BBa_R0080">part R0080</a>). It will contain the LuxR gene (<a href = "http://partsregistry.org/Part:BBa_C0062">part C0062</a>) to induce the "paper" strain.</p>
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<h3>Notebook</h3>
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<h4>May 20</h4>
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<p>Andrew Buss began <a href="http://casp.agency-stem.org/media/G13-LP0012-25.pdf">a protocol to prepare competent cells</a>. This protocol omitted the final step of resuspension.</p>
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<h4>May 21</h4>
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<p>Andrew Buss completed the cell competency protocol from the previous
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day, spinning down 8 mL total in sequence.
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With the arrival of ampicillin, Andrew Buss streaked BBa_S01022 onto a
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quadrant of an ampicillin agar plate.
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Andrew Buss began transforming DNA for later assemblies into cells,
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using 5 uL of resuspended DNA per sample in water as suggested by the
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<a href="http://partsregistry.org/Help:Distribution_Kits">Parts Registry protocol</a>. If the transformation failed with 5 uL, then 40
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uL containing 1 ug could be used in another protocol. 50 pg of DNA from
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the transformation efficiency kit was used as an RFP control. The second
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and last centrifugation in the protocol was carried out using 0.6 mL
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tubes. Unfortunately, the rotor in the centrifuge was designed for 1.5
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mL tubes. When centrifuging, three of the tubes were forced open, and
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fell through the holes in the rotor. These three samples were lost.
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Andrew Buss restarted transformation, using the same protocol. Cells
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were left to incubate overnight in LB containing the appropriate
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antibiotics: the transformation control and pSB1C3 used chloramphenicol,
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while the remainder used ampicillin.</p>
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<h4>May 22</h4>
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<p>The chloramphenicol plate from the previous day displayed faint streaks (possibly simply indentations in the agar) but no distinct colonies. The ampicillin plate containing BBa_S01022 displayed dense growth. Andrew Buss extended some streaks onto the next quadrant to reduce the density to individual cells.</p>
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<p>Andrew Buss streaked two quadrants of an ampicillin plate with BBa_R0080 and BBa_B0010. BBa_J37033 was transformed on the previous day, but was accidentally omitted from the streaking step. </p>
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<a id="paper"></a>
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<h2> Paper </h2>
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<p>The "paper" bacteria strain will have an GFP (<a href = "http://partsregistry.org/Part:BBa_E0040">part E0040</a>) and promoter (<a href = "http://partsregistry.org/Part:BBa_I1051">part I1051</a>). It will contain the Lambda cl repressor gene (<a href = "http://partsregistry.org/Part:BBa_C0051">part C0051</a>) to induce the "scissors" strain.</p>
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<h2> Notebook</h2>
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<p>Grant Hassinger 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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<a id="scissors"></a>
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<h2> Scissors </h2>
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<p>The "scissors" bacteria strain will have an RFP (<a href = "http://partsregistry.org/Part:BBa_E1010">part E1010</a>)and promoter (<a href = "http://partsregistry.org/Part:BBa_I12006">part I12006</a>). It will contain the AraC gene (<a href = "http://partsregistry.org/Part:BBa_C0080">part C0080</a>) to induce the "rock" strain.</p>
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<h3>Notebook</h3>
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<h4>May 20</h4><p>Vansh Singh began <a href="http://casp.agency-stem.org/media/G13-LP0012-25.pdf">a protocol to prepare competent cells</a>. This protocol omitted the final step of resuspension. Vansh caught this mistake in the protocol and continued the process of making competent cells from the protocol's source.</p>
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<h4>May 21</h4><p>Vansh Singh completed cell competency from yesterday.</p>
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<p>Vansh Singh resuspended BBa_B0015, BBa_I12006, BBa_I13502,pSBIA3, and pSBIC3. He transformed these parts into competent cells. Unfortunately, a thermocycler program which was supposed to hold parts at 42 degrees Celsius for two minutes went up to 92 degrees Celsius. He stopped the program as it was climbing up to 92 degrees when it was at 90 degrees. Transformation was compromised. However, Vansh Singh continued the transformation process of those cells. Afterwards, he also found it fit to start another round of transformation. Vansh Singh inoculated the transformed cells from his first batch onto an agar plate. However, he had forgotten to add the respective antibiotics to the plates beforehand. Therefore the plates had to be disposed.</p><p>Vansh Singh moved the cells into the fridge before he left</p>
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<h4>May 21</h4><p>Vansh Singh superficially added Ampicillin to an agar plate and added Kanamayacin to another agar plate. Because of limited plates, he divided each plate into fifths and inoculated cells from each batch into its respective agar plate in its respective "fifth." Vansh Singh made LB agar.</p>
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Latest revision as of 04:15, 22 June 2013