Team:Yale/Our Project/Notebook/Week 2

From 2010.igem.org

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<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li>
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li>
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li>
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li>
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<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li>
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<!------------- Monday ------------->
<!------------- Monday ------------->
<h4> Copper Growth Assay Work Continues </h4>
<h4> Copper Growth Assay Work Continues </h4>
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2010/8/8f/Yale-growthfail.jpg" />
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</div>
<ul>
<ul>
<li>As the growth assay of <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook">6/11</a> showed uniformly poor growth even at the lowest copper concentrations (see Dh5alpha growth above), redid it with care not to let the liquid cultures overgrow, a possible source of the cultures' previous poor performance. (add plot)</li>
<li>As the growth assay of <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook">6/11</a> showed uniformly poor growth even at the lowest copper concentrations (see Dh5alpha growth above), redid it with care not to let the liquid cultures overgrow, a possible source of the cultures' previous poor performance. (add plot)</li>
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<!------------- Tuesday ------------->
<!------------- Tuesday ------------->
<h4> Results and Conclusions of Narrow Concentration Range Growth Assay of 6/14</h4>
<h4> Results and Conclusions of Narrow Concentration Range Growth Assay of 6/14</h4>
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(Show graph)
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2010/d/d1/Yale-dh5alpha-narrow.jpg" />
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</div>
<ul>
<ul>
<li> Negative readings for the 4 mM samples suggest that there was some sort of irregularity with the blank solution, so will disregard those results. </li>
<li> Negative readings for the 4 mM samples suggest that there was some sort of irregularity with the blank solution, so will disregard those results. </li>
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<tr>
<tr>
<td>1</td>
<td>1</td>
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<td>IPTG-inducible pSB1A2 (well 6G plate 1)</td>
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<td>IPTG-inducible BBa_R0011(well 6G plate 1)</td>
<td> phsABC </td>
<td> phsABC </td>
<td> BBa_B0015 </td>
<td> BBa_B0015 </td>
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</table>  
</table>  
*This plan was eventually altered, as promoter choices were altered and no light-inducible promoter was found. <br/>
*This plan was eventually altered, as promoter choices were altered and no light-inducible promoter was found. <br/>
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<br />
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<div id="right">
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<img src="https://static.igem.org/mediawiki/2010/7/71/Yale-biobrick.jpg" />
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</div>
To make these plasmids, we will rely on the standard iGEM assembly protocol involving restriction enzymes EcoRI, XbaI, PstI, and SpeI shown in the diagram at right, but in the first ligation the B0015 terminator will take the place of C0010 and the phsABC  gene in pSB74 will take the place of B0034. <br/>
To make these plasmids, we will rely on the standard iGEM assembly protocol involving restriction enzymes EcoRI, XbaI, PstI, and SpeI shown in the diagram at right, but in the first ligation the B0015 terminator will take the place of C0010 and the phsABC  gene in pSB74 will take the place of B0034. <br/>
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<b> Minimal Medium Work </b> <br/>
<b> Minimal Medium Work </b> <br/>
In Jay Keasling's work with thiosulfate reductase he used a variation of MOPS minimal medium designed to avoid background metal presence from interfering with measurement of bacterial metal removal potential. Began making the various necessary component solutions as detailed in this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/minimal_medium">recipe </a> <br/>
In Jay Keasling's work with thiosulfate reductase he used a variation of MOPS minimal medium designed to avoid background metal presence from interfering with measurement of bacterial metal removal potential. Began making the various necessary component solutions as detailed in this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/minimal_medium">recipe </a> <br/>
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<b>Additional <i> E. coli </i> strain </b> <br/>
 
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Began a culture of BL21 as another hardy option for future assays. <br/>
 
<b>Survival of bacteria in copper growth assay </b> <br/>
<b>Survival of bacteria in copper growth assay </b> <br/>
Previously had plated a drop of each culture from the narrow concentration range copper growth assay on 6/14 to see if the cultures that had stopped growing/never grew were still alive.  Observed the following results, where g signifies growth and - signifies no growth:<br/>
Previously had plated a drop of each culture from the narrow concentration range copper growth assay on 6/14 to see if the cultures that had stopped growing/never grew were still alive.  Observed the following results, where g signifies growth and - signifies no growth:<br/>
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</tr>
</tr>
</table>
</table>
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It is unsurprising that all cells should die at 1 M copper sulfate concentrations, but the lack of growth of DH5alpha at 0 M, 900 uM, etc. seems anomalous, given that the cells clearly survived at higher copper levels and may reflect an insufficient amount of culture spotted in those cases. <br/>
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<i>This information is also recorded on page 11 of the hard copy lab notebook. </i> <br/>
<!------------- Wednesday ------------->
<!------------- Wednesday ------------->
</div>
</div>
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<!------------- Thursday ------------->
<!------------- Thursday ------------->
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Extra content
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<b>Modified MOPS minimal medium</b><br/>
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Finished creation of a stock solution according the this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/minimal_medium">recipe </a> <br/>
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<b>Additional <i> E. coli </i> strain </b> <br/>
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Began a culture of BL21 as another hardy option for future assays. <br/>
<!------------- Thursday ------------->
<!------------- Thursday ------------->
</div>
</div>
<li>
<li>
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Friday 6/18--plasmid pSB74 arrived (!), already in some unspecified E coli, so plated them out. Transformed promoters (constitutive & IPTG inducible), terminator, and repressor (for inducible promoter) into BL21. Also ran BL21 copper growth assays for wide and narrow concentrations ranges.
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Friday 6/18--Arrival of plasmid pSB74, transformation of Biobricks, & copper growth assays for BL21 strain.  
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June the 19th--in which it is revealed that there was a transformation-fail
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-
Redid transformation, this time into LE392, set aside pSB74 containing cells in fridge
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</li>
</li>
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<div style="display:none;" id="friday">
<div style="display:none;" id="friday">
<!------------- Friday ------------->
<!------------- Friday ------------->
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Extra content
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<ul>
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<li>Plasmid pSB74, which contains the thiosulfate reductase gene, arrived from Addgene, already in some unspecified <i>E coli</i>, so plated them out on an ampicillin LB plate and put in incubator at 37.</li>
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<li>Transformed constitutive promote J23114, IPTG-inducible promoter R0011, terminator B0015, and repressor C0012(for inducible promoter) into BL21 according to standard <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">transformation protocol </a> and plated onto ampicillin LB plates</li>
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<li>Also ran BL21 copper growth assays for wide and narrow concentrations ranges according to the modified protocol used on 6/14, starting with dilution of a culture of OD 0.873.</li>
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</ul>
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<i> The activities of this day are also recorded on pages 13-15 of the hard copy lab notebook </i>
<!------------- Friday ------------->
<!------------- Friday ------------->
</div>
</div>
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<li>
<li>
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Sunday 6/20--in evening inoculate 5 mL liquid cultures
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Saturday 6/19--Redo of Biobrick transformations & analysis of BL21 copper growth assay
</li>
</li>
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a>
<a id="link" href="javascript:ReverseDisplay('sunday')">See more/less</a>
<div style="display:none;" id="sunday">
<div style="display:none;" id="sunday">
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<!------------- Sunday ------------->
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<!------------- Saturday ------------->
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Extra content
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<b>Transformation troubles</b> <br/>
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<!------------- Sunday ------------->
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While the pSB74 culture grew, none of the BL21 transformants from 6/19 did, so repeated the transformations of Biobricks B0015, R0011, C0012,and J23114 into LE392 cells, once again following this <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols/transformation">transformation protocol</a> and plating onto ampicillin LB plates. The remaining transformant solutions were set aside in case of another failure.<br/>
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<b>BL21 copper growth assay analysis</b> <br/>
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Retrieved the following data regarding BL21's growth in copper solution: <br/>
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2010/f/f6/Yale-bl21.jpg" />
</div>
</div>
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It appears that BL21 is slightly more sensitive to copper than the other two strains, since 3 mM levels of copper(II) sulfate are enough to almost completely inhibit BL21's growth. <br/>
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<i> The activities of this day are also recorded on page 15 of the hard copy lab notebook </i>
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<!------------- Saturday ------------->
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</div>
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<li>
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Sunday 6/20--Observed growth of  all transformants from 6/19, so used them and the culture containing pSB74 to inoculate 5 mL liquid cultures in LB with ampicillin.  Left to grow overnight on shaker at 37˚C for miniprep the following morning. <br/>
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<i> The activities of this day are also recorded on page 16 of the hard copy lab notebook </i>
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</li>
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<!------------- LAB NOTEBOOK ------------->
<!------------- LAB NOTEBOOK ------------->

Latest revision as of 02:49, 28 October 2010

iGEM Yale

lab notebook: week 2 (6/14-6/20)

  • Monday 6/14--Redo of 6/11 assay of bacterial growth within a narrow copper(II) concentrations after analysis of data showed uniformly poor growth.
  • See more/less
  • Tuesday 6/15--Results and analysis of the 6/14 narrow concentration range growth assay as well as planning for the creation of a standard iGEM plasmid bearing the thiosulfate reductase operon (phsABC).
  • See more/less
  • Wednesday 6/16--Checked on spotted cell survival assay, collected MOPS minimal media materials & started making component solutions
  • See more/less
  • Thursday 6/17--more minimal media work and meeting, started BL21 culture
  • See more/less
  • Friday 6/18--Arrival of plasmid pSB74, transformation of Biobricks, & copper growth assays for BL21 strain.
  • See more/less
  • Saturday 6/19--Redo of Biobrick transformations & analysis of BL21 copper growth assay
  • See more/less
  • Sunday 6/20--Observed growth of all transformants from 6/19, so used them and the culture containing pSB74 to inoculate 5 mL liquid cultures in LB with ampicillin. Left to grow overnight on shaker at 37˚C for miniprep the following morning.
    The activities of this day are also recorded on page 16 of the hard copy lab notebook