Team:Clemson/Notebook

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             <li><a href="/Team:Clemson"><img src="http://f.cl.ly/items/1U3g202r2L1n3v1w3x0u/home.png" alt="" width="100" /></a><br />Home</li>
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<div id = "weeklylog">
<div id = "weeklylog">
<br>
<br>
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<font size = "5"><div align = "center"><u><a name="don">Weekly Log<hr></u></a></div></font>
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<font size = "5"><div align = "center"><u><a name="don">Notebook<hr></u></a></div></font>
<br>
<br>
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<td>
<td>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" width=50px height=50px/>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" width=50px height=50px/>
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<a href="#week1">Week 1</a><br><br>
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<a href="#week1">Primer Design</a><br><br>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg"  width=50px height=50px/>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg"  width=50px height=50px/>
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<a href="#week2">Week 2</a><br><br>
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<a href="#week2">Competent Cells</a><br><br>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" width=50px height=50px/>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" width=50px height=50px/>
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<a href="#week3">Week 3</a><br><br>
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<a href="#week3">DNA Purification</a><br><br>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg"  width=50px height=50px/>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg"  width=50px height=50px/>
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<a href="#week4">Week 4</a><br><br>
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<a href="#week4">Using Standard Parts</a><br><br>
</td>
</td>
<td>
<td>
<img src ="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
<img src ="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
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<a href="#week5">Week 5</a><br><br>
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<a href="#week5">Polymerase Chain Reaction (PCR)</a><br><br>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
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<a href="#week6">Week 6</a><br><br>
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<a href="#week6">PCR Purification</a><br><br>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
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<a href="#week7">Week 7</a><br><br>
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<a href="#week7">Joining DNA Fragments</a><br><br>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
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<a href="#week8">Week 8</a><br><br>
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<a href="#week8">Transformation</a><br><br>
</td>
</td>
<td>
<td>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
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<a href="#week9">Week 9</a><br><br>
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<a href="#week9">Testing Biphenyl Degradation Genes</a><br><br>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
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<a href="#week10">Week 10</a><br><br>
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<a href="#week10">Testing Rhamnolipid Biosynthesis Genes</a><br><br>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
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<a href="#week11">Week 11</a><br><br>
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<a href="#week11">BioBricks</a><br><br>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
<img src="http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg" height=50px width=50px>
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<a href="#week12">Week 12</a><br><br>
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<a href="#week12">References</a><br><br>
</td>
</td>
</table>
</table>
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<hr>
<hr>
-
<a name="week1">This Is Week One</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
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<a name="week1"><u>Primers</u></a><br><br>Primers were designed using sequences obtained from the <a href="http://www.ncbi.nlm.nih.gov/"> National Center for Biotechnology Information</a>. To design the primers, we used <a href="http://www.clustal.org/ "> ClustalX </a> to align sequences and observe conserved regions of DNA. 
-
<a name="week2">This Is Week Two</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
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<br><br>Organisms:<ul><br>
-
<a name="week3">This Is Week Three</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
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<li>&nbsp; • Pseudomonas aeruginosa</li>
-
<a name="week4">This Is Week Four</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
+
<li>&nbsp; • Sphingomonas yanoikuyae</li>
-
<a name="week5">This Is Week Five</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
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<li>&nbsp; • Sphingomonas paucimobilis</li>
-
<a name="week6">This Is Week Six</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
+
<li>&nbsp; • Sphingomonas aromaticivorans</li>
-
<a name="week7">This Is Week Seven</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
+
<li>&nbsp; • Novosphingobium pentaromativorans</li>
-
<a name="week8">This Is Week Eight</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
+
</ul>
-
<a name="week9">This Is Week Nine</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
+
<br>Operons:<ul><br>
-
<a name="week10">This Is Week Ten</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
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<li> &nbsp;• Rhamnolipid synthesis genes:</li>
-
<a name="week11">This Is Week Eleven</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
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<li> &nbsp;&nbsp; o rmlBDAC</li>
-
<a name="week12">This Is Week Twelve</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
+
<li> &nbsp;&nbsp; o rhlABG</li>
 +
<li> &nbsp;&nbsp; o MFST-rhlC</li>
 +
<li> &nbsp;&nbsp; o algC</li>
 +
<br>
 +
<li> &nbsp;• Biphenyl degradation:</li>
 +
<li> &nbsp;&nbsp; o bphA1a</li>
 +
<li> &nbsp;&nbsp; o bphA2</li>
 +
<li> &nbsp;&nbsp; o bphA3</li>
 +
<li> &nbsp;&nbsp; o bphA4</li>
 +
<li> &nbsp;&nbsp; o bphB</li>
 +
<li> &nbsp;&nbsp; o bphC</li>
 +
<li> &nbsp;&nbsp; o bphD</li>
 +
</ul>
 +
 
 +
<br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
 +
<a name="week2"><u>Competent Cells</u></a><br><br>
 +
 +
Electrocompetent cells were created with Escherichia coli JM109 following previously published protocol (Sambrook, 2001).
 +
 +
<br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
 +
<a name="week3"><u>DNA Purification</u></a><br><br>
 +
 +
DNA was extracted and purified from P. aeruginosa, S. yanoikuyae, and S. paucimobilis using Promega’s Wizard Genomic DNA Purification Kit. 
 +
 
 +
<br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
 +
<a name="week4"><u>Using Standard Parts</u></a><br><br>
 +
 
 +
The following standard parts were used:<ul><br>
 +
<li>&nbsp; • BBa_I13521 – RFP</li>
 +
<li>&nbsp; • BBa_I13522 – GFP</li>
 +
<li>&nbsp; • BBa_E0435 – lacZ alpha</li>
 +
</ul><br>
 +
Standard kit instructions were followed to rehydrate the DNA.  The rehydrated plasmid DNA was then transformed into electrocompetent E. coli JM109 and plated onto selective media.  Pure cultures were obtained and stock cultures made.
 +
<br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
 +
<a name="week5"><u>Polymerase Chain Reaction (PCR)</u></a><br><br>
 +
PCR was done using colony PCR or from template DNA extracted from genomic DNA isolation.  Various PCR conditions were used to optimize the PCR products:
 +
<ul><br>
 +
<li>&nbsp; • Varied annealing temperatures</li>
 +
<li>&nbsp; • Varied magnesium levels to optimize polymerase activity</li>
 +
<li>&nbsp; • Varied buffers</li><br></ul>
 +
The following polymerases were used:
 +
<ul><br>
 +
<li>&nbsp; • GoTaq polymerase from Promega</li>
 +
<li>&nbsp; • GoTag Long PCR Master Mix from Promega</li>
 +
<li>&nbsp; • Q5 High Fidelity from New England BioLabs</li>
 +
<li>&nbsp; • OneTaq Master Mix from New England BioLabs</li>
 +
<br>
 +
</ul>
 +
PCR reactions were checked by DNA gel electrophoresis.  An example of one such gel is below:
 +
[INSERT GEL PIC AND LABEL]
 +
<br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
 +
 
 +
<a name="week6"><u>PCR Purification</u></a><br><br>
 +
PCR products were purified using two different protocols based on the quality of the electrophoresed DNA gels:
 +
<ul><br>
 +
<li>&nbsp; • QIAGEN QIAquick PCR Purification Kit was used for DNA that gave one clear band on the gel.  </li>
 +
<li>&nbsp; • QIAGEN QIAquick Gel Extract Kit was used to extract DNA that show multiple PCR products.  </li>
 +
</ul>
 +
<br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
 +
 
 +
 
 +
<a name="week7"><u>Joining DNA Fragments</u></a><br><br>
 +
 
 +
PCR fragments were joined by two methods:<ul><br>
 +
<li>&nbsp; • Chimeric PCR – Fragments of DNA are often joined together by restriction endonuclease digestion followed by ligation, but the fragments of DNA can also be joined together using just PCR. We used the following method to join multiple fragments of double-stranded DNA products together:</li><br><br>
 +
<li>&nbsp;&nbsp;&nbsp; • PCR products rhlA and rhlG (for Rhamnolipid biosynthesis) had already been amplified from P. aeruginosa, and we wanted to join them together so they could be part of a single operon on our recombinant plasmid. A new primer was designed that would serve as the forward primer for rhlG such that it contained a 5’ extension with the ending sequence (approximately the last 20 bp) of rhlAB. The original reverse primer for rhlG could be used to amplify, and the resulting PCR product contained a sequence identical to the end of rhlAB.</li>
 +
<br><br>
 +
<img src="http://f.cl.ly/items/1H0k3u2l2w3J2A3Z2g1T/note1.png" height="200px" width="800px">
 +
<br><br>
 +
<li>&nbsp;&nbsp;&nbsp; • Next the two PCR fragments were combined along with only the forward primer for rhlAB and the reverse primer of rhlG. The denaturing step of PCR separates the double-stranded DNA, and during the annealing step, the overlapping sequences of rhlAB and rhlG anneal to each other along with the primers. Then during the elongation step, a single double-stranded DNA product is formed that contains both of the original DNA fragments.
 +
<br><br>
 +
<img src="http://f.cl.ly/items/2k2V2l3E3V3J1a38011C/note2.png" height="200px" width="800px">
 +
<br><br>
 +
 
 +
</li>
 +
<li>&nbsp; • Restriction digest and ligation – Restriction enzymes from New England BioLabs and Promega, andT4 ligase from Promaga were used.</li>
 +
<br>
 +
</ul>
 +
 
 +
<br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
 +
 
 +
 
 +
<a name="week8"><u>Transformation</u></a<br><br>
 +
 
 +
DNA fragments were ligated into plasmids [INSERT NAME(S) of plasmids] and transformed into E. coli JM109.  Plasmids were purified and sequenced using Sanger’s sequencing to verify successful cloning.
 +
 
 +
<br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
 +
<a name="week9"><u>Testing Biphenyl Degradation Genes</u></a><br><br>
 +
 
 +
 
 +
</a><br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
 +
<a name="week10"><u>Testing Rhamnolipid Biosynthesis Genes</u><br><br>
 +
 
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<br><br><br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
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<a name="week11"><u>BioBricks</u></a><br><br>
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Three BioBricks were designed for rhamnolipid biosynthethis.  Each BioBrick contains a promoter, three to four coding sequences for rhamnolipid biosynthethis enzymes, a reporter gene, ribosome binding sites for all coding sequences, and a double terminator. 
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<br><br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>
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<a name="week12"><u>References</u></a><br><br>
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Sambrook, J.  2001. Molecular Cloning.  Cold Spring Harbor, Ney York: Cold Spring Harbor Laboratory Press. 
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<br><a href="#don"><p align="center"><img src="http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png" height="20px" width="20px" ><font size="2">top</a></font></img></p><hr>

Revision as of 23:03, 30 September 2012

CU iGEM




Primers

Primers were designed using sequences obtained from the National Center for Biotechnology Information. To design the primers, we used ClustalX to align sequences and observe conserved regions of DNA.

Organisms:

  •   • Pseudomonas aeruginosa
  •   • Sphingomonas yanoikuyae
  •   • Sphingomonas paucimobilis
  •   • Sphingomonas aromaticivorans
  •   • Novosphingobium pentaromativorans

Operons:

  •  • Rhamnolipid synthesis genes:
  •    o rmlBDAC
  •    o rhlABG
  •    o MFST-rhlC
  •    o algC

  •  • Biphenyl degradation:
  •    o bphA1a
  •    o bphA2
  •    o bphA3
  •    o bphA4
  •    o bphB
  •    o bphC
  •    o bphD


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Competent Cells

Electrocompetent cells were created with Escherichia coli JM109 following previously published protocol (Sambrook, 2001).

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DNA Purification

DNA was extracted and purified from P. aeruginosa, S. yanoikuyae, and S. paucimobilis using Promega’s Wizard Genomic DNA Purification Kit.

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Using Standard Parts

The following standard parts were used:

  •   • BBa_I13521 – RFP
  •   • BBa_I13522 – GFP
  •   • BBa_E0435 – lacZ alpha

Standard kit instructions were followed to rehydrate the DNA. The rehydrated plasmid DNA was then transformed into electrocompetent E. coli JM109 and plated onto selective media. Pure cultures were obtained and stock cultures made.

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Polymerase Chain Reaction (PCR)

PCR was done using colony PCR or from template DNA extracted from genomic DNA isolation. Various PCR conditions were used to optimize the PCR products:

  •   • Varied annealing temperatures
  •   • Varied magnesium levels to optimize polymerase activity
  •   • Varied buffers

The following polymerases were used:

  •   • GoTaq polymerase from Promega
  •   • GoTag Long PCR Master Mix from Promega
  •   • Q5 High Fidelity from New England BioLabs
  •   • OneTaq Master Mix from New England BioLabs

PCR reactions were checked by DNA gel electrophoresis. An example of one such gel is below: [INSERT GEL PIC AND LABEL]

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PCR Purification

PCR products were purified using two different protocols based on the quality of the electrophoresed DNA gels:

  •   • QIAGEN QIAquick PCR Purification Kit was used for DNA that gave one clear band on the gel.
  •   • QIAGEN QIAquick Gel Extract Kit was used to extract DNA that show multiple PCR products.


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Joining DNA Fragments

PCR fragments were joined by two methods:

  •   • Chimeric PCR – Fragments of DNA are often joined together by restriction endonuclease digestion followed by ligation, but the fragments of DNA can also be joined together using just PCR. We used the following method to join multiple fragments of double-stranded DNA products together:


  •     • PCR products rhlA and rhlG (for Rhamnolipid biosynthesis) had already been amplified from P. aeruginosa, and we wanted to join them together so they could be part of a single operon on our recombinant plasmid. A new primer was designed that would serve as the forward primer for rhlG such that it contained a 5’ extension with the ending sequence (approximately the last 20 bp) of rhlAB. The original reverse primer for rhlG could be used to amplify, and the resulting PCR product contained a sequence identical to the end of rhlAB.




  •     • Next the two PCR fragments were combined along with only the forward primer for rhlAB and the reverse primer of rhlG. The denaturing step of PCR separates the double-stranded DNA, and during the annealing step, the overlapping sequences of rhlAB and rhlG anneal to each other along with the primers. Then during the elongation step, a single double-stranded DNA product is formed that contains both of the original DNA fragments.



  •   • Restriction digest and ligation – Restriction enzymes from New England BioLabs and Promega, andT4 ligase from Promaga were used.



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Transformation
DNA fragments were ligated into plasmids [INSERT NAME(S) of plasmids] and transformed into E. coli JM109. Plasmids were purified and sequenced using Sanger’s sequencing to verify successful cloning.

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Testing Biphenyl Degradation Genes





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Testing Rhamnolipid Biosynthesis Genes





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BioBricks

Three BioBricks were designed for rhamnolipid biosynthethis. Each BioBrick contains a promoter, three to four coding sequences for rhamnolipid biosynthethis enzymes, a reporter gene, ribosome binding sites for all coding sequences, and a double terminator.

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References

Sambrook, J. 2001. Molecular Cloning. Cold Spring Harbor, Ney York: Cold Spring Harbor Laboratory Press.

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