iGEM 2012 — Team Clemson

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# Team:Clemson/Notebook

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Notebook

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![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [Primer Design](#week1)   ![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [Competent Cells](#week2)   ![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [DNA Purification](#week3)   ![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [Using Standard Parts](#week4) | ![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [Polymerase Chain Reaction (PCR)](#week5)   ![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [PCR Purification](#week6)   ![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [Joining DNA Fragments](#week7)   ![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [Transformation](#week8) | ![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [Testing Biphenyl Degradation Genes](#week9)   ![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [Testing Rhamnolipid Biosynthesis Genes](#week10)   ![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [BioBricks](#week11)   ![](http://f.cl.ly/items/2b3W2f0R3p3l0w2b1c1z/dna2.jpg) [References](#week12) |

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Primers
Primers were designed using sequences obtained from the  [National Center for Biotechnology Information](http://www.ncbi.nlm.nih.gov/). To design the primers, we used  [ClustalX](http://www.clustal.org/%20)  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

[![](http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png)top](#don)

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

[![](http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png)top](#don)

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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.

[![](http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png)top](#don)

---

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.

[![](http://f.cl.ly/items/3j2S3D0q3l3j1a2f143m/ua.png)top](#don)

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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:
![](https://dl.dropbox.com/u/42151864/iGEM/Sample_gel_pic.png)