Team:SDU-Denmark/labwork/Notebook/week7

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     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week8">8th week      </a></span>    </regulartext></td>
     <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week8">8th week      </a></span>    </regulartext></td>
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    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week9">9th week</a></span>    </regulartext></td>
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    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week10">10th week</a></span>    </regulartext></td>
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    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week11">11th week</a></span>    </regulartext></td>
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    <span class="classred"><a href="https://2012.igem.org/Team:SDU-Denmark/labwork/Notebook/week12">12th week</a></span>    </regulartext></td>
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<p><b>13-08-2012 to 19-08-2012</b><br></p>
<p><b>13-08-2012 to 19-08-2012</b><br></p>
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<h2>Preparing material for sequencing</h2>
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<p>
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<p>The concentration of the FFT plasmid was low. Too low to be sent for sequencing, so a liquid culture was prepared for overnight incubation in the hopes of obtaining a higher concentration.</br></br>
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We needed to address a problem with our genes and this was where our lack of experience finaly hit us. The transition from eukaryotic cells to bacterial cells has come with an untill now unforseen complication: the ribosomal binding site for eukaryotic cells(Kozak sequence), does not bind bacterial ribosomes, so we need to insert the bacterial version(Shine-Dalgarno sequence). <br/>
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From nanodrop:</br>
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Bacterial ribosomes bind to different sequences AGGAGG in a different distance, 6-7bp, from the coding sequence start codon ATG, while eukaryotic cells like plant cells have a ribosomal binding site CACC just in front of the start codon. Due to the original primers we designed for the coding sequences of both 1-FFT and 1-SST, both sequences still contained the Kozak sequences. We decided to let the genes hold on to the kozak sequence and just mutate the shine-dalgarno sequence hardcoded into the sequence between the restriction site and the start codon. This way our gene will be able to be transcribed in BOTH eukaryotic and prokaryotic cells.<br/><br/>
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SST 1: 116ηg/μL</br>
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SST 2: 135ηg/μL</br>
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New primers were designed and ordered to accommodate the changes needed for the Shine-Dalgarno sequence in appropriate distance from the start codon for both genes.<br/><br/>
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FFT 3: 57ηg/μL</br>
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FFT 3: 41ηg/μL</br></br>
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Meanwhile, we achieved high enough concentration for the one 1-FFT colony without the undesired EcoRI and sent it for sequencing along with 1-SST.<br/>
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We need to address a problem with our genes. The transition from eukaryotic cells to bacterial cells has come with an untill now unforseen complication: the ribosomal binding site for eukaryotic cells(Kozak sequence), does not bind bacterial ribosomes, so we need to insert the bacterial version(Shine-Dalgarno). <h1>LARS SKAL UDDYBE!!</h1></br></br>
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<p>The cultures from overnight had immensely low concentrations:</br>
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</p>
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SST 1: 13ηg/μL</br>
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SST 2: 22ηg/μL</br>
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FFT 3: 23ηg/μL</br></br>
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A new liquid culture was prepared from the former liquid cultures for overnight incubation. To get high concentrations, they were incubated at 37°C for 22 hours.</br>
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New primers were designed and ordered to accommodate the changes needed for the Shine-Dalgarno sequence in appropriate distance from the start codon (6-7 nucleotides)</br>
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The O.N. cultures was purified using the GeneJET Plasmid Miniprep Kit.</br></br> The results from nanodrop were excellent:</br>
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SST 1:        76 ηg/μL</br>
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SST 2: too low    ~25ηg/μL</br>
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FFT 3:            179 ηg/μL</br></br>
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To reach a concentration of 50-100 ηg/μL we dilluted the 49μL buffer+plasmid with another 40μL buffer, leaving it at around 98ηg/μL.</br>
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For SST, the material in high concentration that were obtained monday were used,
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and the material was sent for sequencing. A spare 5-7 μL were saved on freezer.</br></p>
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Latest revision as of 02:14, 27 September 2012

iGEM TEAM ::: SDU-DENMARK

Laboratory Notebook

1st week 2nd week 3th week 4th week
5th week 6th week 7th week 8th week
9th week 10th week 11th week 12th week

13-08-2012 to 19-08-2012

We needed to address a problem with our genes and this was where our lack of experience finaly hit us. The transition from eukaryotic cells to bacterial cells has come with an untill now unforseen complication: the ribosomal binding site for eukaryotic cells(Kozak sequence), does not bind bacterial ribosomes, so we need to insert the bacterial version(Shine-Dalgarno sequence).
Bacterial ribosomes bind to different sequences AGGAGG in a different distance, 6-7bp, from the coding sequence start codon ATG, while eukaryotic cells like plant cells have a ribosomal binding site CACC just in front of the start codon. Due to the original primers we designed for the coding sequences of both 1-FFT and 1-SST, both sequences still contained the Kozak sequences. We decided to let the genes hold on to the kozak sequence and just mutate the shine-dalgarno sequence hardcoded into the sequence between the restriction site and the start codon. This way our gene will be able to be transcribed in BOTH eukaryotic and prokaryotic cells.

New primers were designed and ordered to accommodate the changes needed for the Shine-Dalgarno sequence in appropriate distance from the start codon for both genes.

Meanwhile, we achieved high enough concentration for the one 1-FFT colony without the undesired EcoRI and sent it for sequencing along with 1-SST.

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