Team:SDU-Denmark/labwork/Notebook/week7

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<p><b>13-08-2012 to 19-08-2012</b><br></p>
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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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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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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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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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<p><b>20-08-2012 to 26-08-2012</b></p><br>
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<p>We made a gel of the SST 1 from earlier and cut out the slice.<br>
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We did miniprep on the liquid cultures from the day before (SST 2 and FFT 9) and found out that there was no product of use from the FFT vial (bacteria must have grown badly) but excellent product from SST 2 (105,3 ηg/μL first time and 82,7 ηg/μL the second time) which was marked N1 and N2. <br>
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N1 was the first run of miniprep product and the N2 was the second. We made a gel where we ran the N1 SST on and cut out the smallest slice of the 2 fragments (approx. 1900 bp). The slice was extracted for DNA.<br><br>
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We did miniprep on the liquid cultures from the day before (again) (on FFT 9) and found out that there was no product of use from the FFT vial (bacteria must have grown badly) only 4.0ηg/μL and 2.9ηg/μL in nanodrop. Therefore did we make new liquid cultures (FFT9).<br>
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We ran nanodrop on the SST PCR from yesterday and got about 412-420 ηg/μL from the nanodrop. <br>
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The liquid cultures form the 24th was taken out of the incubator, (after approximately 47 hours) and put in the refrigerator.<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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