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Team:Copenhagen/Results
From 2012.igem.org
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<table id="graa" cellpadding=20px><td width="890px" height="100%" valign="top" ><p align="justify"><h2>ProC and YFI</h2> | <table id="graa" cellpadding=20px><td width="890px" height="100%" valign="top" ><p align="justify"><h2>ProC and YFI</h2> | ||
| - | We performed USER cloning and managed to assemble ProC and YF1 in the pSB1C3 backbone. This has been submitted as a BioBrick. | + | We performed USER cloning and managed to assemble ProC and YF1 in the pSB1C3 backbone. This has been submitted as a BioBrick. <br><br> |
| - | <h2>Lux Cassette</h2> | + | <h2>Lux Cassette</h2> |
| - | After trying to amplify the Lux cassette from the BioBrick plate several times, we checked the sequence from the parts registry using BLAST and found that it was an <i>E.coli</i> surface protein and not the Lux cassette. | + | After trying to amplify the Lux cassette from the BioBrick plate several times, we checked the sequence (from sequencing) in the parts registry using BLAST and found that it was an <i>E.coli</i> surface protein and not the Lux cassette. |
We contacted Thomas Knight from the iGEM headquarter who kindly provided us with a Lux Cassette. This contained one of the “banned” Xbal restriction sites, which we have removed and the Lux cassette is submitted as a BioBrick. | We contacted Thomas Knight from the iGEM headquarter who kindly provided us with a Lux Cassette. This contained one of the “banned” Xbal restriction sites, which we have removed and the Lux cassette is submitted as a BioBrick. | ||
We have transformed the Lux cassette from Thomas Knight into E.cloni® 5α (purchased from Lucigen). | We have transformed the Lux cassette from Thomas Knight into E.cloni® 5α (purchased from Lucigen). | ||
| - | As a control we exploited that the gene cassette had a LacI promotor and we confirmed, after adding IPTG, that the Lux cassette is able to make the bacteria bioluminescent. | + | As a control we exploited that the gene cassette had a LacI promotor and we confirmed, after adding IPTG, that the Lux cassette is able to make the bacteria bioluminescent. <br><br> |
<h2>Experiences with USER cloning</h2> | <h2>Experiences with USER cloning</h2> | ||
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For ProC (35 bp) and YF1 (1131bp) a x10.000 dilution of ProC was necessary in order to assemble the two pieces. | For ProC (35 bp) and YF1 (1131bp) a x10.000 dilution of ProC was necessary in order to assemble the two pieces. | ||
This very problem has made it hard to assemble our construct since we have pieces ranging from 35bp to almost 6000bp. | This very problem has made it hard to assemble our construct since we have pieces ranging from 35bp to almost 6000bp. | ||
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Revision as of 10:42, 26 September 2012
ProC and YFIWe performed USER cloning and managed to assemble ProC and YF1 in the pSB1C3 backbone. This has been submitted as a BioBrick.Lux CassetteAfter trying to amplify the Lux cassette from the BioBrick plate several times, we checked the sequence (from sequencing) in the parts registry using BLAST and found that it was an E.coli surface protein and not the Lux cassette. We contacted Thomas Knight from the iGEM headquarter who kindly provided us with a Lux Cassette. This contained one of the “banned” Xbal restriction sites, which we have removed and the Lux cassette is submitted as a BioBrick. We have transformed the Lux cassette from Thomas Knight into E.cloni® 5α (purchased from Lucigen). As a control we exploited that the gene cassette had a LacI promotor and we confirmed, after adding IPTG, that the Lux cassette is able to make the bacteria bioluminescent.Experiences with USER cloningWe have found, that in order to make USER cloning work, it is very important that the molecular concentration of the parts, that are to be assembled, is as equal as possible. This means that a small piece must be of lower concentration in order to assemble it to a larger piece so the number of each piece is approximately the same. For ProC (35 bp) and YF1 (1131bp) a x10.000 dilution of ProC was necessary in order to assemble the two pieces. This very problem has made it hard to assemble our construct since we have pieces ranging from 35bp to almost 6000bp. |
