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Team:TU-Eindhoven/Notebook/Week11
From 2012.igem.org
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<p>This was the last full week of lab work before the wiki-freeze. We had to round up our work, culturing the last batches of yeast and E. coli for this project. The yeast was meant for another test with the device and the E. coli were used to characterize the BioBricks and produce more GECO-protein for characterization of the fluorescent response.</p> | <p>This was the last full week of lab work before the wiki-freeze. We had to round up our work, culturing the last batches of yeast and E. coli for this project. The yeast was meant for another test with the device and the E. coli were used to characterize the BioBricks and produce more GECO-protein for characterization of the fluorescent response.</p> | ||
| - | <p>The BioBricks were finished last week and we sent them for sequencing. The results came back and there was a little surprise, though not a pleasant one: The GECOs contain a PstI site in the middle of the gene. This is a bit of a problem, since PstI cannot be used in building BioBricks from GECOs. Fortunately there are no other restriction sites of the BioBrick restriction enzymes in the GECO genes, so it is still possible to build the BioBricks, although they will not be fully compatible with the BioBrick standard <a href="http://tools.ietf.org/html/rfc10" target="_blank">RFC 10</a>.</p> | + | <p>The BioBricks were finished last week and we sent them for sequencing. The results came back and there was a little surprise, though not a pleasant one: The GECOs contain a PstI site in the middle of the gene. This is a bit of a problem, since PstI cannot be used in building BioBricks from GECOs. Fortunately there are no other restriction sites of the BioBrick restriction enzymes in the GECO genes, so it is still possible to build the BioBricks, although they will not be fully compatible with the BioBrick standard <html><a href="http://tools.ietf.org/html/rfc10" target="_blank">RFC 10</a></html>.</p> |
<p>We can still build BioBricks from GECOs: We have to cut the BioBrick suffix-side of the backbone pSB1C3 and the tail of the GECO-DNA with SpeI, the other enzyme on the BioBrick suffix side, and the ligate these together. This would yield a BioBrick that can be amplified in E. coli and used by other teams. Ideally, the PstI-sites would be removed by site-directed mutagenesis. This will be left as a challenge to later teams.</p> | <p>We can still build BioBricks from GECOs: We have to cut the BioBrick suffix-side of the backbone pSB1C3 and the tail of the GECO-DNA with SpeI, the other enzyme on the BioBrick suffix side, and the ligate these together. This would yield a BioBrick that can be amplified in E. coli and used by other teams. Ideally, the PstI-sites would be removed by site-directed mutagenesis. This will be left as a challenge to later teams.</p> | ||
Revision as of 10:53, 25 September 2012
Working hard
In order to finish the presentation before Friday, we worked hard and stayed late at the university. On Friday we showed our presentation to our supervisors and they gave feedback. Furthermore, two of us went to Oud-Beijerland (near Rotterdam) to visit a high-school, give a lecture about synthetic biology and supervise an experiment about bacteria.
Progression in the lab
This was the last full week of lab work before the wiki-freeze. We had to round up our work, culturing the last batches of yeast and E. coli for this project. The yeast was meant for another test with the device and the E. coli were used to characterize the BioBricks and produce more GECO-protein for characterization of the fluorescent response.
The BioBricks were finished last week and we sent them for sequencing. The results came back and there was a little surprise, though not a pleasant one: The GECOs contain a PstI site in the middle of the gene. This is a bit of a problem, since PstI cannot be used in building BioBricks from GECOs. Fortunately there are no other restriction sites of the BioBrick restriction enzymes in the GECO genes, so it is still possible to build the BioBricks, although they will not be fully compatible with the BioBrick standard RFC 10.
We can still build BioBricks from GECOs: We have to cut the BioBrick suffix-side of the backbone pSB1C3 and the tail of the GECO-DNA with SpeI, the other enzyme on the BioBrick suffix side, and the ligate these together. This would yield a BioBrick that can be amplified in E. coli and used by other teams. Ideally, the PstI-sites would be removed by site-directed mutagenesis. This will be left as a challenge to later teams.
In order to characterize the BioBricks we monitored the growth rate of BL21 E. coli carrying GECO constructs and plotted a growth curve. The growth rate seems to be unaffected by the construct. We induced to cells to produce GECO protein for futher characterization of the in vitro fluorescent response. The cells that produced protein were lysed with BlockBuster, pelleted and filtered on a Ni-colum (this takes a lot of time, days actually).
