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Team:TU-Eindhoven/Notebook/Week1
From 2012.igem.org
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<h3>Our lab work</h3> | <h3>Our lab work</h3> | ||
| - | To <span class= "red"> isolate the bacteria and yeast </span> in which the genes are inserted, the selection marker of the vector is used. We had to put the fluorescent proteins in a new vector <span class= "red">(pYES3)</span> instead of the | + | To <span class= "red"> isolate the bacteria and yeast </span> in which the genes are inserted, the selection marker of the vector is used. We had to put the fluorescent proteins in a new vector <span class= "red">(pYES3)</span> instead of the initial one (pYES2), because the calcium channels were transported in a vector with the same immunity as the pYES2 vector. During the first attempts, we failed to put the B-GECO in the old vector so we tried it again with the new vector. We also put the proteins in a gel to test whether the B-GECO protein was produced by the E. coli. Furthermore, the <span class= "red">fluorescence</span> of R- and G-GECOs are <span class= "red">measured</span> at different Ca<sup>2+</sup> concentrations. |
The E. coli BL21 transformed with pET28a and B-GECO are subcultured and their plasmids are extracted. These plasmids are used for analysis on agarose gel. This analysis shows which colonies contain the vector plus insert. The ones which contain all necessary content will be used for the production of the B-GECO protein, which is needed to analyze the protein kinetics. We use the pET28a vector, because it is suited for production of proteins in E. coli. Protein expression with pYES2 or pYES3 is only possible in yeast. | The E. coli BL21 transformed with pET28a and B-GECO are subcultured and their plasmids are extracted. These plasmids are used for analysis on agarose gel. This analysis shows which colonies contain the vector plus insert. The ones which contain all necessary content will be used for the production of the B-GECO protein, which is needed to analyze the protein kinetics. We use the pET28a vector, because it is suited for production of proteins in E. coli. Protein expression with pYES2 or pYES3 is only possible in yeast. | ||
Latest revision as of 00:50, 27 September 2012
This week's general work
Our wiki is up and running, but the content is still sober. Parts of the project description are written, combined and corrected in English and Dutch. In the next weeks, more parts of the project description will be finished and published at the wiki. Furthermore, the budget is made for the organization. To end this first productive week properly, some of us went out for dinner and ate some delicious burgers!
Our lab work
To isolate the bacteria and yeast in which the genes are inserted, the selection marker of the vector is used. We had to put the fluorescent proteins in a new vector (pYES3) instead of the initial one (pYES2), because the calcium channels were transported in a vector with the same immunity as the pYES2 vector. During the first attempts, we failed to put the B-GECO in the old vector so we tried it again with the new vector. We also put the proteins in a gel to test whether the B-GECO protein was produced by the E. coli. Furthermore, the fluorescence of R- and G-GECOs are measured at different Ca2+ concentrations.
The E. coli BL21 transformed with pET28a and B-GECO are subcultured and their plasmids are extracted. These plasmids are used for analysis on agarose gel. This analysis shows which colonies contain the vector plus insert. The ones which contain all necessary content will be used for the production of the B-GECO protein, which is needed to analyze the protein kinetics. We use the pET28a vector, because it is suited for production of proteins in E. coli. Protein expression with pYES2 or pYES3 is only possible in yeast.
About the device
We received the device and it looks good! The initial software is created, so now we can set a specific voltage on each electrode.
Model research
Since we had to conclude that the bullfrog model does not fit the calcium homeostasis in yeast cells, we had to start new literature research. A complete week of reading articles, reading articles and sometimes we read some articles.
