"
Team:Linkoping Sweden/Results
From 2014.igem.org
| Line 100: | Line 100: | ||
This Biobrick was created by using our linearized pSB1C3 backbone and one of our ordered pUC57 plasmids. We designed primers specific only for the His-TEV sequence in our pUC57 plasmids and an overnight PCR experiment was set up for the amplification of our His-TEV sequence. The result from the PCR was controlled on an agarose-gel which indicated strongly that our PCR experiment was successful (Fig.4). Furthermore, our His-TEV sequence was digested with EcoR1 and Pst1 as well as our linearized pSB1C3 backbone and finally ligated.</p> | This Biobrick was created by using our linearized pSB1C3 backbone and one of our ordered pUC57 plasmids. We designed primers specific only for the His-TEV sequence in our pUC57 plasmids and an overnight PCR experiment was set up for the amplification of our His-TEV sequence. The result from the PCR was controlled on an agarose-gel which indicated strongly that our PCR experiment was successful (Fig.4). Furthermore, our His-TEV sequence was digested with EcoR1 and Pst1 as well as our linearized pSB1C3 backbone and finally ligated.</p> | ||
| - | <p> All 5 of our Biobrick constructs were analyzed by transforming our plasmids into electro-competent E.Coli cells followed by a PCR colony screening. The following PCR result was controlled by running an agarose-gel which indicated plasmid weights around > 3000 | + | <p> All 5 of our Biobrick constructs were analyzed by transforming our plasmids into electro-competent E.Coli cells followed by a PCR colony screening. The following PCR result was controlled by running an agarose-gel which indicated plasmid weights around > 3000 bp for all the epitope and RFP/MCherry constructs as well as a weight around > 2000 bp for our His-TEV biobrick (Fig.5). Furthermore, all biobricks were analyzed by PCR screening in combination with our His-TEV sequence specific primers. This was done to prove that the ligation was successful by the appearance of a band at the weight for His-TEV on another agarose gel (Fig.6). All 5 biobricks were sent to sequencing to further prove that the cloning of our Biobricks had worked. However, unfortunately there was a problem when we were about to send our samples for sequencing. This means that we have not received any response regarding the results yet. Furthermore, since we lack information regarding the sequencing result, we have no other choice than to wait until we can begin to express proteins.</p> |
</div> | </div> | ||
Revision as of 21:39, 16 October 2014
Our vision is to create a Biobrick which includes the sequence of the Ara h1 protein linked to a red fluorescent protein. We want to accomplish this so that we in turn can express the protein and thus provide an interaction of this protein complex with the antibodies. To ensure that the epitope-red fluorescent protein complex will bind to the Ara h1 specific IgG antibodies several ideas for Biobrick-design was brought to mind. Since we use both monoclonal antibodies specific for epitope 2 on Ara h1 and polyclonal antibodies specific for several epitopes on Ara h1 we decided to create a Biobrick consisting of epitope 2 of Ara h1 linked to a red fluorescent protein as well as a biobrick consisting of five wisely chosen epitopes (epitope 22, epitope 1, epitope 3, epitope 4, and epitope 17) from Ara h1 linked to a red fluorescent protein. However, since there are two different mutants of red fluorescent protein (called RFP and MCherry respectively) we decided to create two setups of every Biobrick combination to ensure that the best possible detection by FRET is attained, as RFP and MCherry differ slightly in their excitation and emission wavelengths. The main idea is to practically test this FRET effect in both epitope-RFP and epitope-MCherry combinations by fluorescence. The data from these tests will show us which mutant is best suited to use and thus prove that our theory works.
