Difference between revisions of "Part:BBa M36242:Experience"
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Our Mature EP-B2 construct provided inconclusive results. We could not confirm whether the mature EP-B2 protein was being secreted. We conducted a fluorescence assay with the media from the cells with the mature EP-B2 sequence. The results of the assay did not show increased fluorescence and therefore, we cannot conclude that the protein was secreted. A possible explanation for these results is that the amount of protein in the media was in such a small concentration that the fluorescence from the mature EP-B2 cleaving the fluorescent part of the quencher could not be detected. | Our Mature EP-B2 construct provided inconclusive results. We could not confirm whether the mature EP-B2 protein was being secreted. We conducted a fluorescence assay with the media from the cells with the mature EP-B2 sequence. The results of the assay did not show increased fluorescence and therefore, we cannot conclude that the protein was secreted. A possible explanation for these results is that the amount of protein in the media was in such a small concentration that the fluorescence from the mature EP-B2 cleaving the fluorescent part of the quencher could not be detected. | ||
| − | [[File:Mature EP-B2 | + | [[File:Media Mature EP-B2 Rhamnose.png]] |
| − | [[File:Mature EP-B2 | + | [[File:Media Mature EP-B2 Glucose.png]] |
Since our fluorescence assay of the media did not provide any clear results, we conducted a second fluorescence assay with lysed cells by using a SDS lysis buffer. The purpose of this assay was to determine if the protein was being produced and was present in the cell. This assay did show that the protein was in the cell. The results all showed increased fluorescence over an hour, proving that the mature EP-B2 protein was present to cleave the fluorescent part of the quencher, which was then detected by the plate reader. | Since our fluorescence assay of the media did not provide any clear results, we conducted a second fluorescence assay with lysed cells by using a SDS lysis buffer. The purpose of this assay was to determine if the protein was being produced and was present in the cell. This assay did show that the protein was in the cell. The results all showed increased fluorescence over an hour, proving that the mature EP-B2 protein was present to cleave the fluorescent part of the quencher, which was then detected by the plate reader. | ||
Revision as of 05:33, 5 December 2015
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Our Mature EP-B2 construct provided inconclusive results. We could not confirm whether the mature EP-B2 protein was being secreted. We conducted a fluorescence assay with the media from the cells with the mature EP-B2 sequence. The results of the assay did not show increased fluorescence and therefore, we cannot conclude that the protein was secreted. A possible explanation for these results is that the amount of protein in the media was in such a small concentration that the fluorescence from the mature EP-B2 cleaving the fluorescent part of the quencher could not be detected.
File:Media Mature EP-B2 Glucose.png
Since our fluorescence assay of the media did not provide any clear results, we conducted a second fluorescence assay with lysed cells by using a SDS lysis buffer. The purpose of this assay was to determine if the protein was being produced and was present in the cell. This assay did show that the protein was in the cell. The results all showed increased fluorescence over an hour, proving that the mature EP-B2 protein was present to cleave the fluorescent part of the quencher, which was then detected by the plate reader.
We subjected the cells to varying levels of rhamnose and glucose levels. For rhamnose, the expected results were found as fluorescence increased in every case. For glucose, we should have found that fluorescence should have stayed the same over the hour, but our results all showed the fluorescence increasing over the course of the hour. A possible explanation for this is that the concentration of the mature EP-B2 protein was already built up very high in the cell and glucose inhibition had no effects. Also there was no clear pattern in the difference in fluorescence over the hour for varying levels of glucose or rhamnose which also could be explained by a large buildup of the mature EP-B2 protein in the cells before the assay was conducted.
Final Conclusions: We cannot conclude that the YebF secretion vector was working properly and secreting the mature EP-B2 but we can confirm that the mature EP-B2 protein was present in the cells.