Difference between revisions of "Part:BBa M36242:Experience"

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12/04/15
 
12/04/15
  
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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.  
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To test the presence of Mature EP-B2 in the cell, we initially performed a 60 minute fluorescence assay of Mature EP-B2 transformed E.Coli media containing substrate quencher 5-FAM at various levels of rhamnose and glucose, at 455 excitation and 515 emission (graphs below). The Mature EP-B2 vector is rhamnose inducible and glucose repressible, so we expected that the fluorescence would show discernible increase with increasing rhamnose over time and a decrease with increasing glucose. However, we could not come to a conclusion on whether or not the protein was present in this assay due to the fluorescence of the 5-FAM in the media showing inconclusive patterns in the presence of both the rhamnose and glucose.
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[[File:Media Rhamnose Mature EP-B2.png]]
 
[[File:Media Rhamnose Mature EP-B2.png]]
 
[[File:Media Glucose Mature EP-B2.png]]
 
[[File:Media Glucose Mature EP-B2.png]]
  
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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 transformed mature EP-B2 E.Coli cells lysed with 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.  
  
 
[[File:Mature EP-B2 Rhamnose 1.png]]
 
[[File:Mature EP-B2 Rhamnose 1.png]]
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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.  
 
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.  
  
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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.
+
Final Conclusions: We cannot conclude that the YebF secretion vector was working properly due to the little effect glucose and rhamnose had on the vector. We can confirm that the mature EP-B2 protein was present in the cells.

Latest revision as of 05:53, 5 December 2015

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12/04/15

To test the presence of Mature EP-B2 in the cell, we initially performed a 60 minute fluorescence assay of Mature EP-B2 transformed E.Coli media containing substrate quencher 5-FAM at various levels of rhamnose and glucose, at 455 excitation and 515 emission (graphs below). The Mature EP-B2 vector is rhamnose inducible and glucose repressible, so we expected that the fluorescence would show discernible increase with increasing rhamnose over time and a decrease with increasing glucose. However, we could not come to a conclusion on whether or not the protein was present in this assay due to the fluorescence of the 5-FAM in the media showing inconclusive patterns in the presence of both the rhamnose and glucose.


Media Rhamnose Mature EP-B2.png Media Glucose Mature EP-B2.png

Since our fluorescence assay of the media did not provide any clear results, we conducted a second fluorescence assay with transformed mature EP-B2 E.Coli cells lysed with 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.

Mature EP-B2 Rhamnose 1.png Mature EP-B2 Rhamnose 2.png

Mature EP-B2 Glucose 1.png Mature EP-B2 Glucose Difference.png

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 due to the little effect glucose and rhamnose had on the vector. We can confirm that the mature EP-B2 protein was present in the cells.