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This DNA sequence for the Mature EP-B2 was the correct sequence for the protein. When conducting a fluorescence assay for the protein in lysed cells, the results proved that the correct protein was being cleaved, therefore the mature EP-B2 protein was present. The assay was successful as the mature EP-B2 protein was necessary to cleave the fluorescent part of the quencher. When designing our constructs, we chose to make two constructs, one with the mature EP-B2 sequence and one with the mature EP-B2 sequence. We were testing whether the inhibitor sequence in the Pro EP-B2 was necessary for the protein to be made. Given our successful results for the Mature EP-B2 (with no inhibitor sequence) from the lysed cells fluorescence assay, we concluded that the inhibitor sequence is not necessary for the protein to be made.
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12/04/15
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Mature EP-B2 was used in comparison to Pro EP-B2 to test whether the inhibitor sequence of the Pro EP-B2 was necessary for the protein to be made. Our fluorescence assay showed that mature EP-B2 fluoresced sufficiently meaning that the inhibitor sequence was not necessary. A fluorescence assay was performed to test presence of Mature EP-B2 in SDS lysed E.Coli. The fluorescence assay showed that Mature EP-B2 was found to be in cells, since the reading detected increasing presence of 5-FAM quencher in the samples, a quencher that is cleaved by the Mature EP-B2 protein. Similar results were also found for the Mature EP-B2. The Mature EP-B2 actually was expressed at much higher RLU than the ProEP-B2, so it is possible that the inhibitor was responsible for the smaller expression found in ProEP-B2 compared to MatureEP-B2 (graphs shown below for comparison). More testing is needed to conclude that the inhibitor was responsible for the difference in expression.
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[[File:Pro EP-B2 Rhamnose Lysed.png]]
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[[File:Mature EP-B2 Rhamnose Lysed.png]]

Latest revision as of 07:11, 5 December 2015

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Applications of BBa_M36142

User Reviews

UNIQe7b18dadb59716e3-partinfo-00000000-QINU UNIQe7b18dadb59716e3-partinfo-00000001-QINU 12/04/15


Mature EP-B2 was used in comparison to Pro EP-B2 to test whether the inhibitor sequence of the Pro EP-B2 was necessary for the protein to be made. Our fluorescence assay showed that mature EP-B2 fluoresced sufficiently meaning that the inhibitor sequence was not necessary. A fluorescence assay was performed to test presence of Mature EP-B2 in SDS lysed E.Coli. The fluorescence assay showed that Mature EP-B2 was found to be in cells, since the reading detected increasing presence of 5-FAM quencher in the samples, a quencher that is cleaved by the Mature EP-B2 protein. Similar results were also found for the Mature EP-B2. The Mature EP-B2 actually was expressed at much higher RLU than the ProEP-B2, so it is possible that the inhibitor was responsible for the smaller expression found in ProEP-B2 compared to MatureEP-B2 (graphs shown below for comparison). More testing is needed to conclude that the inhibitor was responsible for the difference in expression.

Pro EP-B2 Rhamnose Lysed.png Mature EP-B2 Rhamnose Lysed.png