Part:BBa_K4181024
Dsup- K198I_V195I
We directed the evolution of the DSUP protein to change lysine at position 198 to isoleucine and valine at position 195 to isoleucine.
Verification of Dsup
In our design, we add his-tag to Dsup protein and this protein is theoretically bind to the bacterial DNA inside the cell. So, it is a practical idea to detect Dsup protein use Western Blot. Anti-his tag antigen from mouse was used as the primary antigen and the secondary antigen is bind with HRP. The result of our WB test is showing below:
Figure 1.The result of Wester blotting, sample names are on the top. It can be clearly recognize that two lines are visible while the WT with no line, despite of the improper position.
From the photo above, we can recgonize a relatively clear line in the middle of the marker ranging from 55kD to 70kD in the IPTG induced sample and only-plasmid samole, while no line can be observed by the control sample, which is the wildtype WB800N with no plasmid transferred. It is not very obvious and this indicates a relatively low propotion of targeted protein. However, before we conducted the WB assay, we use Bradford protein assay to determine the concentration of total protein in solution, which is extracted directly after treatment of lysozyme. It provided a standard curve of our samples compared to the the standard concentration. Therefore, we indicate that this is because of the following reasons：firstly, as a bacteria chassis known for its production of proteins, B.subtilis had lots of proteins intrinsically; second, we added 30μl protein marker because of the total protein quntity is beyond our expectation so we decided to add more, but this in turn made the marker so bright when we took photo, causing troubles to modulate the time of light exposure; thirdly, there may need some adjustment to our IPTG induction time or to our plasmid. However, Dsup protein, which is 1338bp in sequence, is 42.8kD if calculated by the rule of thumb. Therefore, we had to find out what is wrong in our experiments or the reasons behind this relatively larger results. After disccustion with our advisors and instructors, we thought the result was produced because of the following reasons: firstly, as we could see from the picture, the standard lines of the markers were also a bit close, which means the time of running the electrophoresis is a little bit short and thus there existed the possibility that the different quantity and quanlity of proteins hadn't seperated enough before we stopping the reaction and beginning membrane transfer. Therefore, our instructor suggested that we can treat this result as a successful positive one which is not so solid and perfect. Besides, he advised us to do another test to fully validate the expression of Dsup, thus we conducted a straight-forward SDS-PAGE to make further improvement. Besides, as the WB indicated the low production rate of our protein, we decided to entend our induction time into 9 hours and do not refer to the total protein quantity as a parameter. By the same time, we tried to look for a stronger-promoter because we suspected the lac operon used now may not be potent and productive enough.
We conducted SDS-PAGE as a complement and verification to the result of Western blot. The procedures were much less and we used Coomassie Brilliant Blue G250 to stain. The result is shown below:
Figure 2.Result of SDS-PAGE to detect Dsup protein.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 515
Illegal PstI site found at 1139 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 515
Illegal PstI site found at 1139 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 163
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 515
Illegal PstI site found at 1139 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 515
Illegal PstI site found at 1139
Illegal AgeI site found at 121 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 197
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