Part:BBa_K1104102:Experience
Expression check
We ligated this part with LacI regulated promoter. In order to know whether this biobrick is correctly expressed in the E. coli, we have done digestion on XbaI and PstI cutting site to proof that the length of our biobrick is right. So there is only the coding sequence of RFP-FimH instead of RFP only. The picture on the right is the result of the electrophoresis after the digestion. Secondly, through observing the fluorescence emitted by the E. coli we can be sure that we have produced the protein that we want, which is the RFP-FimH fusion protein. The result is as the picture below.
RFP-FimH fusion protein functional check
After cloning RFP-FimH gene into E. coli, it is a must to examine whether our part is properly combined and well-functional. Therefore we decide to extract RFP-FimH fusion protein from the cytoplasm of the E. coli and test its function by observing whether it will bind to the mannose. We originally want to test directly on the N. ceranae polar tube which has mannose polymer on the surface, however, we found it hard to cultivate under no matter sugar water or mosquito cell line, so we changed our material into E. coli to be the material that carries the mannose to conduct the functional test.
Experimental process is listed below
1.Sonicate the E. coli that can produce RFP-FimH fusion protein
2.Centrifuge under 13200rpm for 1 min. and keep the supernatant
3.Cultivate Escherichia coli K-12 substr MG1655 for 2 hours
4.Take 100λ of liquid
5.Centrifuge under 13200rpm for 2 min.
6.Discard the supernatant and add in 100λ of the protein extraction
7.Fully mix the E. coli with the protein extraction
8.Put the mixture under 4℃ for 30 min.
9.Centrifuge the mixture under 13200rpm for 1.5 min.
10.Observe the pellet under the fluorescence microscope and see if there is fluorescence emitted
experiment group:
control group | experimental group | |
protein added | RFP | RFP-FimH |
Experiment data
We anticipate that the group adding the RFP-FimH will express red fluorescence and the group adding RFP will not express red fluorescence in the pellet. Since the protein with FimH will bind to the mannose on the E. coli, so it will express red fluorescence. The picture on the right shows that the group with FimH has a red fluorescence. Which means RFP-FimH that we cloned really works.
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