Part:BBa_K1104102:Experience

Expression check

Check for the Length of the RFP-FimH
The electrophoresis gel shows the result after the digestion. The central well is the 100 bp marker.The two well on both sides of the marker is the digestion sample of RFP-FimH from different colony. The lower band is the part with the promoter and the RFP-FimH coding sequence, which has the length of 1,485 nt. The upper band is the backbone, pSB1A2, we use.

　　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.

Fluorescence Check
Both eppendorf are the liquid culture of the E. coli with RFP-FimH coding sequence cloned. We can obviously see the red light fluorescence emitted, showing that the fusion protein have successfully been produced and the RFP is still well-function.

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

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RFP-FimH Functional Test Result
The eppendorf on the right is the control group with RFP protein added in the E. coli and 1x PBS mix. On the left is the experimental group with RFP-FimH fusion protein added. We can see that the eppendorf on the left shows a red color at the pellet, while it shows white on the right eppendorf. This indicates that the fusion protein we produce can actually binds to the mannose, which is the target that we want to block on N. ceranae.

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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