Team:WHU-China/Contribution
Contribution
BBa_K1321348
We confirmed the function of dCBD fused with reporter sfGFP by testing the combination strength of dCBD with bacteria cellulose.
We did experiments from two aspects----the qualitative verification and quantitative verification.
- Qualitative experiment
The experimental group is the mixture of dCBD sample with bacteria cellulose homogenate. We also set a negative control which mixed sfGFP with bacteria cellulose homogenate.
After a series of operations such as mixing, centrifugation and washing, the following conclusions were obtained:
When we used ddH2O (0.1% TWEEN 20) to wash the mixture and centrifugated it, we found that the residual amount of sfGFP on BC was less than that of dCBD, which is pretty visible to the naked eyes.
- Quantitative experiment
We did the quantitative test of fluorescence intensity by using a multifunctional microplate reader. We got the results as follows:
BBa_K2485001
We confirmed the sequence and function of BirA with 6x His Tag (BBa_K2485001) in vivo.
It is universally acknowledged that Avi-tag can be specifically recognized by BirA enzyme in vitro/vivo and attach a biotin to the protein. So we constructed three plasmids and express them separately: pet28a-BirA (pB), pet28a-sfGFP+Avitag (pGa) and pet28a-sfGFP+Avitag-BirA (pBGa). And the sequence of BirA is from BBa_K2485001.
After purification by Ni resin and chromatography, we got purified products from pB, pGa and pBGa.
According to the result of SDS-PAGE below, we can know that the BirA enzyme has 6x His-tag since it can purified by Ni-resin. Besides, we got sfGFP from pGa and pBGa.
(M: Marker; BirA: the lysate supernatant of pB purified by Ni-resin; GFP: the lysate supernatant of pGa purified by Ni-resin; LS: the lysate supernatant of pBGa; Ni: the lysate supernatant of pBGa purified by Ni-resin; Chro: the eluent of Ni-resin of pBGa purified by chromatography; M: Marker)
Then we measured protein concentration by Bradford method and tried to measure the biotin levels of products of pGa and pBGa by HABA/Avidin reagent under OD500.
HABA/Avidin is suitable for the spectrophotometric determination of in a diverse range of sample types. This method is based on the binding of the dye HABA to avidin and the ability of biotin to displace the dye in stoichiometric proportions. This displacement of dye is accompanied by a change in absorbance at A500 which has a known extinction coefficient. Here is the method ：
The results of measurement of HABA/Avidin reagent:
According to the results, we know that the sfGFP produced by pBGa are biotinylated well. The results are convincing. Therefore, the function of BBa_2485001—BirA with 6x His Tag is confirmed.
BBa_I746916
We confirmed that sfGFP (BBa_I746916) is available in constructs driven by the pR (BBa_R0051) and determined the relationship between sfGFP protein concentration and fluorescence intensity. Besides, we made a comparison fluorescence intensity of between BBa_K3098015 and BBa_I746916.
We constructed a recombined plasmid composed of pR (BBa_R0051)+RBS (BBa_B0030)+sfGFP (BBa_I746916)+6xHis-tag to express the sfGFP (BBa_I746916). By the way, there was not repressor CI of pR in the expression system.
We also constructed a recombined pet28a plasmid composed of T7 promoter and sfGFP (BBa_I746916) to express the sfGFP with avi-tag.
After purification by Ni resin, we made it a series of gradient dilutions of BBa_K3098015 and BBa_I746916 respectively. Then we measured the sfGFP protein concentration by Bradford method and the fluorescence intensity under 475nm as emission wavelength and 545nm as excitation wavelength.
Due to the fluorescence intensity of blank group without any sfGFP is 113 A.U., the ordinate at the origin should be 113. Then we fit data into a straight line: y=kx+b. It is easy to understand that the higher the value of k is, the stronger the sfGFP fluorescence is.
Therefore, we draw following conclusions:
