The first thing we should do is to transform a plasmid into E. coli and express the protein we want. So we get our target sequence synthesized by IDT and cloned into pSB1C3.Finally, we transform it into E. coli and get the target protein. In order to make the protein more effective, we purify the protein by his-tag column.
Double Heat Shock
Transform is an important part of the experiment. It determines whether your plasmid can be amplified or not. There are two ways to transform our plasmid in our lab, mix and go and heat shock. First, mix and go is a convenient and fast way, but the success rate is low.Heat shock is more effective but it causes more time to do. However, in some experiments, even using heat shock also has a low success rate. Therefore, we change a small part of heat shock protocol, which is to incubate the competent cells at 42 degrees for two times.
Degradation test ( HRP , K2354012)
In order to verify whether our enzymes (HRP) can degrade EDCs or not, we added HRP, diluted H2O2, which is a cofactor for HRP to react, and BPA ( NP ) to 1 mL water totally. H2O2 and BPA (NP) will be degraded by HRP in 40°C incubator and shocked smoothly for 24 hours for the purpose of a complete reaction. After a day, we denatured and spun down HRP for fear that it may degrade BPA ( NP ) continuously. By taking 3µL supernatant of the sample and detect the compounds via LC PDA, we can determine the ability of enzymes to decompose BPA (NP). We will add HRP and various concentrations of BPA (NP) together to test the decomposition efficiency of HRP in different environments.(Results)
In the detection part of our experiment, we used two composite proteins to build our detector, including ERα (Estrogen Receptor alpha) which will be expressed on the surface of modified E. coli to capture BPA (NP) and monobody that is assembled on a gold electrode surface. Before detection test, we need to prepare modified E. coli with Erα and the samples of Monobody.
The following are the steps we used to produce proteins :
Erα : We incubated 5 mL culture to reach OD=0.5 and induced with IPTG. After expressing the culture for 5 hours, the cells were centrifuged and resuspended in PBS buffer. To reduce the activity of E. coli, we freeze our cells in -80°C.
Monobody : We incubated 300 mL culture to reach OD=0.5 and induced with IPTG. After expressing the culture for 12 hours, the cells were harvested by centrifugation at 4 °C. Proteins will be obtained after purification and be stored at 20°C.
As long as we mix the sample with BPA (NP) solution and E. coli expressing ER-alpha together, ERα will capture BPA (NP) and change their structures of complexes. On the other hand, we dropped 10µL of the solution of monobody on the gold surface and store it in 4°C for 5 hours. When the time was up, we blotted the solution of monobody gently and dropped 10µL of the sample on the place containing monobody for 30 minutes to finish our samples.
Theoretically, Erα which captured BPA (NP) can bind to monobody, causing some E. coli to stick on the gold surface. We can check our samples via IR test to prove the experiment.(Figure 1.)(Results)
In order to estimate the corresponding concentration of EDCs, we washed the sample by ddH2O and dropped crystal violet solution to stain E. coli on the gold surface for 2 min, then we washed crystal violet solution by ddH2O and get the density of E. coli under a microscope.(Results)
Function of ice nucleation protein (INP)
INP is a protein which can bring other proteins to the membrane surface of E. coli. To check the function of INP, we transformed E. coli which contained RFP with INP and the other sample which contained RFP without INP. The results can be obtained by comparing the difference between RFP and RFP-IP after we spun down lysis of cells.(Results)
Function of gold binding protein (GBP)
In our experiment, we used GBP to bind Monobody on the surface of gold flakes. We dropped 10µL of the solution of purified monobody with GBP on the gold surface, then stored it in 4°C for 5 hours to let monoboby bind to the gold surface as far as possible.
We can prove the function of GBP by comparing the results between the gold surface and the gold surface contained Monobody via IR test.(Results)
Functional test of ER-alpha
To ensure the function of ER-alpha on the surface of E. coli, we compared the difference between BL-21 E. coli and E. coli which expressed ER-alpha in the different concentration of BPA and NP. From the results, we found that BL-21 can’t affect the outcomes of the biosensing chip.