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NOS cloning

NOS cloning In our experiment, we first cloned NOS gene in Bacillus subtilis by PCR and attached it to Pet28-a for determination using double restriction enzyme and DNA ligase. Then the ligated vector was transformed into the BL21(DE3) for protein expression and DH5α for plasmid application. The plasmid was sequencing by company. It accords with our design.

Figure1: The result of NOS cloning PCR.

NO detection

We first prepared standard curves using standard samples of different concentrations NaNO2 and verified the linear relationship between concentration 1μl to 100μl concentrationand OD540.So OD540 can be used to reflect the content of NO in the sample，so OD540 can be used to reflect the content of NO in the sample

Then we separately measured the change of NO over time in the NOS transformed BL21 (DE3) culture. We found that the nitrite content actually decreased over the initial period of time. The change of nitrite in the bacterial solution without IPTG induction with time was measured. We found that the nitrite content in the bacterial solution also decreased at the initial stage. This may be caused by the consumption of nitrite by the growth of bacteria, so we want to reflect the production of NO by the difference value of nitrite content between IPTG induced and no IPTG induced. The results are as followed.

Figure3: The difference value of NO between IPTG induced and no IPTG induced.

As we can see, the difference value of two culture become bigger as time goes

Western blot

To demonstrate that our expression system does work and to explore NOS proteins as time goes by, we performed Western blot. The culture conditions are the same as those described previously. Due to the operation, only 0-4h was detected. At the same time, GFP-His was added as the positive control.

Figure4: The result of Western blot.

We can see that the expression of NOS-HIS gradually increased with the change of time, indicating that there was NO problem with our expression system, which also explains why the production of NO gradually increased, because in the first hours induced by IPTG, the protein expression was low, and the protein amount did not increase significantly until four hours.