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NO detection

We first prepared standard curves using standard samples of different concentrations NaNO2 and verified the linear relationship between concentration and OD540. As can be seen from the figure, OD540 has a good linear relationship with the 1μl to 100μl concentration, 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. Then at the 5h, the NO concentration decrease.

Figure1: Fluorescence Loss Assay. Amount of GFP fluorescence under the induction of dCas9 targeting GFP.

The expression of dCas9 is regulated by an IPTG inducible expression system. Sp1 - 3 represent three different spacers targeting GFP or the associated promoter. SpNT represents a non-targeting spacer serving as a control.

File:The difference in NO production by time.tiff Fig1. The NO change in IPTG induced cell culture.

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, at the same time,we culture the bacteria with our vector Pet28-a and IPTG induced. The results are as followed.

Fig2. 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.

Fig3. 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.