Part:BBa_K3369000

Background

Nitric oxide (NO) is a key molecule that induces biofilm degradation. In Bacillus Subtilis，nitric oxide is produced by Bacterial nitric-oxide synthases (bNOSs), which converts arginine into NO. In bacteria, since there is no corresponding reductive domain, additional reductive substances are needed for reduction. ALA is a heme precursor that acts as a corresponding reducing substrate.

Design

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. The Pet28-a has a T7 promoter, which is a strong promoter. It also has a kanamycin resistance to help us to screen the transformed cell. It also has a lacI gene and lac operator, which make us to use IPTG to induce NOS expression. Meanwhile，there is a his tag in the vector, which can be used for the further detection. After cloning and transforming, we induce the bacteria under 0.5mMIPTG with 0.5mMArg and 0.45mM ALA.

NOS detection

In water, NO is rapidly oxidized to NO2- and NO3-. NO3- will be reduced to nitrite under the action of E. coli nitrite reductase. The NO2- can be detected by Griess reagents. Under acidic conditions, NO2- promotes the Griess reaction to produce diazo compounds. The compound has an absorption peak at 540nm. And there is a linear relationship between the products and NO2-. Therefore, NO production can be determined indirectly by measuring OD540. In addition to Griess reagent, in order to reduce the oxidized NO3- to NO2-. NADPH and FAD were added and catalyzed by Nitrate Reductsae. After that, the excess reductive material is removed by LDH. At the same time, we also diluted NaNO2 in LB to make standard samples of different concentrations, so as to make standard curves for quantitative analysis.