At the design stage, we first did the preliminary research for our project. Through this, necessary enzymes and substances were identified, and expression vectors and genes were selected.
The four enzymes we selected are as follows. Nitrite Reductase, Hydrazine Synthase, and Hydrazine Oxideneductase will be used for Nitrite (NO2-) and Ammonium (NH4+) removal.
And superoxide dismutase enzymes will be used to perform H2O2 production.
And we used this to construct parts. First, BBa_K4371000 (Anammox cluster: HZS + HZO + NIR) was created by clustering three enzymes used in Nitrite (NO2-) and Ammonium (NH4+) removal.
It was inserted into pEXP5-NT, a specific cell free expression (CFE) vector.
Similarly, BBa_K38007 (SOD1 protein), a parts of Superoxide dismutase, an enzyme that conducts H2O2 production, was inserted into the expression vector pEXP5-NT.
In order to obtain the sequence found in the previous design stage, we ordered the sequence through Twist Bioscience. However, there was a problem that the sequence of the alpha subunit of the hydrazine synthase was longer than 1.8 kb. So we decided to get the gene by sequencing in the plasmid method.
And we construct cell free expression system's experiment methods.
The T7 promoter shows the most active protein expression at 25°C.
Figure. pT7 maximum Temperature (http://parts.igem.org/Help:Cell-free_chassis/Modelling#References)
When checked through modeling, it can be seen that it is generated to the maximum at 25°C.In order to obtain more production, it is necessary to replace it with plasmid origin with a higher CN value or with a stronger RBS. Through this, more enzymes can be obtained and it can have advantages in producing filters.