Part:BBa_K3926006
amilCP report napA expression
uses a strong promoter (BBa_J23100), strong RBS (BBa_B0034), napA coding region (BBa_K3926004), and a double terminator (BBa_B0015). Will generate amilCP when napA expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NotI site found at 2415 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1263
Illegal BglII site found at 1407
Illegal XhoI site found at 1130
Illegal XhoI site found at 2240 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 107
Illegal NgoMIV site found at 1357
Illegal NgoMIV site found at 1645
Illegal NgoMIV site found at 1801
Illegal NgoMIV site found at 2185 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1594
Illegal BsaI.rc site found at 1553
Illegal SapI site found at 1858
Usage and Biology
Introduction
In order to enhance the denitrification of rhizobia, we designed an enhanced denitrification system. We designed napA-pBBR1MCS2. To realize the function of J23100-RBS-napA circuit, we assembled this circuit on the pBBR1MCS2 plasmid backbone and connected to amilCP for characterization (Figure 1).
Figure 1 Design of Enhanced denitrification system plasmid (napA-pBBR1MCS2)
Usage and Biology
Theoretically, the transformation of napA-pBBR1MCS2 will increase the copy number of napA genes, increase the content of denitrification enzymes, and improve the denitrification ability of the strain.
To enhance the denitrification effect of Rhizobium, we chose the strong promoter J23100 as the promoter of the system to maximize the copy number of the target gene. The napA gene is related to denitrification. The backbone we selected is an expression vector that can be expressed in E. coli and Rhizobium (Figure 1).
Characterization
We introduced the napA-pBBR1MCS2 into the engineered bacteria. After a single colony grew, we found that our engineered bacteria successfully expressed the blue color protein amilCP. (Figure 2, Figure 3)
Figure 2 Single colony with napA-pBBR1MCS2
Figure 3 Color changes after introducing napA-pBBR1MCS2 into the engineered bacteria
We cultivated strains containing napA-pBBR1MCS2 and wild-type strain in LB liquid medium. And the content of amilCP was measured by OD588 every 30 minutes, which results are shown in (Figure 4).
Figure 4 Changes in amliCP concentration of LB liquid medium
Conclusion
We have successfully constructed an enhanced denitrification system. Through our system, we increased the number of copies of napA successfully. We verified the system that expresses napA-anilCP fusion protein over time.
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