Part:BBa_K3926007
amilCP report nirK expression
Uses a strong promoter (BBa_J23100), strong RBS (BBa_B0034), nirK coding region (BBa_K3926005), and a double terminator (BBa_B0015). Will generate amilCP when nirK expression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 101
Illegal NgoMIV site found at 764 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Introduction
In order to enhance the denitrification of rhizobia, we designed an enhanced denitrification system. We designed nirK-pBBR1MCS2. To realize the function of J23100-RBS-nirK 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 (nirK-pBBR1MCS2)
Usage and Biology
Theoretically, the transformation of nirK-pBBR1MCS2 will increase the copy number of nirK 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 nirK 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 nirK-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 nirK-pBBR1MCS2
Figure 3 Color changes after introducing nirK-pBBR1MCS2 into the engineered bacteria. Left: nirK-pBBR1MCS2, Right: Control
We cultivated strains containing nirK-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 nirK successfully. We verified the system that expresses nirK-anilCP fusion protein over time.
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