Composite

Part:BBa_K3926006

Designed by: Zhipeng Gao   Group: iGEM21_XHD-Wuhan-A-China   (2021-10-19)
Revision as of 13:35, 20 October 2021 by Gzp1994 (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NotI site found at 2415
  • 21
    INCOMPATIBLE 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
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE 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.

[edit]
Categories
Parameters
None