Coding

Part:BBa_K4632005

Designed by: Haolong Lai   Group: iGEM23_SCAU-China   (2023-10-09)
Revision as of 14:31, 12 October 2023 by LHL scau (Talk | contribs) (→‎Construction and Characterization)

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dapA

Construction and Characterization

Growth of the dapA mutant wm3064 is strictly dependent on exogenously supplied DAP; hence, the removal of DAP provides an efficient counterselection against this donor.(Dehioet al.,1997)

Diaminoheptanedioic acid (DAP) is an α-amino acid. It is an ε-carboxy derivative of lysine. It is a component of cell walls in certain prokaryotes, especially in the peptidoglycan chains of Gram-negative bacteria. These bacteria require DAP for normal growth. Without DAP, they can still grow but are unable to synthesize the peptidoglycan essential for their cell walls.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 64
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Construction and Characterization

Biological safety device efficacy testing

E. coli wm3064 is a dapA mutant strain with diaminopimelic acid (DAP) nutritional defect. The dapA gene were ligated to the downstream of pnirB and cloned to pUC18T-mini-Tn7T plasmid, then transformed to wm3064. The growth of ΔdapA/dapA- pUC18T-mini-Tn7T and ΔdapA strains were tested under aerobic or anaerobic conditions. (Figure. 4)The results showed that ΔdapA could only grow in the presence of DAP no matter in aerobic or anaerobic conditions, while ΔdapA/dapA- pUC18T-mini-Tn7T could grow under anaerobic conditions without the supplementation for DAP.

These results(Figure. 1) indicate that this strain can only grow under anaerobic conditions and possesses biological safety. During production, growth can be ensured by adding DAP.

part-6-5-1.png

Figure. 1 Results of culture experiment of dapA gene defective strain

This work qualitatively confirms that the pnirB element can indeed exhibit specific expression under anaerobic conditions. Furthermore, by utilizing pnirB to drive the essential diaminopimelic acid gene dapA, it restricts bacterial growth to anaerobic environments, such as the gut of the S. invicta. These findings provide a theoretical foundation for the next steps in the development of the biological safety device, specifically the recombination of pnirB into the E. coli chromosome. See more detail in BBa_K4632012[1]

References

[1] Dehio C, Meyer M. Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli[J]. J BACTERIOL, 1997,179(2):538-540.


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