Coding

Part:BBa_K3595001

Designed by: Ying Li   Group: iGEM20_GZ_HFI   (2020-10-24)
Revision as of 17:00, 26 October 2020 by Liyingying (Talk | contribs)


argA-Wildtype


The ammonia that enters into E.coli is converted to glutamate along with α-Ketoglutarate . The series of reactions that convert glutamate into arginine are catalyzed by N-acetyl glutamate synthetase (NAGS), encoded by gene argA, to acetylize glutamate

Usage and Biology

This part can be used as a coding sequence after the promoter pTac and RBS B0034. The argA protein can be translated under the induction of IPTG. We constructed plasmids pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA using argA and argAfbr, respectively. The constructed plasmid was transformed into E.coli DH10b-ΔargR host cell to test its ammonia degradation efficiency.

The structure of the plasmid pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA

Experimental Setup

  • Genetic design principle of argA fbr was described on the page of Part:BBa_K3595082
  • Plasmid pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA ^ fbr was transfered into the E.coli DH10b -ΔargR host cell,respestively. Meanwhile,plasmid pBR322-KanR-pTac-argA was transfered into the E.coli DH10b.
  • Single colonies were selected from the experimental LB-agar plate , then inoculated into test-tube tubes with 4000 μL LB liquid medium with 4uL kanamycin for overnight growth at 37 °C and 200 rpm.
  • Inoculate 15 uL of culture solution overnight into a 24-well plate containing 3 mL M9 medium with 3 ul kanamycin with 1.5 uL 1M IPTG for overnight growth at 37 °C and 200 rpm.
  • Detecting ammonia concentration in culture medium

Results

  • The DH10b with plasmid pTYT-argAfbr and ∆argR showed the most significant increase in conversion of ammonia, 40.47% more conversion rate compared to the wildtype
Detection results of the effect of the ammonia pathway. (A) The least-squared regression line of NH4+. (B)The concentration of NH4+. (C) The percentage of decrease in NH4+ compared with M9 medium.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 626


[edit]
Categories
//cds/enzyme
Parameters
None