Revision as of 19:10, 27 October 2020

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 theE.coli DH10b as a negative control, and M9 media as a blank control
• 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.
• Inoculating 15 uL of culture solution overnight into a 24-well plate containing 3 mL M9 medium for overnight growth at 37 °C and 200 rpm.. The media contained 3 ul kanamycin ， 1.5 uL 1M IPTG and 5mM ammonia.
• 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

Reference

Cunin R, et al. Biosynthesis and metabolism of arginine in bacteria. Microbiol Rev 1986;50(3):314-52.
Rajagopal BS, et al. Use of Inducible Feedback-Resistant N-Acetylglutamate Synthetase (argA) Genes for Enhanced Arginine Biosynthesis by Genetically Engineered Escherichia coli K-12 Strains. Appl Environ Microbiol 1998;64(5):1805-11.