Difference between revisions of "Part:BBa K3595001"
 
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==Experimental Setup==
 
==Experimental Setup==
 
*Genetic design principle of argA fbr was described on the page of [[Part:BBa_K3595082]]
 
*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.
+
*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<i>E.coli DH10b </i>  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.
 
*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.
+
*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
 
*Detecting ammonia concentration in culture medium
 
==Results==
 
==Results==
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3595001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3595001 SequenceAndFeatures</partinfo>
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<!-- Add more about the biology of this part here-->
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=Reference=
 +
Cunin R, et al. Biosynthesis and metabolism of arginine in bacteria. Microbiol Rev 1986;50(3):314-52.<br>
 +
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.<br>
 +
Vyas S, et al. Feedback inhibition of acetylglutamate synthetase by arginine in Escherichia coli. Arch Biochem Biophys 1963; 100(3), 542-6.
  
  

Latest revision as of 19:11, 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


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

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.
Vyas S, et al. Feedback inhibition of acetylglutamate synthetase by arginine in Escherichia coli. Arch Biochem Biophys 1963; 100(3), 542-6.