Part:BBa_K4410004
argJ
This part is the sequence of argJ gene from the Genome of Corynebacterium glutamicun（ATCC13032）. ArgJ gene encodes bifunctional glutamate N-acetyltransferase/amino-acid acetyltransferase ArgJ . ArgJ converts L-glutamate into N-acetyl-L-glutamate and can function well at high concentration of arginine. In our project, we introduced the argJ gene into Escherichia coli. In E. coli, the first step of arginine biosynthesis is the acetylation of glutamate catalyzed by N-acetylglutamate synthase (NAGS) encoded by gene argA. A large amount of arginine feedback inhibits the activity of NAGS. In order to reduce the feedback regulation of arginine on bifunctional glutamate N-acetyltransferase/amino-acid acetyltransferase encoded by ArgJ gene, which is not inhibited by arginine feedback, is introduced to remove the arginine feedback inhibition pathway.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 247
Illegal PstI site found at 932 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 697
Illegal PstI site found at 247
Illegal PstI site found at 932 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 247
Illegal PstI site found at 932 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 247
Illegal PstI site found at 932 - 1000COMPATIBLE WITH RFC[1000]
Results
We first obtained a linearized pGLO-J23100 vector without GFP gene by PCR cloning. The rest part of the pGLO-J23100 vector contains the promotor, RBS gene, and the terminator sequence. Gel electrophoresis showed that the length of pGLO-J23100 was about 4524bp, which was within expectation (figure 1-B). We then extract out ArgJ fragment by PCR from BW25113 template. As figure 1-A shown, the result of gel electrophoresis for ArgJ indicated that the length was about 1223bp, as expected. The linearized pGLO-J23100 vector and the ArgJ fragment are ligated by One-step cloning. The constructed plasmid is introduced into EcN1917-PKD46 by electrotransformation. The result of gel electrophoresis for pGLO-J23100-argJ bacterial colony showed that the whole gene sequence length is about 2025bp, which corresponded to our expectations (Figure 1-C).
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