Part:BBa_K4644014
ygay-50trc-gdhA
One of Plasmids in the Polyglutamic acid synthesis module.
Biology
gdhA(glutamate dehydrogenase) catalyze the production of ammonia from L-glutamate.
Glutamate dehydrogenase(Gdh) is a kind of oxygen-free dehydrogenase dependent on nicotinamide coenzyme NAD(P)+/NAD(P)H, with GTP and ATP as allosteric inhibitors.GDP and ADP were allosteric activators.Gdh is widely found in various animals, plants and microorganisms, and plays an important role in carbon and nitrogen metabolism.In different organisms,Gdh can catalyze the oxidative deamination reaction from L-glutamate to α-ketoglutaric acid and ammonia,and can catalyze reductive amination from α-ketoglutaric acid and ammonia to L-glutamate as well, depending on different cofactors.
Characterization
Because this module requires glutamic acid as a precursor, and in the metabolic system of E. coli, the gdh pathway is one of the major pathways for glutamic acid production. Therefore, we identified the gene encoding glutamate dehydrogenase, gdhA, in E. coli. We replaced the constitutive promoter -50trc. Additionally, to reduce the burden on the bacterial cells, we used CRISPR-Cas9 technology to target the ygaY locus in the genome for integration. After integration was completed, we induced the elimination of the PT plasmid using L-arabinose and cultured the cells at 42°C for 12 hours to eliminate the pCas9 plasmid.
- Fig 1. ygay-50trc-gdha
- Fig 2. Integration Strain Plate Streaking Chart
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 36
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 36
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1425 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 36
Illegal BamHI site found at 1407 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 36
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 36
Illegal AgeI site found at 645
Illegal AgeI site found at 1001 - 1000COMPATIBLE WITH RFC[1000]
| None |