Part:BBa_K4644013
pCS-lac-racE
One of Plasmids in the second expression box.
Because the synthesis of polyglutamic acid requires equal amounts of L-glutamic acid and D-glutamic acid, the addition of glutamate racemase can increase the yield of polyglutamic acid. Therefore, we identified the glutamate racemase gene racE in Bacillus amyloliquefaciens LL3 and directly ordered it from a genetic company. We amplified the gene fragment for homologous recombination directly from the obtained PMV plasmid containing this gene. This gene fragment was then homologously recombined with the pcs27 plasmid backbone to obtain pcs-lac-racE.
- Fig 1. pcs-lac-race
Usage
The carrier strain to be used, Nissle 1917 (EcN), was pre-cultured overnight at 37°C in LB medium without antibiotics. plasmids（BBa K4644013 and BBa K4644012） were co-transformed using electroporation, and positive colonies were selected on solid LB medium with double resistance to ampicillin and kanamycin. The selected colonies were then inoculated into liquid LB medium with double resistance to ampicillin and kanamycin and cultured overnight at 37°C. Subsequently, they were inoculated into 50 ml of M9 medium with double resistance to ampicillin and kanamycin, induced with IPTG, and supplemented with the substrate glutamic acid (5g/L). The cultures were shaken at 30°C for 48 hours. At the 12th, 24th, 36th, and 48th hours of fermentation, samples of the fermentation broth were taken respectively. After processing the samples, they were subjected to High-Performance Liquid Chromatography (HPLC) analysis to obtain fermentation data for this module.
- Fig 2. The experimental group's production of polyglutamic acid
- Fig 3. The blank group group's production of polyglutamic acid
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 56
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 56
Illegal NheI site found at 920 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 56
Illegal BamHI site found at 902 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 56
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 56
- 1000COMPATIBLE WITH RFC[1000]
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