Part:BBa_K3875007
Usage and Biology
Design:
Firstly, we selected the gene
gadB-E89Q+z452-466 ( BBa_K3875000) and
gdhA ( BBa_K3875008), and used the promoter
pLlacO ( BBa_R0011) to promote them and
terminator BBa_B0015 to terminate the procedure[1]. After that, the entire sequence
( BBa_K3875007)
was introduced into pCS27 and yielded the recombinant plasmid pCS-lac-gadB(mut)-lac-gdhA-T1. Bellows are a picture of our plasmid profile.
Method&Testing:
Firstly, we transfer the plasimid into DH5a and culture for 12 hours, then we can pick individual colonies and shake flask to cultivate under the circumstance that the temperature is 37℃. Then we performed enzyme validation.
Meanwhile, electrophoresis of the PCR products was identified, and finally PCR product was purified by gel extraction kit. Then, we eletrotransformation the plasmid into E.coli Nissele 1917, and culture it for 12 hours. Then we select a single colony to continue to cultivate and prepare for fermentation.
In order to detect the expression of recombinant gene, the plasmid containing pCS-lac-gadB(mut)-gdhA was transformed into E. coli DH5α. for fermentation.
Testing
We tried to verify whether the plasmid was successfully constructed. It demonstrated the success of plasmid construction.
Below is the result of fermentation and it shows that we have successfully producing GABA from simple carbon sources.
GABA was produced by fermentation with glycerol as substrate
GABA was produced by fermentation with plam oil as substrate
GABA was produced by fermentation with soybean oil as substrate
Based on these statistics, it is obvious that we have successfully produce GABA. Also, considering that normal synthesis pathways, which consisted of gadB(wild), of producing GABA usually produce GABA for 1mg/ml, so it is obvious that our gadB(mut) gene plays its special function and helps overproducing GABA！
Reference
[1] Sheng, L., Shen, D., Yang, W., Zhang, M., Zeng, Y., Xu, J., Deng, X., & Cheng, Y. (2017). GABA Pathway Rate-Limit Citrate Degradation in Postharvest Citrus Fruit Evidence from HB Pumelo (Citrus grandis) × Fairchild (Citrus reticulata) Hybrid Population. Journal of agricultural and food chemistry, 65(8), 1669–1676. https://doi.org/10.1021/acs.jafc.6b05237
Sequence and Features
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