Part:BBa_K5235010
GMAS-PPK-BsAld-CsAlaDC-GNP1
In 2024, SHSBNU-China aims to use synthetic biology techniques to genetically edit the E. coli strain BL21 to produce L-theanine. We designed a plasmid containing five genes (BsAld, CsAlaDC, GMAS, PPK, and GNP1) responsible for the production and transport of theanine on the common vector pET28a. The plasmid is designed to be expression in E. coli with IPTG controlled induction, followed by HP-LC testing of the supernatant to determine the yield.
We used five genes in total: BsAld, CsAlaDC, GMAS, PPK, and GNP1. - BsAld, CsAlaDC, and GMAS are responsible for the de novo synthesis of theanine, starting from glucose. BsAld and CsAlaDC carry out the conversion of pyruvate to ethylamine, and GMAS synthesizes theanine from ethylamine and glutamate, which is produced by the generic bacterial TCA cycle. - PPK provides ATP to aid the synthesis pathway. - GNP1 is responsible for transporting the synthesized theanine out of the bacterial cells.
Usage and Biology
by SHSBNU-China 2024
In 2024, SHSBNU-China aims to use synthetic biology techniques to genetically edit the E. coli strain BL21 to produce L-Theanine. L-theanine is a non-protein amino acid found in tea leaves that can be used to alleviate anxiety and improve sleep quality. We designed a plasmid containing five genes (BsAld, CsAlaDC, GMAS, PPK, and GNP1) responsible for the production and transport of theanine on the common vector pET28a. The plasmid is designed to be expression in E. coli with IPTG controlled induction, followed by HP-LC testing of the supernatant to determine the yield.
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Fig. 1(a) Function of BsAld, CsAlaDC, and GMAS.
Fig. 1(b) Function of PPK and GNP1
This composite part is composed of five basic parts: BsAld, CsAlaDC, GMAS, PPK, and GNP1. - BsAld, CsAlaDC, and GMAS are responsible for the de novo synthesis of theanine, starting from glucose. BsAld and CsAlaDC carry out the conversion of pyruvate to ethylamine, and GMAS synthesizes theanine from ethylamine and glutamate, which is produced by the generic bacterial TCA cycle. - PPK provides ATP to aid the synthesis pathway. - GNP1 is responsible for transporting the synthesized theanine out of the bacterial cells.
Fig. 2 Gene circuit for expression of this part in E. coli.
We controlled the expression of the enzymes with one single T7 promoter, aided with a lac operator (LacO), all carried on a pET28a vector. Each part has its own RBS (part B0034), but all connected in series and controlled by a common T7 promoter. The plasmid was transformed into E. coli (BL21). The bacteria strains were first cultured in normal LB medium with proper antibiotics till the OD600 reached 0.6 - 0.8. Then, centrifuge at 4000 rpm for 5 minutes to collect the bacteria, resuspend in the special fermentation medium, add 0.3 mM IPTG to induce enzyme expression, and culture at 30°C with 220 rpm for 24 hours.
Fermentation medium: 10 g/L tryptone, 5 g/L yeast extract, 10 mmol/L MgCl2·6H2O, 150 mmol/L (NaPO3)6, 0 - 20g/L glucose.
Centrifuge the fermented broth at 12,000 rpm, and collect the supernatant.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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