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.

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.

Fig. 3 HPLC results verified the successful production of L-Theanine around retention time 2.4s.

In the HPLC data, the standard L-Theanine sample used analytical grade L-Theanine purchased from Aladdin Chemicals with greater than 98% purity. The standard sample solution was prepared at a concentration of 200mg/L. The HPLC result showed a single peak of standard L-Theanine around retention time 2.4s. The control group was prepared using the E. coli culture with the correct expression plasmid, but with no iPTG induction, nor added glucose. There is a small hump observed in the HPLC results for the control group, suggesting possible leaking expression of the designed enzymes. For the sample group, proper iPTG induction was conducted at OD600 around 0.6 – 0.8, and 15g/L glucose was added. The HPLC result demonstrated a clear rising around retention time 2.4s, indicating the production of L-Theanine.

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References

[1] Cao, R., Hu, S., Lu, Y., Wang, W., Fu, Z., & Cheng, J. (2023). Fermentative Production of L-Theanine in Escherichia coli via the Construction of an Adenosine Triphosphate Regeneration System. Fermentation, 9(10), 875–875.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]