Contribution

This year, our team constructed a modified EcN1917 (EcN) with ArgR gene deleted and ArgJ gene inserted. This engineered EcN can produce high concentration of arginine without arginine feedback inhibition, and can be used as a treatment vehicle for solid tumors.

In this project, we have made the following achievements.

 

1 Characterization of lysin part BBa_K2277000

We constructed a pGLO-lysin plasmid and transformed it into DH 5α, E. coli MG1655 and EcN1917 (E. Coli Nissle 1917) strains, and characterized the effect of lysin on the growth of these strains. The growth of E. coli DH5α, E. coli MG1655 and EcN1917 treated with 30mM arabinose showed that lysin expression induced by arabinose inhibited the growth of these bacteria.

Please refer to BBa_K2277000 registry page for detailed results.

2 Construction of an argR knockout EcN strain and exploration of a successful gene knock-out method[1]

In this project, we successfully knocked down the argR gene in EcN1917 to increase the production of arginine in the strain. The gene knockout strain can be used for other subsequent projects about EcN strains.

 

Besides, the construction method of gene knockout strains explored by our team provides a set of methods for other teams to use in the construction of various gene knockout strains. The method for gene knockout used by our team is as follows:

We first linearized the H1-argR-H2 fragment (BBa_K4410005), which is the argR gene with its upper and lower homologous arms, by PCR from EcN1917 WT. 

Using the TA cloning method, the pMD19T vector and H1-argR-H2 were connected to form recombinant pMD19T. 

H1-pMD19T-H2 was then linearized by PCR. The Kan^R gene was linearized from pKD4 vector by PCR. Kan^R gene and H1-pMD19T-H2 gene were combined into recombinant pMD19T by one-step cloning. 

The recombinant pMD19T was heat transformed into a DH5α colony.

Donor DNA (H1-Kan^R-H2, BBa_K4410006) was linearly extracted from DH5α by PCR. 

Donor DNA was precipitated by alcohol and then electro-transformed into EcN1917 WT.