Part:BBa_K4410007
J23100-argJ
It is the key part that is responsible for the expression of N-acetyl glutamate synthase, which is one of the necessary enzymes in the process of arginine production. The expression of the sequence is arginine feedback-inhibition resistant, which means that it can largely increase the expression amount of enzyme. This part includes the constitutive promoter J23100, ribosome binding site (RBS) sequence and bifunctional glutamate N-acetyltransferase/amino-acid acetyltransferase ArgJ.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 319
Illegal PstI site found at 1004 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 769
Illegal PstI site found at 319
Illegal PstI site found at 1004 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 319
Illegal PstI site found at 1004 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 319
Illegal PstI site found at 1004 - 1000COMPATIBLE WITH RFC[1000]
Results
1. Viability of native EcN1917 at relatively high arginine concentration
Before engineering EcN1917, we first used MTT assay to test whether high arginine concentration might inhibit the growth of native EcN1917. The results showed that EcN1917 survived and grew well at arginine concentration ranging from 0 µg/mL to 10 mg/mL (Figure 1). ≥20 mg/mL arginine suppressed the survival of EcN1917. This tolerance range of arginine concentration indicated that EcN1917 would be viable when it was engineered to be an arginine synthesizing factory in vivo.
2. Construction of EcN1917∆argR (argJ)
We first obtained a linearized pGLO-J23100 vector without GFP gene by PCR cloning. The rest part of the pGLO-J23100 vector contains the promotor, RBS, and the terminator sequence. Gel electrophoresis showed that the length of pGLO-J23100 was about 4524bp, which was within expectation (Figure 2-B). We then extracted out ArgJ fragment by PCR from BW25113 template. As figure 2-A shown, the result of gel electrophoresis for ArgJ indicated that the length was about 1223bp, as expected. The linearized pGLO-J23100 vector and the ArgJ fragment were ligated by One-step cloning. The constructed plasmid was introduced into EcN1917-PKD46 by electrotransformation. The result of gel electrophoresis for pGLO-J23100-argJ bacterial colony showed that the whole gene sequence length is about 2025bp, which corresponded to our expectations (Figure 2-C).
3 Functional Verification of EcN1917∆argR (argJ)
The production of L-arginine of the engineered bacteria was detected by Hitachi amino acid analyzer. Wild type EcN1917 was used as control. The 24- and 48-hour fermentation broth of the bacteria were collected and broken by ultrasonic crusher, and detected by automatic amino acid analyzer (Figure 3). The results showed that EcN1917∆argR (argJ) had a yield of 3.6 mM L-arginine.
4 Real sample test
We also co-cultured CT26 cells with the EcN1917△argR strain at MOIs of 25, 50,100, and 200 (number of bacteria cells: number of CT26 cells) for 3 hours, and evaluated the viability of the CT26 cells by MTT assay, with wild type EcN1917 (EcN1917) used as control. As shown in Figure 4, EcN1917△argR-argJ could significantly decrease the viability of CT26, as compared to EcN1917, which indicating that arginine producing EcN1917 was capable to exert anti-cancer effect against colon cancer cells.
To verify the anti-cancer effect of arginine. We had several cellular experiments on colon cancer CT 26 cells. MTT assay showed that arginine affected the viability of colon cancer cells through concentrations dependent manner (Figure 5-A). As a substrate for endogenous NO synthase, arginine can regulate the tumor microenvironment through the NO pathway. It is generally believed that increasing NO level can increase the blood flow to tumors and alleviate hypoxia in tumors. In order to further explore the effect of L-arginine on the anaerobic metabolism of tumor cells, different concentrations of L-arginine were incubated with CT26 cells to detect the level of lactic acid in the cell supernatant. The experimental results showed that arginine at low concentrations (10 and 20 mM) down-regulated the level of lactate metabolism in tumor cells without affecting cell viability (Figure 5-B). When the concentration of arginine reached 50 mM, it had a certain inhibitory effect on cell viability, and the cells’ regulation of lactate level was limited at this time. Since tumor metabolism is closely related to the tumor immunosuppressive microenvironment, tumor cells can promote T lymphocyte apoptosis by upregulating PD-L1 expression. To verify that arginine can regulate the tumor immunosuppressive microenvironment, we detected the mRNA expression level of PD-L1 in CT26 cells after arginine exposure by RT-qPCR. The results showed that arginine at a concentration of 50 mM could significantly down-regulate the mRNA expression level of PD-L1 in CT26 cells (Figure 5-C). These experiments indicated that arginine at high concentrations (100 and 200 mM) could exert a direct tumor-killing effect. At low concentrations, arginine could regulate tumor microenvironment by down-regulating the expression level of lactic acid and the mRNA expression level of PD-L1 in tumor cells, which was beneficial for tumor immunotherapy.
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