Engineering

Summary

Cancer is currently the trickiest enemy for human beings. it is said that colorectal cancer ranks second among the most common types of cancer in terms of mortality worldwide.^{(https://www.spandidos-publications.com/10.3892/mco.2021.2433)}Our project aims to develop a modified E. coli Nissle 1917 (EcN1917) that can persistently produce arginine in the tumor microenvironment (TME). There are two reasons to increase the arginine concentration in the TME: First, arginine can kill cancer cells. Plus, supplement of arginine activates the immune system that can attack cancer cells.^{(https://www.x-mol.com/paper/1262894917545467904/t?recommendPaper=1311354128826601472)} Native EcN1917 has pathways for arginine synthesis but they are inhibited by the feedback of arginine in the environment. Therefore, to reach our goals, we constructed EcN1917 to maximize arginine production by avoiding these feedback inhibitions.

Here are the project steps:

(1) We knocked out the argR gene which encodes ArgR protein. ArgR represses the expression of enzyme genes important for arginine biosynthesis in response to the arginine level in the cell.

(2) We added the argJ gene derived from Corynebacterium to remove the arginine inhibition on NAGS enzyme encoded by argA gene. NAGS is responsible for the acetylation of glutamate, an important step in arginine biosynthesis in EcN1917, but is inactivated by arginine. argJ encodes bifunctional glutamate N-acetyltransferase/amino-acid acetyltransferase, which can also acetylate glutamate and can function well at high concentration of arginine.

(3) To ensure biosafety, we inserted the lysin gene into our designed EcN1917 so that it would be lysed in response to arabinose after the bacteria have done their work.

In our project, we successfully constructed nine parts to fulfill the above goals (Table 1).

Table 1 Part list

No.	Name	Type	Description	Designer	Length
1	BBa_K4410000	Basic	Fragment H1 from E. coli Nissle 1917	Ruyi Shi	1000bp
2	BBa_K4410001	Basic	Fragment H2 from E. coli Nissle 1917	Ruyi Shi	1000bp
3	BBa_K4410002	Basic	argR	Ruyi Shi	472bp
4	BBa_K4410003	Basic	KanR	Ruyi Shi	795bp
5	BBa_K4410004	Basic	argJ	Ruyi Shi	1167bp
6	BBa_K4410005	Composite	H1-argR-H2	Ruyi Shi	2472bp
7	BBa_K4410006	Composite	H1-KanR-H2	Ruyi Shi	2795bp
8	BBa_K4410007	Device	J23100-argJ	Ruyi Shi	1334bp
9	BBa_K4410008	Basic	Terminator	Ruyi Shi	87bp

1. Natural EcN1917 survivability under arginine

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.

Figure 1: The effect of arginine on the growth of native EcN1917

2. Engineering EcN1917

Step 1: Construction of EcN1917^△^argR strain: Knock out argR

ArgR protein acts as an arginine repressor of the arginine synthetic genes in EcN1917, we successfully knocked down the argR gene in EcN1917 to increase the production of arginine in the strain.

We first linearized the H1-argR-H2 fragment with upper and lower homologous arms by PCR from EcN1917 WT. The pMD19T vector and H1-argR-H2 were connected to form Recombinant pMD19T by TA cloning. H1-pMD19T-H2 was 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 DH5a colony (Figure 4-E). All above steps were validated by electrophoresis (Figure 4 A-D).

Donor DNA (H1-Kan^R-H2) was linearly extracted from successfully transformed DH5a by PCR. Donor DNA was precipitated by alcohol and then electrotransformed into EcN1917 WT. All above steps were validated by electrophoresis (Figure 5 A-B). We used kanamycin to verify that the argR gene was knocked out (Figure 5-C). Hence, EcN1917^△argR strain, EcN1917 with argR gene knocked out, was constructed successfully.

Figure 3:Schematic illustration of the λ red recombination system

Figure 4: (A) The electrophoretogram of H1-argR-H2; (B) The electrophoretogram of recombinant pMD19T(2680bp); (C) The electrophoretogram of the H1-pMD19T-H2(4757bp); (D) Electrophoretogram of the donor DNA (3589bp); (E) DH5α bacteria colony transformed with the recombinant pMD19T

Figure 5: (A) The electrophoretogram of donor DNA (H1-Kan^R-H2) linearly extracted from DH5a by PCR (3589bp); (B) Electrophoretogram verification of the successful transformation of EcN1917 with the donor DNA (2021bp); (C) EcN1917 colonies electrotransformed with the donor DNA.

Step 2: Construction of EcN1917^∆^argR (argJ): Insert argJ gene into EcN1917^△^argR

We first constructed a pGLO-J23100-argJ vector containing the argJ gene. To construct this vector, we used PCR to clone out a linearized pGLO-J23100 vector abandoning the GFP gene sequence. Gel electrophoresis showed that the length of the linearized pGLO-J23100 was about 4524bp, which was within expectation (Figure 6-A). We then used BW25113 as template to extract argJ gene fragment by PCR. Gel electrophoresis indicated that the length of the argJ fragment was about 1223bp, as expected (Figure 6-B). Finally, we constructed the pGLO-J23100-argJ vector by directly ligating the linearized pGLO-J23100 and the argJ fragment via one-step cloning. The pGLO-J23100-argJ vector was then electrotransformed into EcN1917^△argR. 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 expectation (Figure 6-C).

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 7). The results showed that EcN1917^∆^argR (argJ) had a yield of 3.6 mM L-arginine.

Figure 6: (A) Nucleic acid gel electrophoresis result of pGLO-J23100. (B) Nucleic acid gel electrophoresis result of argJ fragment. (C) Nucleic acid gel electrophoresis result of pGLO-J23100-argJ bacterial colony.

Figure 7 L-Arginine production of ECN1917 WT (upper) and ECN1917^∆^argR (argJ) (bottom) after incubation for 48h.

Step 3: Insert lysin gene into EcN1917

We constructed a pGLO-Lysin plasmid by adding the lysin gene into a pGLO vector. Gel electrophoresis showed that the length was as we expected (Figure 8-A). The pGLO-Lysin plasmid was successfully electrotransformed into EcN1917 (Figure 8 B-D). EcN1917 transformed with pGLO-Lysin were lysed significantly by 10-hour treatment of 30mM arabinose (Figure 9).

Figure 8 (A) Nucleic acid gel electrophoresis result of pGLO-lysin plasmid (5530bp); (B-D) EcN1917 colonies after transformation (Experimental group: EcN1917 transformed with pGLO-Lysin plasmid, Positive control: EcN1917 transformed with pSU plasmid, Negative control: competent EcN1917)

Figure 9 Effect of arabinose on EcN1917 transformed with pGLO-Lysin plasmid (Left: bacteria solution without arabinose, right: bacteria solution treated with 30mM arabinose for 10h)

3. Real sample test

EcN1917 is one of the kinds of probiotics that is extremely proper to be used in medicine. This kind of bacteria will not trigger some inflammations in human bodies. Thus, we choose it as our engineering-based probiotics. The final bacterial product is equipped to produce large amount of arginine without feedback inhibition, and can lyse in response to arabinose. Our product can be used as a treatment approach for cancer patients by increasing arginine concentration in the tumors and help the immune system to kill cancer cells. When the treatment is done, arabinose can be taken to get rid of the bacteria in the body.

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 10-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 10-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 10-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.

Figure 10: (A) Effect of arginine on the viability of CT26 cells (colon cancer cells): the higher concentration of arginine, the lower survival rate in percent of CT26 cells ; (B) Effect of arginine on the lactic acid level of CT26 cells ; (C) Effect of arginine on the PD-L1 mRNA expression of CT26 cells

We also co-cultured CT26 cells with the EcN1917^△^argRstrain 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 11, 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.

Figure 11: Effect of EcN1917^△^argR on the viability of CT26 cells (MOI: multiplicity of infection, the ratio of the number of bacteria to the number of target cells)

4. Future work

During the whole progress of the experiments, the function-testing part is not that efficient to examine how our engineered probiotics works. We may further do some trials on laboratory mice and deduce the function on human bodies, which can be better for us to ensure that arginine will work successfully and the bacteria will not cause any other damage to human.