1.Gene cloning from Acetobacter xylinus

To realize the production of bacterial cellulose (BC) in E. coli, we cloned 6 genes related to the synthesis of BC from acetobacter xylinus. Some genes were amplified using PCR methods, and one of them was synthesized directly. In order to insert these genes to the bi-expressing vector pETDuet-1, which contains two separate promoters, some genes were connected together to form polycistron using the overlapping PCR method.

1.1 Glucose hexokinase (GHK)

This enzyme catalyzes Glucose converted to Glucose-6-phosphate (G-6-P), which is the glycolytic intermediate and the origin of substrate synthesis for bacterial cellulose production.

Glucose hexokinase (GHK) was amplified using PCR method, and inserted into the vector pSB1C3. Figure 1 shows the identification result.

Fig.1. The result of Glucose hexokinase (GHK) gene cloning.

M: Marker; 1: PCR result of GHK; 2: Digestion of pSB1C3 containing GHK.

1.2 Phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGPase)

These two enzymes are original polycistron in the acetobacter xylinus, so we cloned them together. The phosphoglucomutase (PGM) can isomerize the glucose-6-phosphate (G-6-P) to glucose-1-phosphate (G-1-P), which is the second reaction for bacterial cellulose production from glucose. Another enzyme is UDP-glucose pyrophosphorylase (UGPase), it can catalyze the glucose-1-phosphate (G-1-P) to react with UTP, forming uridine-5’-phosphate-α-D-glucose (UDPG), which is the third reaction for bacterial cellulose production from glucose. The reactions catalyzed by them are as follows:

These two enzymes were amplified using PCR method, and inserted into the vector pSB1C3. Figure 2 shows the identification result.

Fig.2. The result of PGM and UGPase gene cloning.

M: Marker; 1: PCR result of PGM and UGPase; 2: Digestion of pSB1C3 containing PGM and UGPase genes.

1.3 Bacterial cellulose synthase (Bcs) complex

Bacterial cellulose synthase (Bcs) complex contains 4 subunits BcsA, BcsB, BcsC and BcsD, which catalyzes the bacterial cellulose synthesis, using uridine-5’-phosphate-α-D-glucose (UDPG). This is the last reaction for bacterial cellulose production from glucose.

Due to the long sequence (9102bp), this enzyme complex was synthesized directly and connected into pUC57 vector firstly, then it was inserted into bi-expressing vector pETDuet-1. The identification result is showed in Figure 3.

Fig.3. The gene cloning result of Bcs and other genes.

M: Marker; 1: Digestion of pETDuet-1 vector containing polycistron of GHK, PGM, UGPase, CMCax, CcpAx, and β-glucosidase; 2: Digestion of pETDuet-1 vector containing Bsc complex; 3: Digestion of pUC57 vector containing Bsc complex.

1.4 CcpAx protein (ORF-2)

The CcpAx protein, also known as ORF-2, functions as a mediator of protein-protein interactions and is important for localization of the Bcs complex to the cell membrane. This gene was amplified using PCR method, and then inserted into the vector pSB1C3. The identification result is showed in Figure 4.

Fig.4. The result of CcpAx gene cloning.

M: Marker; 1: PCR result of CcpAx; 2: Digestion of pSB1C3 containing CcpAx.

1.5 Endoglucanase (CMCax)

Endo-β-1,4-glucanase(CMCax) can hydrolyze the glucan chain and help straighten out the glucan, which is important for bacterial cellulose synthesis. When antibodies to recombinant CMCax are added to the culture medium, the formation of cellulose fibre is severely inhibited. This gene was amplified using PCR method, and then inserted into the vector pSB1C3. The identification result is showed in Figure 5.

Fig.5. The result of CMCax gene cloning.

M: Marker; 1: PCR result of CMCax; 2: Digestion of pSB1C3 containing CMCax.

1.6 β-glucosidase

β-glucosidase is encoded by bglxa gene in Gluconacetobacter xylinus. β-glucosidase and endoglucanase (CMCax) both can hydrolyze tangled glucan chains when there is a failure chain arrangement and are both crucial for bacterial cellulose synthesis. This gene was also amplified using PCR method, and then inserted into the vector pSB1C3. The identification result is showed in Figure 6.

Fig.6. The result of β-glucosidase gene cloning.

M: Marker; 1: PCR result of β-glucosidase; 2: Digestion of pSB1C3 containing β-glucosidase.

1.7 Construction of polycistron

Polycistron is one of the characteristics in Prokaryote. It is common that some genes are regulated by the same promoter. So, some cloned genes were connected to form a polycistron using overlapping PCR in our project. Then this polycistron was inserted into the expression vector pETDuet-1. This process was identified in figure 7 and figure 8.

Fig.7. The PCR results of GHK, (PGM and UGPase), CMCax, CcpAx, and β-glucosidase.

M: Marker; 1: GHK(969bp); 2: PGM and UGPase (2277bp); 3: CMCax(1080bp); 4: CcpAx(936bp); 5: β-glucosidase(2202bp).

Fig.8. The overlapping PCR results of some genes.

M: Marker; 1: GHK + PGM + UGPase + CMCax + CcpAx + β-glucosidase (7464bp); 2: CMCax + CcpAx + β-glucosidase (4215bp); 3: CMCax + CcpAx (2013bp); 4: GHK + PGM + UGPase (3249bp).

At last, the two polycistrons containing all the gene required for bacterial cellulose production were cloned successfully into the expressing vector pETDuet-1, and transferred to the E. coli. Figure 9 shows the identification result.

Fig.9. The gene cloning result of two polycistrons.

M: Marker; 1: Digestion of pETDuet-1 vector containing polycistron of GHK, PGM, UGPase, CMCax, CcpAx, and β-glucosidase; 2: Digestion of pETDuet-1 vector containing Bsc complex (BcsA, BcsB, BcsC, and BcsD).

2.The optimization of culture conditions of Acetobacter xylinus

When we were cloning these genes, we cultured the acetobacter xylinus to optimize some conditions for high BC production yield. This strain was donated friendly by iGEM team BNDS_China, which collaborated with us all the time in this season.

We searched some references, and figure out that the temperature, pH value, fermentation time, inoculum size, and the carbon sources are the important conditions. So, we tried to culture the acetobacter xylinus for BC production with different conditions. And the results are showed in the following figures (Fig. 10-Fig. 14).

Fig.10. The effect of temperature on BC production.

Fig.11. The effect of pH value on BC production.

Fig.12. The effect of fermentation time on BC production.

Fig.13. The effect of inoculum size on BC production.

Fig.14. The effect of carbon sources on BC production.

The optimization experiment results indicated that 30°C, pH6.8, fermentation 7 days, 10% inoculum size, and glucose serving as the carbon source are the best culture conditions for high BC production yield in acetobacter xylinus. And we know that for the BC production in E. coli, these conditions should be optimized again. But these optimized conditions can be referred for the following experiments.

Team:JNFLS/Results