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Novel Coronavirus was first epidemic and first gain control in China. So we are lucky to have the opportunity to go into the lab and do research for our project. We have successfully cloned some genes involved in bacterial cellulose synthesis and transferred them into E. coli for obtaining more stable, higher-yielding bacterial cellulose. We have created some parts using these genes, and here are three of them.

BBa_K3605004

This part is Endo-β-1,4-glucanase(CMCax) with 1080bp, from Gluconacetobacter xylinus. It can hydrolyze the glucan chain and help straighten out the glucan, which is important for bacterial cellulose. 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 1.

Fig.1. The result of CMCax gene cloning.

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

We used the software SnapGene Viewer to analyze the sequence of this part to make sure that there are no some key restriction enzyme sites in it. The result is showed in the follows (figure 2):

Fig.2. The sequence feature of CMCax gene.

BBa_K3605005

This part contains the CcpAx protein, also known as ORF-2, encoded by ccpax gene, functions as a mediator of protein-protein interactions and is important for localization of the bacterial cellulose synthase complex to the cell membrane in Gluconacetobacter xylinus. CcpAx gene, containing 936bp, was amplified using PCR method, and then inserted into the vector pSB1C3. The identification result is showed in Figure 3.

Fig.3. The result of CcpAx gene cloning.

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

We used the software SnapGene Viewer to analyze the sequence of this part to make sure that there are no some key restriction enzyme sites in it. The result is showed in the follows (figure 4):

Fig.4. The sequence feature of CcpAx gene.

CcpAx protein is crucial for the production of bacterial cellulose in the culture medium, because it helps the bacterial cellulose synthase to localize to the cell membrane. To make sure CcpAx protein is overexpressed in the transferred E. coli, we purified this protein using Ni-NTA affinity chromatography. The result indicated that CcpAx was successfully overexpressed, showed in the figure 5.

Figure 5. Expression of CcpAx and purification by Ni-NTA affinity chromatography.

M: protein marker; 1: precipitation samples in the cell lysates; 2: supernatant samples in the cell lysates; 3: 50 mM imidazole eluent; 4: 100 mM imidazole eluent; 5: 200 mM imidazole eluent; 6: eluent after purification. This figure showed that 200mM imidazole eluent is the best concentration for elution expressed CcpAx.

BBa_K3605006

This part is β-glucosidase encoded by bglxa gene with 2202bp in Gluconacetobacter xylinus. Both of the β-glucosidase and endoglucanase (CMCax, BBa_K3605004) 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.

The software SnapGene Viewer was used to analyze the sequence of this part to make sure that there are no some key restriction enzyme sites in it. The result is showed in the follows:

Fig.7. The sequence feature of β-glucosidase gene cloning.

As we stated in the design of our project, there are 4 enzymes involved in the synthesis of bacterial cellulose starting with glucose. In addition, CcpAx protein helps the Bcs localize to the cell membrane; Endoglucanase (CMCax) and β-glucosidase can hydrolyze tangled glucan chains when there is a failure chain arrangement. Using overlapping PCR, all these genes are connected to form two polycistrons, which are inserted to the expression vector pETDuet-1. The identification result is showed in figure 8.

Fig.8. 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).

After completion gene cloning work, the transferred E. coli were cultured in the medium for bacterial cellulose production. We used the optimized conditions acquired from the acetobacter xylinum culturing for producing bacterial cellulose. The E. coli were cultured for 7days at 30°C, pH6.8, 10% inoculum size, and glucose as the carbon source, then the cellulose in the medium was harvested. The result is showed in the figure 9.

Fig.9. The production of bacterial cellulose in transferred E. coli.

The result showed that the bacterial cellulose was produced successfully in the transferred E. coli, indicating that the parts transferred to the E. coli worked well. The result also showed that the production yield is not as high as in the acetobacter xyilus (showed in the result of our project on the wiki), suggesting that the culture conditions still need optimization.