Part:BBa_K3605005

CcpAx protein from Gluconacetobacter xylinus.

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.

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 Fig.1.

Fig.1. The result of CcpAx gene cloning. M: Marker; 1: PCR result of CcpAx; 2: Digestion of pSB1C3 containing CcpAx.

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 Fig.2.

Fig.2. 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.

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 f.3.

Fig.3. 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 441
  Illegal AgeI site found at 204
  
• 1000
  COMPATIBLE WITH RFC[1000]