Part:BBa_K3089013

csgA-linker-mfp5-mfp5-His fusion protein for yeast expression

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 274
  Illegal PstI site found at 23
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 274
  Illegal PstI site found at 23
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 274
  Illegal XhoI site found at 244
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 274
  Illegal PstI site found at 23
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 274
  Illegal PstI site found at 23
  
• 1000
  COMPATIBLE WITH RFC[1000]

This recombinant protein is our own new design which has a property of both cohesion (CsgA) and adhesion (Mfp5). CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing mechanical cohesive strength. Mfp5 is mussel foot proteins from Mytilus galloprovincialis responsible for interface adhesion. By adding two Mfp5s to CsgA, we expect it to have a stronger adhesive property. The results show, this recombinant protein can adhere to plastics and glasses better than any otherparts in our toolbox. The sequence of this composite part is optimized to achieve better expression in Pichia pastoris.

Sequence and Features BBa_K3089013 SequenceAndFeatures

Figure 1. Protein distribution in barnacle cements

Characterization

Three different experiments were done to characterise the BBa_K3089013 biobrick:

• Molecular cloning
• Protein expression

Molecular cloning

Figure 2. Illustration of gene insertion into Pichia pastoris GS115 genome. Box in blue indicates the recombinant protein CsgA-linker-mfp5-mfp5.

CsgA-linker-mfp5-mfp5-His was synthesized and cloned to yeast Pichia pastoris and 1 strain was verified by gel electrophoresis and sequencing (Figure 2).

Figure 3. Determination of gene insertion into the Pichia genome by gel electrophoresis.

Protein expression

Proteins were expressed in small scale induced by methanol. Unfortunately, no proteins of interest were found in culture medium of CsgA-Mfp5-Mfp5 after induced expression for 48 hours at 30℃(Figure 3).

Figure 4. SDS-PAGE confirm expression(30℃，48h) of CsgA-mfp5-mfp5 and rBalcp19k-mfp5(4 colonies) expression. P, cell pellets. S, supernatant.