Part:BBa_K4430009

TFP

It is the key part that is responsible for expressing tail fiber protein (TFP). TFP is a receptor binding protein (RBP) from the genome of Escherichia coli (EC) phage T7. Researches have demonstrated that GFP-TFP can bind strongly to the cell surfaces of E. coli strains.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1464
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1432
  Illegal AgeI site found at 1087
  Illegal AgeI site found at 1525
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 526
  

Usage and Results

Plasmid Construction

We designed our functional part TFP and cloned into pET28a backbone plasmid. As mentioned on our design page, in order to separate EC from food samples, pET28a-TFP (Figure 1) plasmid was synthesized by Tsingke Biotechnology Co., Ltd.

Figure 1 Plasmid map of pET28a-TFP.

Protein expression test

SDS-PAGE electrophoresis was used to check the expression of TFP protein (64.1 kDa). As shown in Figure 2, this protein has been successfully expressed and purified.

Figure 2 Protein SDS-PAGE electrophoresis results of TFP.

Magnetic separation effect

To determine how efficient magnetic beads-TFP (MBs-TFP) were for capturing bacteria in solution, the capture efficiencies were calculated. Colonies on the plates were counted after incubation for 16 h at 37 °C. The number of colonies before (nbefore) and after (nafter) magnetic separation were compared to calculate the capture efficiency using the equation. The calculated capture efficiency was 95.65%, decreasing as bacteria in mix (Table 1). E.coli strain K12 MG1655 is used in this experiment.

Table 1 Magnetic separation effect of E. coli by MBs-TFP