Part:BBa_K5048068

Neae-LAP-6xHis

Codes for one of our team’s fusion proteins, presenting the LAP protein on the surface of E.coli BL21(DE3) to enable the bacteria-intestine adhesion and the bacteria-bacteria adhesion(Fig 1). Codon optimized for expression in E.coli.

Fig 1 The expected effect for the Adhesion module, which adheres to the engineering bacteria and small intestine

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1990
  Illegal PstI site found at 3427
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1990
  Illegal PstI site found at 3427
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1990
  Illegal BglII site found at 4470
  Illegal BglII site found at 4568
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1990
  Illegal PstI site found at 3427
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1990
  Illegal PstI site found at 3427
  Illegal NgoMIV site found at 2676
  Illegal NgoMIV site found at 3942
  Illegal AgeI site found at 2308
  Illegal AgeI site found at 3964
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1244
  Illegal SapI site found at 4290
  

Design

We constructed this part by restriction endonuclease cleavage and T4 ligation. During the process of design, we had to consider the enzyme and the cleavage site we needed to use. When inserted into the pET-28a backbone, this part is included in Adhesion Construction(Fig 2).

Fig 2 The genetic circuit respectively for pET-28a-[Neae-LAP] (right)

Source

See our parts: BBa_K5048047, BBa_K5048058

Experiments

This part acts as an adhesin construct that can display protein LAP on the outer membrane of E. coli and adhere to HSP60.

The overall experiments we conducted on BBa_K5048058 include:

1. Verification of PCR products
2. Verification of transformation of the plasmid

Results

Verification of PCR products

For the Neae-LAP plasmid, although the PCR primers lack specificity, we still obtain the targeted band(Fig 3) and implement purification to acquire the targeted segments.

Fig 3 The input for T4 ligase.
The target gene segments are marked in the red box. Segment LAP is marked in the red box below while the segment Neae backbone is marked in the upper box. Neae backbone: 7259bp; LAP: 2649bp

Verification of the transformation

For pET-28a-[Neae-LAP], we ultimately successfully transferred the plasmid into E. coli BL21(DE3) and had a corresponding sequencing result(Fig 4).

Fig 4 Neae-LAP successfully transferred into E. coli BL21(DE3)
A. The colony PCR for E. coli BL21(DE3) strains. Neae-LAP 3 has the segment corresponding to the estimated DNA length. Neae-LAP: 4831bp; B. The sequencing result for Neae-LAP in E. coli BL21(DE3), demonstrates the successful transformation.

Applications of BBa_K5048068

A part of Adhesion Construct that can display protein LAP on the outer membrane of E. coli and adhere to HSP60 in engineered bacteria and small intestine.