Part:BBa_K1897011

Invasin

This part was made up of promoter pLuxR BBa_R0062, ribosome binding site (RBS) BBa_B0030, Invasin coding sequence BBa_K1897010 and double terminator BBa_B0015. It is derived from Yersinia pseudotuberculosis, and codes for a 103-kDa membrane surface protein that recognises and binds to β1-integrin on the surface of eukaryotic cells (Isberg and Leong, 1988; McCormick et al., 1997).

Usage and Biology

Invasin allows Yersinia to invade mammalian cells and hide from the immune system. Once invasin binds to β1-integrin on the surface of eukaryotic cells, it is internalised into the cell (Isberg and Leong, 1988). It has been shown by Isberg and Falkow (1985) that with just the invasin gene alone, Escherichia coli can invade mammalian cells expressing β1-integrin. This allows for the entry of E. coli into mammalian cells, and allows for potential delivery of molecules into targeted mammalian cells when used in conjunction with other systems (for example, with the use of quorum sensing, where only in an area of high bacterial cell density will invasin be produced).

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 4
  Illegal XhoI site found at 2791
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 976
  Illegal AgeI site found at 2485
  
• 1000
  COMPATIBLE WITH RFC[1000]

Construction and verification of composite Invasin

Invasin was sent for synthesis, and colony screening was done to the transformed Escherichia coli colonies to identify those with the synthesized plasmid (Figure 1).

Figure 1: Gel electrophoresis photo of colony screening done to identify the transformed clones that consisted of the plasmid with invasin. Each lane indicates one screening for one colony. As seen in the gel picture, colonies 1, 2, 4, 5, 7 and 8 had the desired band size of around 3313 bp (Box A).

As seen in Figure 1, colonies 1, 2, 4, 5, 7 and 8 were positive clones for invasin plasmid. The bacteria was co-transformed with two plasmids BBa_K1897017 and BBa_K1897008, and invasin protein induction was done by adding 100 μM of N-(3-oxo-hexanoyl)-homoserine lactone (AHL) to the transformed bacteria and incubated overnight (at 37 degrees with shaking). The cells were then lysed and centrifuged. Western blot for invasin was done on both the lysate and pellet obtained, and a control was also done without addition of AHL and also lysed and centrifuged. The expected protein band size is approximately 108 kDa, slightly larger than the theoretical size since there are also two HA-tags.

The protein expression of invasin was also tested (Figure 2).

Figure 2: Induction of invasin with pLuxR. The bacteria was co-transformed with two plasmids BBa_K1897017 and BBa_K1897008, and invasin protein induction was done by adding 100 μM of N-(3-oxo-hexanoyl)-homoserine lactone (AHL) to the transformed bacteria and incubated overnight (at 37 degrees with shaking). The cells were then lysed and centrifuged. Western blot for invasin was done on both the lysate and pellet obtained, and a control was also done without addition of AHL and also lysed and centrifuged. The expected protein band is boxed up (expected size: around 108 kDa), and two clones (#2 and #17) were used.