Part:BBa_K1897017

Invasin + GFP

This part is made by 3A ligation of Invasin BBa_K1897011 and GFP BBa_K1897014. Invasin is derived from Yersinia pseudotuberculosis, while green fluorescent protein (GFP) is derived from Aequeora victoria.

Usage and Biology

This part was intended for use in conjunction with BBa_K1897015 to allow for invasion of the co-transformed bacteria into mammalian cells expressing β1-integrin on their surface. Activation of transcription of invasin and pLuxR requires BBa_K1897015 or externally added LuxR + N-(3-oxo-hexanoyl)-homoserine lactone (AHL). The presence of GFP allows for the viewing of bacteria under the microscope after an invasion assay to visually see if the bacteria has successfully invaded the cell or not. It also allows for the verification that genes under the control of pLuxR (BBa_R0062) has been expressed.

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
  Illegal AgeI site found at 3346
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4002
  

Construction of Invasin+GFP

LuxR+LuxI was made via 3A ligation of invasin BBa_K1897011 and GFP BBa_K1897014. This was done using the restriction enzyme sites SpeI on the biobrick suffix of invasin (BBa_K1897011) and XbaI on the biobrick prefix of GFP (BBa_K1897014). After cutting those two restriction enzyme site, ligation was performed using T4 ligase and a scar site is formed, joining the two fragments together. At the same time, the EcoRI site on the biobrick prefix of invasin (BBa_K1897011) and the PstI site on the biobrick suffix of GFP (BBa_K1897014) was also cut, and joined to a pSB1K3 backbone that was also cut with EcoRI and PstI. This resulted in an end product of a plasmid with invasin and GFP joined together.

Verification of Invasin+GFP

Figure 1: Gel electrophoresis photo to check that the 3A ligation was successful for invasin+GFP. A restriction enzyme (RE) digestion was performed using EcoRI and SpeI on the individual plasmids. A single digest control was done to ensure that the digestion was successful. The expected band size for invasin+GFP was around 4200 bp, which is seen in Box B (a band between 4000 and 5000 bp) on the gel. Each lane in Box B is a duplicate digest reaction.

A restriction digest (single cut as positive control and double cut) was done on the plasmid obtained to check for the insert size. The expected insert size was around 4200 bp, which is shown in Box B of Figure 1. The band below Box B is the pSB1K3 backbone. As the band size appears to be between 4000 and 5000 bp, this likely indicates that the 3A ligation was successful.