Part:BBa_K1809034

Const. Promoter-RBS-BclA-CfAFP-DT

This part contains the constitutive promoter BBa_J23100, an RBS, the cell surface expression tag BclA, Choristoneura fumiferana antifreeze protein (CfAFP), and a double terminator. It encodes an antifreeze protein that attaches to the cell surface.

Antifreeze proteins (AFPs) are molecules with the unique property of binding and shaping ice crystals, preventing their growth into relatively large cell-lysing structures (Davies and Sykes 1997). They exhibit a property known as thermal hysteresis, whereby their activity decreases the freezing point of water without changing the melting point. This property allows organisms which produce AFPs to survive colder conditions. Additionally, an AFP from the deer tick Ixodes scapularis has been shown to have biofilm inhibiting properties, potentially giving these AFPs uses in both commercial and medical applications (Heisig et al. 2014).

We tested 16 different AFPs for both freeze survival and biofilm activity in two different constructs. The first construct simply expressed the antifreeze protein strongly, while the second construct included a BclA tag that caused the antifreeze protein to bind to the cell surface. This AFP, taken from the eastern spruce budworm Choristoneura fumiferana (CfAFP), was incorporated into only the BclA construct for testing.

To test freeze survival, a liquid culture of E. coli was grown up overnight and three samples were taken. An MTS assay was performed for the first sample to determine the amount of live E. coli present, while the other two samples were placed in a slow-freeze box at -20C and -80C respectively. After 24 hours MTS assays were then performed on these two samples to determine what percentage of cells survived the freezing.

Figure 1: Freeze survival for an empty vector control and CfAFP at -20C and -80C.

BclA-CfAFP showed no conclusive freeze survival in either strain at either temperature.

To test biofilm activity, each construct was transformed into the biofilm forming strain EMG2:Kλ E. coli and the strain was grown on a 96 well plate. Crystal violet was added to each well and washed, then acetic acid was used to break down the remaining crystal violet so that biofilm formation could be measured colorimetrically. These results were then normalized to an EMG2:Kλ without plasmid control.

Figure 2: Biofilm activity for an empty vector control and CfAFP.

BclA-CfAFP showed significantly decreased biofilm formation compared to the control as expected.

References

Davies, P. L., and B. D. Sykes. 1997. Antifreeze proteins. (0959-440X (Print)).

Heisig, M., N. M. Abraham, L. Liu, G. Neelakanta, S. Mattessich, H. Sultana, Z. Shang, J. M. Ansari, C. Killiam, W. Walker, L. Cooley, R. A. Flavell, H. Agaisse, and E. Fikrig. 2014. Antivirulence properties of an antifreeze protein. (2211-1247 (Electronic)).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 405
  Illegal AgeI site found at 343
  
• 1000
  COMPATIBLE WITH RFC[1000]