Part:BBa_K1809013

Const. Promoter-RBS-TiAFP-DT

This part contains the constitutive promoter BBa_J23119, an RBS, Typhula ishikariensis antifreeze protein (TiAFP), and a double terminator. It produces a high level of expression of the antifreeze protein.

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 gray snow mold Typhula ishikariensis (TiAFP), was incorporated into both constructs 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 TiAFP at -20C and -80C.

TiAFP was shown to have no effect on the freeze survival of E. coli in either construct or 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 TiAFP.

The untagged, normally expressed TiAFP showed a significant decrease in biofilm formation, while the tagged BclA-TiAFP showed significant increased biofilm formation relative to the control.

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 719
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 152
  Illegal AgeI site found at 281
  Illegal AgeI site found at 311
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 572