Part:BBa_K1809022
Const. Promoter-RBS-ZeAFP-DT
This part contains the constitutive promoter BBa_J23119, an RBS, Zoarces elongatus antifreeze protein (ZeAFP), 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 eastern viviparous blenny Zoarces elongatus (ZeAFP), was incorporated into only the untagged constructs for testing. This part was originally taken from Yale 2011's part BBa_K652004[1], and was made into this basic coding part for easier integration into plasmids.
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.
Our ZeAFP construct showed no increased freeze survival at either -20C or -80C.
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.
ZeAFP showed a significant increase in the formation in biofilms.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 77 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 266
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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