Part:BBa_K652004

RBS-ZeAFP-Term

This part contains an RBS, the type III Zoarces elongatus antifreeze protein (ZeAFP), followed by a terminator.

Note: Becuase this protein is so small (7kDa), expression may be difficult to detect on a Tris-Glycine gel. Using a Tris-Tricine system where small proteins are easier to see (not so diffuse) is advisable.

WPI-Worcester 2015 Characterization

As part of our antifreeze protein library, we characterized ZeAFP by measuring both its freeze survival and biofilm activity, as well as creating the BioBricks BBa_K1809021[1] and BBa_K1809022[2].

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 ZeAFP at -20C and -80C.

We found no conclusive evidence for ZeAFP promoting freeze survival in E. coli.

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 ZeAFP.

We found that ZeAFP significantly increased biofilm formation relative to our control.

WPI iGEM 2018 Additions

This graph shows the concentrations of purified proteins, including ZeAFP, labeled as "Ze" on the graph.

These are graphs of biofilm assays that included the protein ZeAFP. The proteins were either mixed into the bacteria or it was used to coat the plate that the biofilm was going to be grown on.

This is a graph of the biofilm production with and without the expression of different antifreeze proteins.The expression of these proteins was proven on the following gel:

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 33
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 222
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]