Part:BBa_K652004:Design

RBS-ZeAFP-Term

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]

Design Notes

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 <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1809021">K1809021</a> and <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1809022">K1809022</a>.

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.

Source

Generously provided to us by Peter Davies and Sherry Gauthier of Queen's University.