Part:BBa_K1423000:Experience

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Applications of BBa_K1423000

WPI-Worcester 2015 Characterization

As part of our antifreeze protein library, we created ten antifreeze constructs using the BclA tag to bind them to the cell surface. We then tested these antifreeze proteins for both freeze survival and effects on biofilm formation.

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 ten antifreeze proteins tagged with BclA at -20C and -80C.

The BclA-tagged antifreeze protein BclA-GaAFP showed increased freeze survival at -20C.

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 ten antifreeze proteins tagged with BclA.

We found that the BclA-tagged antifreeze proteins BclA-DcAFP, BclA-IAFGP, BclA-RiAFP and BclA-TiAFP all increased biofilm formation, while BclA-CfAFP was the only construct to show decreased biofilm formation.

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