Difference between revisions of "Part:BBa K652004"

Revision as of 03:47, 19 September 2015

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]https://parts.igem.org/wiki/index.php?title=Part:BBa_K1809021] and [1].

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.

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.

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

Sequence and Features