Team:WPI-Worcester/Description
Project Description
Project Goals
The goal of this project is to generate an Antifreeze Protein Library, built from a diverse range of organisms and quantitatively characterized in E. coli. Antifreeze Proteins (AFPs) have demonstrated biofilm inhibition, a recently discovered property which we thought beared investigation (Heisig et al. 2014). Biofilms are an important part of antibiotic resistance in some bacteria, their disruption could be an important mechanism to combat the rise of antibiotic resistant strains (Donlan and Costerton 2002). Heisig et al. described the properties of a single AFP, IAFGP, and we knew that many more existed. We decided to focus our project on gathering and characterizing a broad group of AFPs into a library. The library is quantitatively characterized with regards to the AFP’s effect on freeze survival and biofilm formation. We believe this library will give synthetic biologists an array of tools to use to control biofilm formation. Overview
We searched the literature, including nucleotide and protein sequence databases, to find known antifreeze proteins. After generating a list of candidates we were able to select a number of AFPs which were well documented, of an appropriate size to clone into a plasmid vector, and which represented a wide range of organisms. We then took their sequences and codon optimized them for our host organism, E. coli. We then had that optimized sequence, with the biobrick prefix and suffix, synthesized. We used Biobrick Cloning to insert our sequences into a plasmid backbone and transformed the final product into E. coli. The transformed strains were used in our Biofilm and Freeze Assays to quantitatively measure the effects of our AFPs on freeze survival and biofilm formation. The freeze assay involved slow freezing cells to -20C or -80C for 24 hours and comparing the survival rate to that of the culture they were taken from. The biofilm assay involved growing the biofilm forming strain EMG2:Kλ E. coli expressing our antifreeze proteins in a 96 well plate, staining the wells with crystal violet after the incubation period, and solubilizing the crystal violet in acetic acid to quantify biofilm formation colorimetrically. We were interested in what effect the localization of the AFP had on its efficacy, and so each AFP was created in two forms, one native and one with an N-terminal BclA tag, which anchored it to the cell surface. Due to time constraints and difficulties with the cloning process not all AFPs were created in both forms; some exist only in the tagged or native form. Improved Characterization of ZeAFP
ZeAFP is an antifreeze protein from Zoarces elongatus. We tested BBa_K652004, created by the Yale 2011 iGEM team, using the promoter J23119 in our freeze survival and biofilm assays. We also created the theoretical coding part BBa_K1809021 for the region of BBa_K652004 which codes for ZeAFP and generated the part BBa_K1809022 using the promoter J23119 and ZeAFP. Our assay data can be found below.
ZeAFP Freeze Assay Data
ZeAFP Biofilm Assay Data
Improved Characterization of BclA
We created eleven AFP parts N-terminally tagged with BclA to localize them to the cell surface. BclA was taken from BBa_K1423005, created by the WPI 2014 team, and in our assays YFP was replaced with the respective AFPs. The BclA tagged parts were tested in our freeze survival and biofilm inhibition assays. Due to time constraints we were unable to test all BclA tagged parts in both assays. A list of the tagged AFPs, as well as our assay data, can be found below. BclA Freeze Assay Data
BclA Biofilm Assay Data
Resources
Donlan, Rodney M., and J. William Costerton. 2002. Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms. Clinical Microbiology Reviews 15 (2):167-193.
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)).
