Team:Minnesota/Design
Minnesota
Our project was designed to engineer E. coli with two different plasmids: one will contain the
genes for the purine auxotrophy system and the other will have the arabinose induced
overexpression vector. The delayed auxotrophy system will serve as the control mechanism by
inhibiting cell replication as the plasmid is further diluted out within the population of bacterial
cells over time. The overexpression system will be made using the inducible AraC promoter,
which only allows for plasmid replication in the presence of arabinose. This system will allow
for confirmation of cloning of cytolysin FitD into the overexpression plasmid and ensure the E.
coli strain would be able to survive with the toxin. After creating the two plasmid systems, they
will be combined and their effectiveness will be tested against zebra mussel viability.
As previously mentioned, the delayed auxotrophy system will serve as a control system so the
replication of the bacteria can be eventually inhibited upon release into the lakes. The plasmid
will consist of a ori6K origin of replication, the associated pir gene under the control of a Ptac
promoter, the lacI repressor under a constitutive promoter, the purA gene, and an ampicillin and
chloramphenicol resistance marker.
The overexpression system will be created using the PBAD18-Kan plasmid which has a pBR322 origin of replication, the AraC gene, and a kanamycin resistance marker.7 See Figure 2 below. In order to assemble the cytolysin FitD toxin, Gibson’s assembly will be used as the nucleic acid sequence is 6,000 base pairs.8 The E. coli strain used will be an AraC knockout mutant; E. coli will be used as a model system for the research into Pseudomonas fluorescens and because it is an easier organism to manipulate.
After the expression of the FitD protein is confirmed, the toxicity of the protein will be tested against zebra mussel. The Minnesota Aquatic Invasive Species Research Center (MAISRC), which works in partnership with the University of Minnesota, has agreed to provide zebra mussels so an experiment to determine LD50 can be run on the engineered E. coli.
