Team:Cornell/wetlab
- WHAT WE DO
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fishPHARM
- DRY LAB
- fishBit
- Heimdall Monitoring
- MODELING
- Math Modeling
- Animation
- DISCUSSION
- Policy and Practices
- Entrepreneurship
- Collaborations
- DEVELOPMENT
- Notebook
- Future Applications
- Safety
- OUTREACH
- OUR TEAM
Overview
In order to determine the most effective probiotic strain of bacteria against Bacterial Coldwater Disease, we engineered the BL21 strain of Escherichia coli for the production of twenty isoforms of the Entericidin B peptide (EcnB). EcnB is toxic to the growth of Flavobacterium psychrophilum, the causative agent of BCWD. To create the optimal treatment system against BCWD, we had to first determine the most effective EcnB peptide against F. psychrophilum. Multiple isoforms of the polypeptide toxin exist, each of which is produced by a different species of bacterium. Trials were thus conducted to decide which species of bacterium produced the most potent EcnB isoform. In essence, the different Entericidin types were placed in plates that were subsequently cultured with F. psychrophilum. The potency of each Entericidin isoform was assessed with Zone of Inhibition (ZOI) measurements. Once a winner was declared, the gene for the isoform was employed in our transformation. Our project relied heavily on the use of BioBricks. We created and documented 20 EcnB BioBricks. In addition, we recognized our EcnB peptides potentially needed additional help for stability in overexpression. We tackled this problem by designing the potentially stabilizing BioBricks for EcnB production. These parts are summarized as follow -- EcnB only series: These parts included a constitutive T7 promoter, a ribosome-binding site, the EcnB genes, a 6X Histidine tag to facilitate protein purification, and a terminator. MBP-TEV-EcnB series: These parts included a fusion protein called Maltose Binding Protein (MBP) for additional stability. They included a constitutive T7 promoter, a ribosome-binding site, the MBP gene, the TEV protease cutsite gene, the EcnB genes, a 6X Histidine tag to facilitate protein purification, and a terminator. EDA-GSG series: These parts included a newly discovered fusion protein called KHG/KDPG adolase (EDA). Since EDA has never been characterized or BioBricked, we wanted to test it with a known stable endoglucanase called cel5a. The first BioBrick included a constitutive T7 promoter, a ribosome-binding site, the EDA gene, the GSG linker sequence, BamHI/NdeI restriction sites for modularity, a 6X Histidine tag to facilitate protein purification, and a terminator. The second BioBrick included a cel5a gene at the modular site. EcnA/EcnB: This BioBrick was created because EcnA is known to be the antidote for EcnB. When they are synthesized together, they are expected to offset the properties of each other. This part included a constitutive T7 promoter, a ribosome-binding site, the EcnA gene, the TEV protease cutsite gene, the EcnB gene, BamHI/NdeI restriction sites for modularity, a 6X Histidine tag to facilitate protein purification, and a terminator. Restriction enzymes were used to place all of these parts on the plasmid delivered to the Escherichia chassis. Plasmids akin to the type employed by our project can be used to treat a host of other illnesses. A variety of organisms cause disease through their very presence in the host organism. Exploitation of toxins that target them in systems similar to the one devised by our team this year could pave the way for a microbiological treatment protocol for a plethora of different infectious diseases including.