Overview

flavocide is fishPHARM’s biodegradeable peptide treatment for BCWD. To create our optimal treatment system, we had to first determine the most effective EcnB peptide against F. psychrophilum, the causative agent of BCWD. Multiple isoforms of the polypeptide toxin exist, each of which is produced by a different species of bacterium in nature. Zone of Inhibition trials and growth assays were then conducted to decide which species of bacterium produced the most potent EcnB isoform. A variety of organisms cause disease through their very presence in the host organism. The exploitation of toxins similar to the system developed by our team this year could pave the way for a microbiological treatment protocol for a plethora of other infectious diseases.

BioBricks

Our project relied heavily on the use of BioBricks. We are in the process of creating 20 different BioBricks for each isoform of EcnB. We have also added our EcnB peptide downstream of stabilization proteins MBP and EDA to help stabilize EcnB production. Our constructs have been summarized as follows:

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 have included a constitutive T7 promoter, a ribosome-binding site, the MBP gene, the TEV protease cutsite gene, the EcnB gene, 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 in iGEM’s history, we wanted to test it with a known 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.

Chassis

BL21 is a cell strain commonly used with the T7 bacteriophage promoter system. In its chromosomal DNA is the T7 RNA polymerase gene, which can be regulated by arabinose induction and glucose inhibition of the araBAD promoter. This allows for efficient and high-level protein expression. Furthermore, the T7 Lysozyme gene in the pLysS plasmid is able to reduce basal expression by suppressing T7 RNA polymerase activity in uninduced cells [1].

EcnB Isoform & Strain List

Each of our 20 BioBrick isoforms of the EcnB polypeptide toxin is naturally produced by a different species of bacterium as shown in the chart below.

Growth & Overexpression

Protein Stabilization

Because ecnB peptide has a relatively small size of approximately 5.3 kDa (or ~48 amino acids), it can be easily degraded within E. coli after inducing overexpression. To avoid this, we introduce the usage of fusion proteins for enhanced stability and yield.

The first fusion protein in question is with the maltose-binding protein (MBP) used in E. coli in the catabolism of maltodextrins. Having a MBP-fusion protein generally increases the solubility of proteins expressed in E. coli, though for us the primary usage is to artificially increase the size of the ecnB protein to avoid degradation. Once expressed and isolated from the system, it then becomes rather simple to recover the ecnB protein through the usage of a TEV protease cut site situated between MBP and ecnB. To this end, we have developed a BioBrick containing a constitutive T7 promoter, a ribosomal binding site, the MBP gene, the TEV protease cut site, the ecnB gene of interest, a 6xHis tag, and a terminator.

The second fusion protein includes the addition of the fusion expression partner KDPG aldolase (EDA), a novel solubility enhancer protein that has yet to be BioBricked in iGEM. Similar to MBP previously, EDA increases solubility of the chimeric proteins as well as limiting aggregation of the fusion partner.

ZOI Assays

Future Work

The Cornell iGEM wet lab subteam is committed to discovering the next generation of medicine for fish. Our goal is to provide our customers the most effective and cost-efficient form of flavocide. To accomplish this, we seek to perform a series of medical trials to further test our product's efficacy and we aim to streamline the manufacturing process for large-scaled production.

From the Zone of Inhibition (ZOI) assays, we have preliminary data for comparing the relative strengths of our EcnB isoforms. To ensure our medicine also provides a long lasting form of protection, we are in the process of testing the functional stability of each isoform. We aim to compare their extracellular degradation rates and hope to lengthen functional lifetimes by successfully linking them downstream of stable fusion proteins like EDA and MBP.

In addition, we understand that fish are dynamic systems. To account for these additional levels of complexity, we seek to develop a proposal for safely and ethically testing the efficacy of flavocide in live salmonids. First, we hope to test for the effective EcnB concentration range against Flavobacterium through ZOI assays. With these results, we could use our drug delivery model to predict the required effective EcnB concentration range in the salmonid bloodstream. This provides a reasonable magnitude for drugs to test in later experimental trials. With this information, we can develop an informed proposal in accordance with the IACUC regulation and code of conduct. Although we don't expect bioaccumulation and toxicity in the fish system, we would like to confirm and verify that our proposed isoform falls within this accepted assumption.

Once our chosen isoform shows progress against bacterial coldwater disease, we can optimize the manufacturing process for large scaled production. This would include characterizing bacterial growth and expression for crucial parameters to minimize time and material expenses while maximizing flavocide yields.

By continuing to advance wet lab on all fronts, we hope to provide fisheries with an even more comprehensive solution against BCWD in the near future.

References

[1]Garcia, L., & Molineux, I. (1995). Rate of translocation of bacteriophage T7 DNA across the membranes of Escherichia coli. Journal of Bacteriology, 177(14), 4066-4076.