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.
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].
Each of our 20 isoforms of the EcnB polypeptide toxin is naturally produced by a different species of bacterium as shown in the chart below.
| Plasmid |
Bacterial Origin |
| A |
Ralstonia pickettii DTP0602:
Raslstonia pickettii is a gram-negative, rod-shaped bacteria. This oligotroph is typically found in damp environments ranging from rivers to soils to biofilm on waterpipes. R. pickettii has the ability to sequester toxic metal ions and degrade harmful hydrocarbon molecules, and has consequently been of great interest in the bioremediation research community. There is no documentation regarding R. pickettii affecting healthy individuals, but if it contacted an individual with an already compromised immune system, it can infect the respiratory tract and bloodstream. |
| B |
Methyloversatilis universalis FAM5:
M. universalis is a gram-negative, facultative methylotroph. It has the ability to grow on single carbon compounds like methane and dimethylsulfide, and consequently have been of interest to greenhouse gas and environmental research. This particular strain was isolated from lake sediments. |
| C |
Xanthomonas arboricola pv. pruni MAFF 301427:
Xanthomonas arboricola is a gammaproteobacteria.These bacteria are responsible for causing the bacterial spot of stone fruit, a prevalent diseases amongst peach, plum, nectarine, apricot, and almond orchards. They are found worldwide, stretching across four continents. All strains of this bacteria are pathogenic and test positive for starch hydrolysis and quinate metabolism |
| D |
Sphingobium yanoikuyae:
Sphingobium yanoikuyae is a soil bacterium. It can also be isolated from clinical specimen and has unique abilities including degrading potent environmental pollutants, such as biphenyl, naphthalene, and phenanthrene. They have therefore been of interest for their bioremediation potential. |
| E |
Bordetella avium:
Bordetella avium is a gram negative, motile, non-spore forming coccobacillus (microbewiki). It is found in patients with cystic fibrosis and have been known to causes bordetellosis (upper respiratory disease) in poultry (Raffel TR 2002). It preferentially binds to ciliated tracheal epithelial cells (Spears PA 2003). The entericidin locus in B. avium is activated under high osmolarity and may function together with hydrolase to affect water concentration. Entericidin A and B are cell-envelope lipoproteins that also allow plasmids to be passed on to daughter cells (wikipedia). |
| F |
Azospirillum brasilense:
Azospirillum brasilense is a soil bacteria that promotes plant growth via nitrogen fixation. Its motile flagellum is critical in attaching to plant roots in rhizosphere (microbewiki). Genome data suggests that this genus transitioned from water to land around the time of the emergence of vascular plants (Azospirillum Genomes Reveal Transition of Bacteria from Aquatic to Terrestrial Environments 2011). Entericidin’s role in osmolarity may play a role in this transition. |
| G |
Escherichia coli str. K-12 substr. DH10B:
Escherichia coli are generally found in “animal feces, lower intestines of mammals, and even on the edge of hot springs.”[1] This gram negative, rod like bacterium produces the ecnB protein by RpoS (a sigma factor) regulation. The protein acts as a toxin to induce “programmed cell death of bacterial populations in stationary phase.”[2] |
| H |
Enterobacter aerogenes KCTC 2190:
Enterobacter aerogenes is generally found in “soil, water, dairy products, and in the intestines of animals” [1]. This highly motile, rod-shaped, gram negative bacterium is commonly found in respiratory, gastrointestinal, and urinary tract infections as well. It tends to be an opportunistic bacterium that infects a host whose immune system is already suppressed. |
| J |
Mannheimia haemolytica D174:
Mannheimia haemolytica has been implicated in bovine respiratory disease. It is the primary cause of epizootic pneumonia in cattle, also commonly known as Shipping Fever. This bacterium can be found in the nasopharynx of hosts and enters the lungs when host defenses are weakened by stress or infection. M. haemonlytica can produce a cytotoxin known as leukotoxin which targets leukocytes, potentially contributing to its pathogenicity. |
| K |
Cedecea neteri:
Cedecea neteri is a rare gram-negative, motile, nonspore-forming bacillus. It has been associated with bacteremia and isolated from human clinical specimens. |
| L |
Klebsiella oxytoca G54:
Klebsiella oxytoca is a gram-negative, rod-shaped bacteria. It is commonly cultured from the healthy skin, mucous membranes, and intestines of healthy humans, but is also a pathogen. K. oxytoca has been found in patients with antibiotic-associated hemorrhagic colitis, urinary tract infections, and celiac disease. |
| M |
Thioclava sp. 13D2W-2:
Thiocava is an aerobic and sulfur-oxidizing bacteria found in sulfidic hydrothermal regions. Thioclava are autotrophic and grow with thiosulfate as an energy source. |
| N |
Sinorhizobium meliloti 1021:
Sinorhizobium meliloti is a gram negative, nitrogen fixing bacterium. It exists symbiotically with legumes and works in the denitrification process (microbewiki). Enzymatic reactions and cellular processes change significantly when bacterium occupies root nodules of hosts (Djordjevic MA 2004). Proteins that play a significant role in the occupation process include osmoregulation proteins and potentially entericidin. |
| O |
Acinetobacter baumannii:
Acinetobacter baumannii is a pleomorphic gram negative organism that is associated with nosocomial infections. It is often found in aquatic environments and is known for its antibiotic resistance. |
| P |
Rhodobacter capsulatus:
Rhodobacter capsulatus is a purple, photosynthetic bacterium with a high capacity for “aerobic chemoautotrophic growth,” helping it to grow in a variety of conditions [1]. It’s use of oxygen as a terminal electron acceptor allows for this ability. It is mainly found in freshwater and marine environments. |
| Q |
Psychrobacter sp. 1501(2011):
Psychrobacter bacteria tends to live in very cold environments, including “Antarctic ice, soil, sediments” and the deep ocean [1]. This bacterium has been found in fish and meat products and, less commonly, in human tissue. It is a gram negative, non-motile bacterium. |
| R |
Agrobacterium:
Agrobacterium is a gram-negative, non-sporeforming, rod-shaped bacterium. It is known to cause gall disease and has been studied in its mechanism to cause tumors. Agrobacterium are usually found on root surfaces and infects wound sites in root tissues. |
| S |
Thalassospira: n/a
|
| T |
Erwinia: E. amylovora is a gram-negative, rod shaped soil bacterium. It is known to be harmful to plants and is pathogenic for orchards including pears and apples. |
| U |
Lautropia mirabilis ATCC 51599 |
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
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.
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
