Cornell iGEM

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.

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

Plasmid	Bacterial Origin
A	Ralstonia pickettii DTP0602
B	Methyloversatilis universalis FAM5
C	Xanthomonas arboricola pv. pruni MAFF 301427
D	Sphingobium yanoikuyae
E	Bordetella avium
F	Azospirillum brasilense
G	Escherichia coli str. K-12 substr. DH10B
H	Enterobacter aerogenes KCTC 2190
J	Mannheimia haemolytica D174
K	Cedecea neteri
L	Klebsiella oxytoca G54
M	Thioclava sp. 13D2W-2
N	Sinorhizobium meliloti 1021
O	Acinetobacter baumannii
P	Rhodobacter capsulatus
Q	Psychrobacter sp. 1501(2011)
R	Agrobacterium
S	Thalassospira
T	Erwinia
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

1. Citations