Part:BBa_K1595024:Experience

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Overview of Characterization of MBP+EcnB BioBrick

The peptide EcnB is capable of inhibiting the growth of F. psychrophillum in order to treat bacterial coldwater disease prevalent in salmonid species. In order to stabilize the EcnB peptide, we have placed EcnB downstream of MBP, a maltose binding protein. We have experimentally validated BBa_K1595024 to work as expected in decreasing the growth of F. psychrophillum as detailed below using lysate assays as well as disk diffusion assays.

All lysate assay tests were run with 20 mL cytophaga media samples. The samples were inoculated from a -80℃ frozen glycerol stock of F. psychrophillum and then incubated at 15℃ for 48-72 hours. An overnight culture of EcnB-BL21 constructs were used to inoculate a preinduction culture consisting of LB and chloramphenicol (25μg/mL). The cultures were incubated at 37℃ for 6 hours and induced with L-arabinose (1% w/w). After 6 more hours of incubation at 37℃, 125 mL of culture were centrifuged at 5000g for 10 minutes. The pellets were resuspended in 1 mL phosphate buffer saline solution and homogenized (4) for 10 minutes. The lysates were centrifuged at 10000g for 10 minutes. Samples were placed either on ice or at 4℃ throughout the lysis process. The supernatants were added to the F. psychrophillum cultures. Optical density readings were performed using a spectrophotometer set at OD600. Readings were performed over 12 hours. The chart below depicts the progression of OD600 over 12 hours after the inoculation of lysate from BBa_K1595024 as compared to lysate from BL21 control and lysate from buffer control. As shown, F. psychrophillum exposed to lysate from BBa_K1595024 experienced a drop in growth as expected, whereas F. psychrophillum exposed to BL21 and buffer lysate did not exhibit a decrease in growth.

We further validated the efficacy of BBa_K1595024 using a series of disk diffusion assays on plates inoculated with by soaking disks in lysate solution from BBa_K1595024, and compared the diameter of the zone of inhibition to that of oxytetracycline soaked disks (the current fish farm industry standard antibiotic used to treat BCWD), BL21 lysate soaked disks as negative control, and a variety of lysates from other BioBricks we have made. The BBa_K1595024 zone of inhibition had an average diameter of 50.7mm, as compared to 55.2mm from oxytetracycline’s zone of inhibition. Pictured below is one example of a ZOI assay for BBa_K1595024 (right), control(left), and oxytetracycline(upper disk).

From the results above, it is clear that BBa_K1595024 functions notably well in inhibition of F. psychrophilum. Below are the Zone of Inhibition mean diameter measurements for two negative controls, empty BL21 and BBa_K1460001, the positive control, oxytetracycline, and the EcnB constructs, BBa_K1595006, BBa_K1595008, BBa_K1595016, BBa_K1595024. As expected, BBa_K1595024 is able to decrease the growth of F. psychrophilum and is comparable if not better than oxytetracycline's antibiotic abilities.