Difference between revisions of "Part:BBa K1595024:Experience"
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All lysate assay tests were run with 20 mL cytophaga media samples. The samples were inoculated from a -80℃ frozen glycerol stock of <i>F. psychrophillum</i> 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 <i>F. psychrophillum</i> 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. | All lysate assay tests were run with 20 mL cytophaga media samples. The samples were inoculated from a -80℃ frozen glycerol stock of <i>F. psychrophillum</i> 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 <i>F. psychrophillum</i> 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. | ||
| − | We further validated the efficacy of | + | We further validated the efficacy of BBa_K1595024 using a series of disk diffusion assays containing lysate from BBa_K1595024, and compared the zone of inhibition to oxytetracycline used in the field as well as BL21 lysate as a negative control. The BBa_K1595024 zone of inhibition had an average diameter of 50.7mm, as compared to 55.2mm from oxytetracycline’s zone of inhibition. A two-sample t-test shows no statistical difference between the two diameters (p > 0.05). From these results, it is rather clear that the fusion protein MBP+EcnB functions notably well in combating <i>F. psychrophilum</i>, with EcnB retaining its effectiveness despite being part of a fusion protein combination. Below is the Zone of inhibition 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. |
<img src="https://static.igem.org/mediawiki/2015/a/a6/Cornell_Lysate_assay_7_new.jpeg"> | <img src="https://static.igem.org/mediawiki/2015/a/a6/Cornell_Lysate_assay_7_new.jpeg"> | ||
Revision as of 03:39, 19 September 2015
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Overview of Characterization of MBP+EcnB BioBrick
As described on the Cornell iGEM wiki, 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 proved the efficacy of BBa_K1595024 against the growth of F. psychrophillum as we expected through the use of lysate 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.
We further validated the efficacy of BBa_K1595024 using a series of disk diffusion assays containing lysate from BBa_K1595024, and compared the zone of inhibition to oxytetracycline used in the field as well as BL21 lysate as a negative control. The BBa_K1595024 zone of inhibition had an average diameter of 50.7mm, as compared to 55.2mm from oxytetracycline’s zone of inhibition. A two-sample t-test shows no statistical difference between the two diameters (p > 0.05). From these results, it is rather clear that the fusion protein MBP+EcnB functions notably well in combating F. psychrophilum, with EcnB retaining its effectiveness despite being part of a fusion protein combination. Below is the Zone of inhibition 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.
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