Confirming the Synthesis of Biomarkers
In order to verify the accurate synthesis of our biomarkers in an E.Coli system, we used qualitative analysis and quantitative analysis to show our success.
For our qualitative analysis, we compared the amount of fluorescence in our transformed colonies to the number of colonies to monitor for success of transformation. Our qualitative analysis can be found below in the detailed document.
For our quantitative analysis, after we purified our recombinant biomarkers following transformation, we quantified the amount of synthesized biomarkers through using a Braford assay. Our results demonstrated that we were able to successfully produce a biomarker from our construct and characterize the efficiency of different promoters and terminators in synthesizing our biomarkers.
To calculate the concentrations of the biomarkers within each well, we first ran the BSA model to create the standard curve, where the x-axis represents absorbance, and y-axis represents concentration in mg/ml. The standard curve later allowed us to convert the measured absorbance into measurements of concentration. The equation and the image of the standard graph we derived from the BSA model is as follows.
Our line of best fit for the BSA standard curve derived from polynomial regression is y = 0.1258x^3 - 0.5139x^2 + 0.848x + 0.2487. We chose to model our equation as a cubic function because it provided a better approximation for our data, with a correlation of 0.9607 and root mean square error (RMSE) of 0.0835 as seen above.
Using the standard curve and the measured absorbance, we interpolated values of concentration for each of the different constructs. We performed this conversion by substituting the absorbance values into the x-variable in our standard curve, to find the corresponding y-variable which represents concentration. The post-conversion concentrations of the biomarkers are listed below.
|T7 Promoter+T7 Terminator||Mucin(A1)||1.83|
|Anderson Promoter+T7 Terminator||Mucin(A2)||N/A|
|Optimized Promoter+T7 Terminator||Mucin(A3)||N/A|
|T7 Promoter+T1 Terminator||Mucin(A4)||0.16|
|Optimized Promoter+T1 Terminator||Mucin(A6)||0.16|
|T7 Promoter+Lux Terminator||Mucin(A7)||0.15|
|Optimized Promoter+Lux Terminator||Mucin(A9)||0.49|