Veryfying the effectiveness of unmodified 0.19 α-amylase inhibitor
Gibson Assembly & Protein Purificaion
We assembled the three types of constructs for protein production. Once the yeast cell grew, the cells without secretion tag construct were lysed to extract and purify the protein. For the ones with secretion tag, media just above the cell pellets were taken for purification. The purified proteins were then used in all of the test mentioned below.
Gel Electrophoresis
We conducted the electrophoresis as a way to collect more evidence to establish that the 0.19 albumin alpha-amylase inhibitor was produced successfully in each construct. By comparing the known size of the 0.19 protein from literature to the proteins isolated and collected from our samples, we were able to evaluate whether or not 0.19 had been correctly produced. The product produced by our yeast was expected to travel the same distance and show the same molecular weight. This method was chosen because the materials were readily available to us and many members of the team have performed it before.
Bradford Assay
The purpose of this is to test whether our modifications were produced by measuring the concentration of 0.19. This concentration value would be used in math modeling as well. However, this assay did not give consistent results (some protein concentration was negative), but we do not know the reason behind this.
Improving the 0.19 amino acid sequence
Starch Solution Inhibition Assay with Iodine
In this experiment, we measured the concentration of starch with alpha-amylase and 0.19 overtime to see if the modified 0.19s successfully inhibited the breakdown of starch by the amylase. The iodine solution was used as a indicator for the presence of starch, which can then be anaylzed with absorbance reader. This Assay gave us unexpected results, as some colors in the 96 well plate did not fit in the gradient. Therefore, we did not analyze the absorbance values from this Assay.
CNP Assay
The CNP (2-chloro-4-nitrophenyl maltotrioside) test was used to measure the enzymatic activity of alpha-amylase. CNP demonstrates a color change from a transparent blue to colorless as it is cleaved by alpha-amylase, thus acting as an indicator that allowed us to observe amylase inhibition via absorption measurements. The CNP assay allowed us to measure inhibition with greater precision than the starch-iodine assay.
iGEM parts improvement for the effective production of alpha-amylase
Gibson assembly, protein purification
For assembling parts, we used the same method as above, except the cells were E.coli competent cells, not yeast. This was because we improved already-existing E.coli part from the iGEM registry.
CNP Assay
The results of the CNP from testing 0.19 proved to have a consistent measurements, therefore we adopted the same method for measuring amylase activity.
