SDS-PAGE gels were run to test the purification after running through the Ni-NTA columns. After several attempts at protein purification in which we were unable to properly isolate protein in our products as the elutions show several bands. We made several adjustments to our protein purification protocol. We adjusted our Lysis, Wash, and Elution buffers to a pH of 7.5 and we suspended our freeze-thaw lysis protocol in favor of using sonication. After meeting with our advisor, Dr. Glynn, we were able to secure workspace in a 4°C cold room, which gave us more control over the temperature at which we purified our proteins and suspended potential degradation that could take place at warmer temperatures.
These two SDS-PAGE Gels show our elutions for GST tagged Epidermicin NI01 and MBP tagged Lacticin Z-Aureocin A53 hybrids. The bands we are looking for are present at the 36kDa mark in the gel with GST tagged Epidermicin NI01, however several other bands with similar intensities are present, marking that it is somewhat impure. In the gel with MBP tagged Lacticin Z-Aurecin A53 Hybrid, the band around 66kDa seems to be present, however, the elutions again show that purity in the elution is somewhat lacking. This was the extent to which we could purify the protein given our limited time frame
To determine if our protein was being expressed, we ran a dot blot. We used a His antibody to check for protein expression because our proteins contain a His tag at the N-terminus. We included non induced and induced samples of the bacteriocins Lacticin Z, Lacticin Z-Aureocin A53 hybrid, Aureocin A53, and Epidermicin NI01. A blot of a positive control that bind well to the chosen antibody and a negative control of non-transformed BL21 E. coli.
Only one of our constructs, Aureocin A53, expressed protein at a level equal to or above the level of the positive control under a 5 second exposure level. However, using a 30 second development, all but Epidermicin NI01 showed some level of expression. Constructs induced with IPTG and not induced showed equal levels of expression, which means that in this case IPTG does not have an effect on protein expression for our constructs. Instead, our constructs show leaky expression, meaning that although our constructs are being expressed, we have no control over the level of expression.
MRSA and S. aureus Spot on Lawn and MIC Assays
Due to constraints with conducting these assays and concerns about the danger of the bacterial strains we were using, testing was only able to taking over a three day period. Since we were given only specific days to test, the proteins that we collected were in the freezer for several days before testing was able to occur. This leads us to believe that our proteins degraded before we tested them. Our MIC and Spot on Lawn assays showed no antimicrobial effect on MRSA or S. aureus, which leads us to believe that the proteins were degraded before testing occurred.