GFP Expression in Cells
To verify that our DNA schema was appropriate for expression with the T7 promoter we decided to validate it with an easy phenotypic readout: GFP in T7 express competent E. coli.
Successful expression of GFP in our DNA system in T7 express E. coli under the long promoter (BBa_I712074) with the 5’UTR (BBa_K1758100) superfolder GFP (BBa_I746916) and Terminator (BBa_B0012) in pSB1C3 vector. The same DNA sequence but as a linear PCR amplicon was expressed using NEBs PURExpress kit
Bacterial Killing Assay by OD600
In the first attempt of the killing assay, we tested all twelve of our AMPs under both long and short promoters and in the presence of MetAP. The goal of this broader assay was to identify which AMP’s under which configuration were effective. Most of the AMPs under all configurations had OD’s within range of the 95% confidence interval of the control reaction. This control reaction contained cell-free buffer and water at the same volume as the AMP reaction in order to account for toxicity. However, ISGCock (BBa_K2431022), BP100 (BBa_K2431002), and Micrococcin (BBa_K2431000) showed slightly lower OD’s than the control at most, if not all, of the promoter and MetAP configurations (Figure 1). However, due to the lack of DNA for replicates of these AMP reactions, no statistical conclusions could be drawn. In addition, for all of the AMPs there was a steady decrease in OD after about 5 hours, leading to widely varied data points that could not be analyzed. This was likely because of the evaporation of the media, due to a lack of a seal on the plate. Therefore, the data shown in Figure 1 only includes data points up to that point. In order to achieve replicates, we proceeded to do a second killing assay using only ISGCock (BBa_K2431022), BP100 (BBa_K2431002), and Micrococcin (BBa_K2431000). Our second assay was run with sealed plates, which helped eliminated cell death due to evaporation. However, this assay indicated no difference in cell death between AMP reactions and control reactions (Figure 2). The OD’s for all reactions were remarkably consistent, indicating that our AMP was most likely not expressed at a meaningful level in our reactions.
In addition, the control containing only cells had a higher OD in general than all reactions containing cell-free buffer components, indicating that any toxicity was most likely due to the presence of this buffer. The discrepancy between the two killing assays could be due to multiple factors. Firstly, the AMP DNA for the second assay was prepared separately from the first assay. Any impurities in the final DNA template, such as ethanol or guanidinium may have contributed to cell death. Another factor that contribute to this discrepancy was the evaporation of the liquid cell cultures. This evaporation could have led to cell death in some reactions early on in the time-course, creating the appearance of a toxic AMP. In addition, the first assay did not have replicates for the AMP reactions, furthering the uncertainty in the effectiveness of the AMPs. In the second assay, all AMP reactions were performed in triplicate and were consistently within 95% confidence interval of all other reactions, save the cells only control. Once again, this points to a lack of sufficient expression of the AMP and a small toxic effect due to cell-free buffer conditions.
AMP expressed via PURExpress Plate Based Killing Assay
In order to determine whether or not it was the AMPs or the cell free expression reaction that was ineffective we expressed BP100 (BBa_K2431002) and Micrococcin C7 (BBa_K2431000) using NEBs PURExpress cell-free protein expression kit using long and short promoters. These reactions were then applied to a BL21 E. coli bacterial lawn on LB plates at reaction concentration and at a 1:4 dilution. Plates were left to grow overnight at 37C. Pictures below show that under both promoters and typically at both dilutions both BP100 and Micrococcin C7 are effecitve at killing BL21 E. coli. 11L and 11S indicate BP100 expressed with the long and short promoters respectively. 12L and 12S indicate Micrococcin C7 expressed with the long and short promoters respectively. “N” indicates the normal undiluted reaction concentration and “D” indicates the diluted reaction mixture. No Temp plates were plates dosed with control PURExpress reaction without any DNA template added.The success of the AMPs in killing bacteria in this assay once again indicates that the expression of AMP in the cell free reaction used in the liquid culture killing assay was not sufficient. However, this plate based assay shows the antimicrobial effects of both Micrococcin and BP100.Control: D & N
Part 11: BP100 (BBa_K2431002)
Part 12: Micrococcin C7 (BBa_K2431000)