Team:NortheasternU-Boston/Results

Results

Figure 1: The OD of liquid cultures at 600nm over time in the presence of cell-free AMP reactions. L and S indicate the long (BBa_I712074) and short promoter (BBa_I719005), respectively. “MetAP” indicates that methionine aminopeptidase (BBa_K2431025) was present in the reaction. The control reaction contained all components of the cell-free reaction except for input DNA. Error bars shown are 95% confidence intervals, n=4.
Figure 2: The OD of liquid cultures at 600nm over time in the presence of ISGCock (BBa_K2431022) , BP100 (BBa_K2431002) , and Micrococcin (BBa_K2431000) AMPs. L and S indicate the long (BBa_I712074) and short promoter (BBa_I719005), respectively. “MetAP” indicates that methionine aminopeptidase (BBa_K2431025) was present in the reaction. The control reaction “Control average” contained all components of the cell-free reaction except for input DNA. Error bars shown are 95% confidence intervals, n=6. The reaction “MetAP Control Average” contained all components of the cell-free reaction as well as MetAP DNA. The reaction “Cells Only Control” contained only media and cells.

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, BP100, and Micrococcin 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, BP100, and Micrococcin. 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.

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