Team:Michigan/Results

MSBT 2017

Results

Experiment #1: How do different promoters in pThermolyze affect cell growth at 37C and 25C?

We performed this experiment three times, but it proved difficult to ensure all cultures began at 0.2-0.4 OD, so for our analysis, we discarded the data for all trial runs that did not start with a time 0 OD of 0.2-0.4. If we had not done this, we would be comparing cultures that were at very different stages of growth. The remaining data are shown in the table below. Unfortunately, this method of pruning the data for consistency meant that we did not have any valid trials for promoter J grown at 37C and only one trial for that promoter at 25C, so we have excluded promoter J from our analysis. As the weakest of the three promoters that we tested, it is unlikely that its exclusion impacts the results. We see no consistent drop in OD at any time point once the cultures with our killswitch and promoters were switched to incubating at 25C. Such a drop would have indicated our killswitch was lysing cells as we had hoped.

Trial Sample Time 0 0:20 0:40 1:00 1:20 1:40 2:00 2:20 2:40 3:00 3:20 3:40 4:00 4:20 4:40 5:00 5:20 5:40 6:00
1 P-25 0.354 0.379 0.497 0.44 0.471 0.497 0.573 0.621 0.601 0.694 0.803 0.912 0.951 1.132 1.077 1.246 1.505 1.362 1.484
2 P-25 0.33 0.365 0.447 0.473 0.507 0.638 0.657 0.777 0.913 0.945 1.045 1.162 1.321 1.301 1.525 1.666 1.782  
1 C-25 0.297 0.346 0.474 0.388 0.46 0.449 0.495 0.59 0.634 0.668 0.717 0.883 0.856 0.964 1.096 1.029 1.207 1.126 1.287
2 C-25 0.243 0.262 0.302 0.311 0.373 0.44 0.462 0.507 0.562 0.585 0.639 0.747 0.806 0.828 0.879 0.977 1.1  
3 C-25 0.268 0.32 0.287 0.306 0.309 0.415 0.409 0.472 0.502 0.545 0.684 0.67 0.763 0.754 0.853 0.929 0.884 0.998 1.06
1 T-25 0.258 0.233 0.287 0.277 0.268 0.335 0.304 0.442 0.33 0.436 0.479 0.463 0.649 0.549 0.555 0.681 0.599 0.794
3 T-25 0.293 0.319 0.307 0.312 0.359 0.498 0.449 0.502 0.535 0.604 0.689 0.697 0.756 0.911 0.935 0.96 0.992 1.062 1.109
2 J-25 0.368 0.505 0.541 0.603 0.712 0.862 0.863 0.962 1.064 1.169 1.301 1.421 1.611 1.734 1.846 2.059 2.236  
1 L-25 0.28 0.371 0.468 0.374 0.413 0.423 0.553 0.581 0.595 0.633 0.715 0.755 0.839 0.984 1.106 1.095 1.225 1.33 1.341
2 L-25 0.36 0.431 0.435 0.533 0.604 0.741 0.776 0.841 0.952 1.123 1.175 1.286 1.517 1.671 1.681 1.921 2.11  
1 P-37 0.354 0.554 0.726 0.929 1.157 1.41 1.642 2.026 2.276 2.43  
1 C-37 0.363 0.646 0.658 0.913 1.025 1.227 1.492 1.718 2.061 2.35 2.452  
2 C-37 0.362 0.513 0.724 0.992 1.126 1.37 1.598 1.896 2.1 2.249  
3 C-37 0.331 0.472 0.773 1.033 1.212 1.428 1.604 1.991 2.187 2.465  
1 T-37 0.235 0.342 0.392 0.512 0.59 0.657 0.893 1.117 1.43 1.595 1.857 1.999 2.385  
2 T-37 0.213 0.249 0.315 0.437 0.53 0.736 0.895 1.093 1.369 1.531 1.893 2.208 2.339  
3 T-37 0.291 0.523 1.003 0.998 1.161 1.41 1.636 1.948 2.283 2.439  
1 L-37 0.332 0.706 0.744 1.023 1.223 1.433 1.718 2.275 2.443                    


For the remaining trials, we calculated generation time from 20 minutes after half the cultures were switched to 25C until the 100 minute time point. We used the formula 80/(3.3*Log(Abs100/Abs20)), to calculate the generation time. We chose these two time points because after 20 minutes at 25C, the cultures should have cooled to their new temperature and stabilized, and 200 minutes was the last time point where no culture went over the measurable limit of 2.5 OD.


We then averaged these generation times across each type of culture (ie: P at 25C, P at 37C, etc.).


There are two main takeaways from these generation times. First, we see negligible differences in generation time between the five types of culture at 37C. This is promising because it tells us that our construct has no negative impact on cell growth at working conditions. In theory, producing holin, endolysin, antiholin, and TlpA36 could have put undue metabolic strain on the cell, but we see this is not the case. Also promising is the fact that there is a noticeable increase in generation time between our two control cultures (P and C), and the two cultures with our kill switch and promoters. This suggests that even though our kill switch did not outright lyse the cells, they do seem to be impacting growth rate, supporting the possibility that the selection of a stronger promoter may yield a viable killswitch.

To further establish that this increase in generation time is due to our killswitch and not just due to the shift in temperature, we can examine the ratio of the 25C cultures’ generation time to their matching 37C cultures’ generation time.


This confirms that promoter T is causing some increase of the generation time at 25C independent of the increase in generation time inherent in growth at a lower temperature. It appears promoter L may possible have an effect. Again, our killswitch did not lyse all of the cells as we intended it to, but if it is reducing growth rate, it may be lysing some of them. Those results suggest that a strong enough promoter could make our killswitch functional.

Experiment #2: Can pThermolyze transformed cells be stored as glycerol stocks and successfully recovered?

A bacterial lawn grew on all five plates in this experiment.

Experiment #3: Experiment #3: Can pThermolyze transformed cells be recovered from liquid cultures after storing at room temperature for 4 hours?