Part:BBa_M36459:Experience
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Applications of BBa_M36459
Method
The cell lysis actuator was paired with a pre-proven sensor that is induced by Rhamnose and suppressed by Glucose. This sensor sends a signal to the actuator using Polymerase per Second (PoPS). The layout of the actuator will be First RBS, First GOI (Holin), Second RBS, Second GOI (Endolysin), Third RBS, Third GOI (Rz Protein), Terminator. This is relatively basic setup and each part comes directly after the previous part; there is no untranslated region within our sequence.
Our plasmid was placed in Escherichia coli as it is the easiest to test the cell lysis actuator in and it has the most practical application for the overall research idea of having dangerous bacteria lyse upon exposure to the environment. The plasmid is Kanamycin resistant since making the plasmid Ampicillin resistant could interfere with the lysis actuator. Ampicillin works by creating pores in the cell membrane leading to cell lysis, similarly to how the 3 protein sequence in the lysis actuator causes cell death. Kanamycin causes cell death by interfering with the 30S subunit in prokaryotic ribosomes. It is unknown whether Ampicillin would actually interfere with the cell lysis actuator designed.
Experimental Description
Upon receiving the lysis actuator contained in a pJ821 plasmid from DNA 2.0, the plasmid was transformed using Calcium Competent cells prepared a month prior. Transformation was done twice by Whitmeyer and twice by Lewis to create a sham control. Unfortunately, none of the transformed colonies grew as expected as 2 did not grow at all and 2 were infected, determined to be fungal based on color and growth patterns. This was determined to be a problem with the Calcium competent cells. The procedure was repeated but this time with Top 10 cells to nearly eliminate the chance of failure due to the cells. Also, to control whether the cell lysis actuator was the problem (eg. it kills the cells before they are able to form colonies), an identical transformation with p81 plasmid was performed. P81 is also kanamycin resistant and compatible with E. Coli. The transformation was successful with both the pJ821 and p81 plasmids as both colonies grew successfully on LB + kanamycin plates in relatively equal numbers. Three colonies from both the pJ821 and the p81 plasmid were picked and allowed to grow up in liquid culture containing LB + kanamycin. After the cultures had grown up they were made into glycerol stocks and the remaining culture was used to perform test number 1.
There were four tests performed with our cell lysis actuator and they are outlined below. Results from each of these tests may be viewed in the results section of the paper. The initial test performed using three different colonies from both the experimental cell lysis actuator (pJ821) and a control, p81, to test whether or not the cell lysis actuator worked. The results would be confirmed by the response from p81, a plasmid that contains the same proven sensor as pJ821, but also a proven GFP actuator. 3 ml of leftover liquid culture from each of the colonies grown up were used (3 containing the experimental pJ821 cell lysis plasmid, 3 containing the p81 control plasmid). Each group of 4ml media was divided into 2 groups of 1.5 ml each. One of the 1.5 ml groups from each colony was given 15 uL rhamnose to create a 1000 uM solution of rhamnose while the other 1.5 group was given nothing. In the end, there were three 1.5 ml tubes containing the pJ821 + Rhamnose, 3 containing just pJ821, 3 containing p81 + Rhamnose, and 3 containing just p81. This test was done for qualitative reasons only as there was not enough media to create a strong test. However, this test would be able to allow us to see if our actuator was actually working.
The second test performed was a logarithmic scale of different amounts of rhamnose to check what dose would be lethal with the cell lysis actuator. This would be done using concentrations of rhamnose of 0 uM, 5 uM, 10 uM, 100 uM, 1000 uM. A liquid culture was grown up using glycerol stock 1 of the cell lysis actuator in 100 ml of LB + Kan. The culture was allowed to grow overnight in a 37 degree shaker incubator with the shake speed set to 275 rpm. The media was then distributed into 25 falcon tubes containing 2 mL of media each. The desired rhamnose was then added to each of the 25 falcon tubes (5 tubes for each of the 5 concentrations of rhamnose mentioned earlier in the paragraph). These tubes were then placed in a 25 degree shaker incubator with the shake speed placed on 200 rpm to allow the mixture to remain well mixed without significant E. Coli growth. The cell density of each culture was read the next day using a spectrophotometer.
The third test performed was done to provide a more accurate understanding of exactly what dose would be lethal when paired with our cell lysis actuator. Knowing that 5ml had a significant impact on the cell density with a non-significant decrease with a greater amount, it was decided to hone in on the 0 to 5 uM rhamnose area. This was done with the same process as the second test, with 5 tests for each value of rhamnose, 0 uM, 1 uM, 2 uM, 3 uM, 4 uM, 5 uM. The liquid culture was prepared and the rhamnose + actuator were stored in identical conditions. The only difference was that the values were read using a 96 well plate reader rather than a spectrophotometer.
Knowing the lethal doses of rhamnose was a critical first step for understanding the cell lysis actuator. However, it is also important to know the time period the actuator takes to work. This test was performed using 36 samples – 12 grown from a recently picked colony, 12 grown from glycerol stock 1, and 12 grown from glycerol stock 2. This was done to make sure the glycerol stocks were not affected in any way compared to the colonial cultures. Within each group of 12, 3 were given no rhamnose, 3 were given 1 uM rhamnose, 3 were given 10 uM rhamnose, and 3 were given 100 uM rhamnose. The amounts of rhamnose were varied so the action of different amounts could be compared over time. The 36 cultures with rhamnose added were placed in the 96 well plate reader along with a blank. The optical densities of the cultures were then measured every half hour for 12 hours.
Results
Experiment 1:
Confirmation of the existence of the cell lysis actuator in our cells was found during the first experiment. Rhamnose is usually a harmless chemical to E. Coli cells, however the cell lysis actuator is designed to take the input signal from a Rhamnose sensor and create holins that ultimately lyse the cell. As shown in Figure 2 and Figure 3 (below), the presence of Rhamnose visibly lowers cell density, and the presence of Glucose has no effect on cell density, thus confirming the existence and functionality of the cell lysis actuator in these E. Coli cells. The control p81 cells confirmed the results by responding to the Rhamnose and Glucose doses as expected; exhibiting fluorescence when exposed to Rhamnose and not exhibiting fluorescence when suppressed by glucose. As stated above, glycerol stocks were made from these three proven samples and labeled Glycerol 1, Glycerol 2, and Glycerol 3. All subsequent experiments (aside from Time Lapse) used the now proven Glycerol 1 as the source for growing cell cultures.
Experiment 2:
As shown here, when the concentration of Rhamnose was increased exponentially the cell density dropped to a fairly consistent average between 0.3 and 0.4 absorbance, as compared to our control value with no added Rhamnose that measured between .8 and 1 absorbance. This signaled that the cell lysis actuator was very sensitive to Rhamnose concentrations, and did not respond much differently at higher concentrations than 5 micromolar. The error bars included on the graph represent the range covered by a 95% confidence interval. As seen from the range encompassed by the confidence intervals, the only change after 5 micromolar concentration is the narrowing of the confidence interval. As a result, the third experiment was conducted linearly between 0 and 5 micromolar in increments of 1 micromolar.
Experiment 3:
The data here followed a different pattern than was observed in Experiment 2. The control, 0 Rhamnose data point is not at the levels observed in the other experiments. We believe that our control was contaminated during the experiment. Further analysis of this data is contained in the discussion.
Experiment 4:
As shown on the left, the actuator was mainly effected by the 100 micromolar concentration. As a result, the effects on the 100 micromolar concentration over time are shown for each inoculation type. Further analysis of the data is contained in the discussion section.
Discussion
After repeated success with the cell lysis actuator, it is safe to conclude that the DNA plasmid was successfully put in the cells and that it produced the proteins desired which worked in lysing the cells. This result carries strong significance because the cell lysis actuator designed can be easily used by others for whatever purpose. Because of the amount of data we assembled, any user could accurately predict both the amount of their signal needed to cause total cell lysis and the amount of time it would take for lysis to happen.
Further tests would need to be performed in order for more accurate amounts and times to be assembled as some data from the tests performed worked with varying efficiencies. However, this does provide a great benchmark for any future users of this biological device.
One troubling result was how we were never able to achieve the same level of success in terms of complete lysis as the original test. Unfortunately, we did not make any quantitative data for those results as there was there was only one data point for each. We expected to be able to replicate the same results each time we performed the experiment, however no lysed cell culture ever looked as clear as those original three. Originally, we believed the problem might have to do with the freezing associated with the glycerol stocks affecting our cells. However, the dose response curves in the fourth test were similar for the colonial and glycerol stock cultures. We believe the most likely reason for this variance is due to overgrown cultures. For each test we allowed the culture to sit with the rhamnose overnight. However, in that time the E. Coli likely grew even more and if it reaches capacity, the cells will die. Unlike cells that die due to our cell lysis actuator and essentially disappear optically, these dead cells will sink to the bottom or sometimes float around solution. It is likely that the only culture that did not have these dead but unlysed cells was the original test, leading to those being the only ones completely clear. A second possibility is that because our actuator is so damaging to the cell it inhabits, a mutation in approximately 1/3 of the cells that survive culture could cause them not to lyse. The reason for this possibility, is that our experimental groups all reached a very similar, .33 OD measurement after the experiment was over, signaling the existence of some systematic response by the cells that prevents them from lysing. Further experimentation would include running an experiment, waiting for most of the cells to lyse, and then placing the cells back in the shaker incubator to grow. If the cells regrow and become 100% resistant to Rhamnose, then the second possibility is true. If the cells regrow slowly (or not at all) and once again can be lysed, then the first possibility is true.
Our third test also seemed to have a problem with our control group. Although the cell density seemed to be the similar among the groups exposed to Rhamnose, the group not exposed to Rhamnose seemed to have suffered a similar drop in cell density. As seen in experiments 2 and 4, a concentration of 0 Rhamnose should produce a cell density between .8 and 1. In experiment 3 the cell density was close to .35, signifying some type of contamination in the cells. Our most likely guess is that we accidentally added Rhamnose to our control group as well, thus bringing the cell density to similar levels as our experimental groups. A repeat of our procedure would have cleared up the issue, however we ran out of time. If that was in fact the only mistake in this experiment, then the lack of a dose response curve would show that the effectiveness of our actuator lies somewhere in between 0 and 1 micromolar of Rhamnose, making our actuator extremely sensitive.
Another set of seemingly contradictory data involves the time response curves done in the fourth test. The 100 uM cultures were the only ones that had a significant response over the 12 hours tested. However, this contradicts our previous results that indicated concentrations of as little as 1 uM had a response overnight. The main difference is that the low dose cultures that responded to the rhamnose were placed in the shaker incubator overnight whereas the cultures in experiment 4 sat in the 96 well plate reader overnight. This likely led to an unequal distribution of the rhamnose thereby not allowing it to affect the cells as quickly.
An interesting test that could potentially be done in the future would involve the plate reader being used to measure various doses of rhamnose over time, but with the plate being agitated for the half hour between measurements. Although tedious, this would give a much more accurate picture of how quickly the cell lysis actuator responds to various doses of rhamnose.
Looking at the graph in experiment 4, the colonial stock starts at a lower concentration (this also effects the percentage column chart, seemingly reducing the effect of Rhamnose between 0 and 1 micromolar in the colonial stock). This is because originally we had planned to put our plate into the plate reader immediately after adding Rhamnose to the falcon tubes, however because we had to wait for another group to use the plate reader, our experiment didn’t go into the reader until an hour after adding Rhamnose. In a full table of our data the absorption difference between the 0 Rhamnose and the Rhamnose exposed samples at the start is plainly visible, with the zero starting at about 0.95 and the other samples at around 0.8 (would have been included however we ran out of space). This leads to the conclusion that the colonial stocks respond faster to the Rhamnose, either because freezing/thawing damage cell metabolic processes, or because freezing/thawing damages some of the plasmids in the cell. Further experimentation could include comparing the amount of metabolites present in glycerol stock/colony cells, and comparing the amount of plasmid obtained from glycerol/colony cells. Any difference would signal a small flaw in the freezing and storing process of cells, a flaw that will have to be considered for the consistency of future experiments (does not affect our experiment because all other measurements were done with Glycerol 1).
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