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Team:Exeter/EColiStressTesting
From 2014.igem.org
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The Toxicity of TNT and Nitroglycerin to E. coli.
Trinitrotoluene and nitroglycerin are both toxic compounds that will kill out lab strain of E. coli at a certain concentration. We needed to know this level in order to find out how our degradation constructs effect the survival rate of our bacteria. We observed the rate of growth of our bacteria when various concentrations of each chemical were present. We did this by observing the growth of cultures in 200ul wells on 96-well plates. We found out the concentrations of each chemical that inhibited growth in unmodified E. coli. These concentrations were between 0.326 mM and 0.364 mM for TNT, with complete inhibition occurring above 0.400 mM, and between 0.086 mM and 0.169 mM for NG, with complete inhibition occurring above 0.169 mM. A secondary result of these tests shows that 96-well plates can be used to efficiently grow E. coli.
Introduction
Trinitrotoluene and nitroglycerin, the compounds our parts are primarily designed to degrade, are known to be toxic to a wide range of organisms, including bacteria, and in order to discover how our parts were affecting the degradation of TNT/NG we first had to find out how our basic E. coli strain would naturally respond. Once we have this information we can find out if our parts improve the survival and growth of E. coli in further experiments.
We tested the bacteria’s tolerance for TNT/NG by observing whether growth occurred in a fresh culture after a set volume of the chemical had been added. A set volume of overnight culture was added to MYE media in a 96 well plate, as well as a volume of TNT/NG. By using a 96-well plate we were able to massively increase the range of concentrations of compounds we were able to test.
We ran a series of increasingly focused experiments to understand E. coli’s response to these chemicals. Our research was constrained by the limited amounts of TNT and NG we had access to.
Experiment One: What approximate level of TNT prevents growth of E. coli in a new cell culture?
This test was carried out on plate 1. E. coli was grown for 48 hours with the addition of a certain volume of TNT (0-40ul, with steps of 2.5-10ul) at t = 0. Over this time its optical density was measured, as it is (until a point) proportional to the sample’s growth. We carried out this test to gain an approximate knowledge of what level of TNT would supress E. coli growth.
Experiment Two: Does a lethal dose of TNT remain lethal as a cell culture grows?
This test was carried out on plate 2. E. coli was grown for 48 hours with the addition of a concentration of TNT found to be lethal in Experiment 1 (20 ul) added every hour for 3 hours. Over this time its optical density was measured. We carried out this test to find out if a lethal concentration of TNT remained lethal as the size of the cell sample grew.
Experiment Three: What level of NG prevents growth of E. coli in a new cell culture?
This test was carried out on plate 2. E. coli was grown for 48 hours with the addition of a certain volume of NG (0-20ul, with steps of 2-4ul) at t = 0. Over this time its optical density was measured. We carried out this test to gain an approximate knowledge of what level of NG would completely supress E. coli growth.
Experiment Four: What precise level of TNT prevents growth of E. coli in a new cell culture?
This test was carried out on plate 1. E. coli was grown for 48 hours with the addition of a certain volume of TNT (0-20ul with steps of 1ul between 10-20ul) at t = 0. Over this time its optical density was measured. We did this experiment to obtain a more accurate view of how TNT effects cells when it is added to the initial cell culture. The concentrations used were based on the information gained from experiment one.
Materials
- TECAM 200 PRO microplate reader.
- Grenier 96 well black plates.
- Top10 E. coli
- 1000 ug ml-1 Trinitrotoluene, or 4.4uM.
- 1000 ug ml-1¬ Nitroglycerin, or 4.4uM.
- LB Media
- MYE Media
Method
These experiments were carried out on 96 well plates, using a TECAM 200 PRO microplate reader. The Top10 strains were grown on LB media overnight and scanned while on MYE media. Each strain was grown overnight in 10 ml of LB media in a shaking incubator at 37 oC.
To create the culture for each well, 200ul of MYE media was mixed with 3 ul of the required strain, as well as a volume of TNT or NG specific to each well. When MYE of LB media was used as a control 200 ul was used. The wells were scanned in the TECAM machine, a process which took around 5 minutes. The cultures were kept at 37oC while this occurred. The plates were then transferred to a shaking incubator (800 rpm), usually for 55 minutes.
In cases where plates where run overnight they were left in the TECAM machine and the shaking function was used. Each plate had a different arrangement of cell cultures. The layout of the plate and location of the cultures used in each experiment is listed below.
Plate 1:
- E1-3 Top10, with 5 ul TNT
- F1-3 Top10, with 10 ul TNT
- G1-3 Top10, with 20 ul TNT
- H1-3 Top10, with 30 ul TNT
- F10-12 Top10, with 0 ul TNT
- G10-12 LB Media.
Plate 2:
- A1-3 Top10, with 0 ul
- B1-3 Top10, with 20 ul TNT added at T=0
- C1-3 Top10, with 20 ul TNT added at T=1
- D1-3 Top10, with 20 ul TNT added at T=2
- E1-3 Top10, with 20 ul TNT added at T=3
- F1-3 Top10, with 20 ul TNT added at T=4
- A7-8 Top10, with 0 ul NG
- B7-8 Top10, with 2 ul NG
- C7-8 Top10, with 4 ul NG
- D7-8 Top10, with 8 ul NG
- E7-8 Top10, with 12 ul NG
- F7-8 Top10, with 16 ul NG
- G7-8 Top10, with 20 ul NG
- F10-12 Top10, with 0 ul
- G10-12 LB Media
- H10-12 MYE Media
Plate 3:
- A1-3 Top10, with 20 ul TNT
- B1-3 Top10, with 19 ul TNT
- C1-3 Top10, with 18 ul TNT
- D1-3 Top10, with 17 ul TNT
- E1-3 Top10, with 16 ul TNT
- F1-3 Top10, with 15 ul TNT
- G1-3 Top10, with 14 ul TNT
- H1-3 Top10, with 13 ul TNT
- A4-6 Top10, with 12 ul TNT
- B4-6 Top10, with 11 ul TNT
- C4-6 Top10, with 10 ul TNT
- D4-6 Top10, with 5 ul TNT
- E4-6 Top10, with 2 ul TNT
- F4-6 Top10, with 0 ul TNT
- G4-6 LB Media
- H4-6 MYE Media
Results
Experiment One: What approximate level of TNT prevents growth of E. coli in a new cell culture?
Figure 1 shows how natural E. coli responded to varying concentrations of TNT. Between 0-7 hours the rate of growth was slower as the volume of TNT added to the culture increased. After 24 hours a clear gap can be seen between 10 and 20 microlitres of TNT, with those at 10 or below all showing an optical density between 0.3400 and 0.4200, while those at 20 or above all showed an optical density between 0.1200 and 0.0800.
Figure 2 compares the growth of natural E. coli in MYE media as the concentration of TNT within the culture increases. It’s clear that as the concentration of TNT increases the rate of growth decreases. It also shows that above 10 l the growth of E.coli is severely limited. At 24 hours the cultures have an optical density around 10% that of uninhibited E. coli, while at and below 10 ul growth is 80-90% of the control culture.
Experiment Two: Does a lethal dose of TNT remain lethal as a cell culture grows?
Figure 3 shows the response of natural E. coli when, what is a lethal dose of TNT (20 l) if added at the start of the culture, is added after E. coli has had an opportunity to grow. TNT was added at t = 0, 1, 2 and 3. When TNT was added at an OD below 0.2500 growth inhibited, and the OD fell to a level similar to that of a culture that had been given a lethal dose. However, when the OD was above 0.2700 and a “lethal” dose was added growth slowed, then continued, eventually matching the OD of the control culture (data in table).
Experiment Three: What level of NG prevents growth of E. coli in a new cell culture?
Figure 4 shows how natural E. coli responded to varying concentrations of NG. Between 1-7 hours all that can be observed is that all cultures that have has NG added to them have their growth inhibited, while the control culture experienced significant growth. After 24 hours a clear gap can be seen between 4 and 8 microlitres of NG, with those at 4 or below all showing an optical density between 0.2800 and 0.4000, while those at 8 or above all showed an optical density between 0.1100 and 0.0800.
Figure 5 compares the growth of natural E. coli in MYE media as the concentration of NG within the culture increases. It’s clear that as the concentration of NG increases the rate of growth decreases. It also shows that above 4 l the growth of E. coli is severely limited. At 24 hours the cultures have an optical density around 5% that of uninhibited E. coli, while at and below 10 l growth is 80-90% of the control culture.
Experiment Four: What precise level of TNT prevents growth of E. coli in a new cell culture?
Figure 6 shows how natural E. coli responded to varying concentrations of TNT. These results target a more specific area than the previous experiment, between 10 and 20 l. Generally the rate of growth of the culture was slower as the volume of TNT added to the culture increased. After 15 hours a clear gap can be seen between 16 and 17 l of TNT, with those at 16 or below all showing an optical density between 0.3900 and 0.5000, while those at 17 or above all showed an optical density between 0.2200 and 0.1400.
Figure 7 compares the growth of natural E. coli in MYE media as the concentration of TNT within the culture increases. This experiment specifically targets the 10-20 l range. At 15 hours it can be seen that toxicity sharply increases between 16-18 ul of TNT added, changing from 75% of the growth of the control culture to 20%.
Discussion
Experiment One: What approximate level of TNT prevents growth of E. coli in a new cell culture?
From this experiment we obtained two approximate values that were used in future experimental design. 20 ul TNT in 200 ul was enough to almost completely inhibit growth, while 10 ul challenged the bacteria and slowed growth, but ultimately resulted in growth close to that of the uninhibited culture.
Experiment Two: Does a lethal dose of TNT remain lethal as a cell culture grows?
20ul of TNT was established in Experiment One to be a lethal dose, so this experiment used that value. The addition of TNT before the OD reached 0.2500 lead to a reduction of growth, causing it to reach a similar value to that of the culture that had TNT added initially. However, when the OD was greater than 0.2700 the culture managed to recover from the addition of the chemical. This shows that the level of TNT required to prevent the growth of bacteria increases as the number of bacteria present increases.
Experiment Three: What level of NG prevents growth of E. coli in a new cell culture?
In this experiment we found that E. coli growth was completely inhibited by the presence of 8 ul or greater of NG, while volumes of 4ul or below allow close-to-normal growth to occur. This corresponds to concentration for toxicity 0.169 mM, while survival occurs in concentrations of 0.086 mM or below.
Experiment Four: What precise level of TNT prevents growth of E. coli in a new cell culture?
In this experiment we found that E. coli growth was completely inhibited by the presence of 8 ul or greater of NG, while volumes of 4ul or below allow close-to-normal growth to occur. This corresponds to concentration for toxicity 0.169 mM, while survival occurs in concentrations of 0.086 mM or below.
Conclusion
We can see that E. coli is clearly effected by the levels of TNT and NG it is exposed to. We have found a fairly well defined line in which cell cultures transition from being able to grow, albeit slowly, to having their growth completely inhibited. With this information we can test if our constructs increase the survival rate of E. coli, or find an appropriate level of pollutant with which to trigger our promoters.
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