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Team:Exeter/EColiStressTesting
From 2014.igem.org
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The Toxicity of TNT and Nitroglycerin to E. coli.
Aim
Trinitrotoluene and nitroglycerin are known to have toxic properties. In order to asses the efficacy of our constructs to either degrade these compounds or detect them we need to know their effect on E. coli. This page describes our findings with regards to the effects of TNT and NG on TOP10 E. coli cells.
We found out the concentrations of each chemical that inhibited growth in 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 are known to be toxic to a wide range of organisms, including bacteria. Before commencing any work with engineered E. coli, we first had to know out how the E. coli strain we are using (TOP10) would respond to the addition of TNT or NG to the media.
To test this we monitored growth of E. coli following addition of a known concentration of TNT or NG. These experiments were performed in a 96-well plate, allowing us to increase the range of concentrations of compounds we were able to test. Moreover, TNT and NG are only available in small quantities, and the smaller culture volumes allowed this to go further.
The following questions were asked:
- What concentration of TNT or NG prevents growth of E. coli in a new cell culture?
- What concentration of TNT or NG is lethal to an established cell culture?
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.
Summary
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.
Materials
- TECAM 200 PRO microplate reader.
- Grenier 96 well black plates.
- Top10 E. coli
- We used One Shot® TOP10 Chemically Competent E. coli from Invitrogen to test the xenobiotic response, as it is the strain that our constructs will be tested in.
- 1000 ug ml-1 Trinitrotoluene, or 4.4uM.
- Supplied by AccuStandard. Dissolved in MeOH:AcCN.
- 1000 ug ml-1¬ Nitroglycerin, or 4.4uM.
- Supplied by AccuStandard. Dissolved in MeOH:AcCN.
- LB Media
- Used to grow overnight cultures of E. coli.
- The recipe for 1l LB media is as follows:
- Tryptone 15g
- Yeast Extract 10g
- NaCl 5g
- Once these components have been added ddH2O should be used to bring the solution to 1l, followed by autoclaving.
- The recipe for 1l LB media is as follows:
- MYE Media
- MYE is a modified minimal media used to grow bacteria in the 96-well plates. MYE was used here as LB has a natural fluorescence that would interfere with our readings. It was created by Howard et al. (2013) and was modified from Schirmer et al. (2010).
- The recipe for 1l MYE media is as follows:
- Na2HPO4 6g
- KH2PO4 3g
- NaCl 0.5g
- NH4Cl 2g
- Tris.HCL (pH 7.25) (1M Solution) 200ml
- Yeast Extract 0.5g
- Autoclave the solution, then added a volume of (previously sterilised) stock solution:
- CaCl2 11mg/l 100ul
- MgSO4.7H2O 0.25g/l 1000ul
- FeCl3.6H2O 27mg/l 100ul
- ZnCl.4H2O 2mg/l 100ul
- Na2MoO4.2H2O 2mg/l 100ul
- CuSO4.5H2O 1.9mg/l 100ul
- H3BO3 0.5mg/l 100ul
- Thiamine 1mg/l 100ul
- Triton X100 0.1% 1000ul
- Finally add a carbon source (filter sterilized or autoclaved):
- Glucose 3% 60ml
Method
All experiments were carried out in 96 well plates, incubated in a shaking incubator at 37C with constant shaking at 800 rpm. Cultures were in MYE media and were inoculated from an overnight culture grown in LB media. 3ul of overnight culture was used to inoculate 200ul of media. TNT or NG was added to the media either at the start of the growth phase, or during growth, as indicated in the results section.
All fluorescence readings were taken for bacteria in MYE media due to the large autofluorescence generated by LB media. The temperature within the TECAN was controlled at 37oC to minimize cooling of the cultures during readings. Where cultures were grown overnight they were left in the TECAN 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.
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.
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.
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.
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.
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
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