The toluene is known to be a volatile compound. This is why we decided to determine its rate of evaporation before proceeding to any subsequent manipulation with it. This will determine the care taken when handling it as well as the time the stock solution will be kept.
1.2mL of pure toluene (244511, Sigma-Aldrich) was added in a 1.5mL Eppendorf tube. The tube was left open under a chemical hood and its weight was determine every 30 minutes for 6 hours.
Results
Time
t = 0 minutes
t = 30 minutes
t = 60 minutes
t = 90 minutes
t = 120 minutes
t = 150 minutes
t = 180 minutes
t = 210 minutes
t = 240 minutes
t = 270 minutes
t = 300 minutes
t = 330 minutes
t = 360 minutes
Weight
1.0513 g
0.9796 g
0.9377 g
0.8894 g
0.8365 g
0.8057 g
0.7782 g
0.7661 g
0.7324 g
0.7172 g
0.6837 g
0.6560 g
0.6325 g
Toluene is definitly a volatile compound, left open under a chemical hood !!! mg of pure toluene have evaporate from the tube within 6 hours. Those results show that toluene evaporte with a rate of 72mg/h. However, this rate could have been increased by the chemical hood suction.
Therefore care have to be taken when manipulating it. Each tube containing toluene have to be open under a chemical hood and any toluene tubes must be left open. Care have to be taken when adding toluene to bacteria culture medium.
The stock solution of toluene will be prepare again for each replicates.
With these results, we can then test the lethality of E.Coli DH5α when exposed to several toluene concentrations.
Survival test to toluene
Protocol and tested concentrations
The protocol 12 (available here ) was used to asses cell survival in presence of toluene. We tested a large range of concentrations (from 5ng/L to 5mg/L) to evaluate the overall impact of toluene on the E.coli DH5α cell growth. 5ng/L is a concentration closed to environmental toluene concentration.
An overnight culture of bacteria tranformed with our biosensor was realized. 300µL of this overnight culture was inoculated into 150mL of LB containing chloramphenicol at 25ng/mL. This culture was homogenized and separated into several falcon tubes. Tolune was then added to the culture medium to reach desired concentrations and the samples OD600 was checked every hours during 8 hours. Three replicates of each concentration were done to ensure statistical results.
Results
The graph 1 shows the evolution of sample optic density (OD600) according to the time and the toluene concentration added in cell culture medium. The curve shows normal growth profile with a lag phase of three hours and then an exponential phase for all samples whether the toluene concentration added in the cell culture medium.
Toluene addition at a concentration of 5ng/L and 500ng/L did not significantly affect cell development. The growth curve shows the same profile than cells grew without toluene. At a concentration of 5mg/L of toluene in the medium, an effect on cells development can be noticed. Four hours after toluene addition, the OD600 of cell grown in 5mg/L of toluene increased slowlier than the OD600 of cell grown without toluene.
Figure 1: Evolution of E.coli DH5α grotwh depending on the toluene concentration added in the cell culture medium
In compliance with those results, bioluminescence test were realized with a toluene concentration in the culture medium comprised between 0 and 10 mg/L.
Biosensor construction
Three parts were synthesized by IDT in order to construct the whole biosensor : BBa_K2023004 composed of Pr-RBS, BBa_K2023005 coding for the XylR protein and BBa_K2023006 made of Terminator-Pu-RBS-Gaussia Luciferase-Terminator. Those three BioBricks are described in the Parts and characterization section.
The biosensor was then assemble using digestion and ligation as described in the notebook Biosensor Construction . The 2nd of August, E.coli bacteria were transformed the whole biosensor genetic construction. Colony PCR were realized to select the recombinant bacteria. The results (Figure 2) indicated that the colony 6 and 7 have integrated the whole biosensor as strips can be seen at 3400bp. Those colonies were miniprep and the collected DNA was sent to sequencing.
Figure 2: Colony PCR results of bacteria transformed with our whole biosensor. Strips are expected at 3400 bp /p>
Sequencing results were not what we expected: a mutation on the start codon of the XylR protein was present in the plasmid of all our clones. Therefore, we performed a "site directed mutagenesis to overcome this problem.
(!!! fichier sequençage !!!)
Biosensor characterization
XylR production by modified E.coli bacteria
We aimed to asses the XylR production by E.coli cells modified with the BioBrick BBa_K2023013 composed of Pr-RBS-XylR-RBS-mRFP-Terminator. This BioBrick was designed to monitor XylR synthesis by our cells.
To do so, we thaw a tubes of transformed cells on ice for 10 minutes, we added different volumes of SOC medium to obtain several dilutions (1/2, 1/4 and 1/8), we plated different concentrations of bacteria cells on petri dish with LB Agar + Chloramphenicol at 25µg/L final and incubate overnight.
We obtained the expected results : red colonies were shown on the petri dish (Figure 3) due to the synthesis of the red protein mRFP meaning that the Pr promoter trigger XylR and mRFP synthesis.
Figure 3: Monitoring of XylR production though control of the activity of the Pr promoter. Modified bacteria containing XylR coding device with mRFP grown on a petri dish. The red color of the bacteria indicate that mRFP protein is produce and therefore that XylR is produce too.
This construction will not allow XylR quantification but those results show that XylR protein was constitutively produce by our genetically modified bacteria containing XYlR coding device with mRFP.
We wanted to realize a XylR-mRFP fusion but this part was too big for IDT synthesis.
We created the BioBrick BBa_K2023010 (XylR-His) to give to future iGEM the opportunity to purify and quantify XylR protein.
Bioluminescence assays
Protocol and tested concentrations
We used the protocol 13 (available here )and decided to test several concentration of toluene (0ng/L, 10ng/L, 100ng/L, 10µg/L and 10mg/L).Bioluminescence assays were realized several times after toluene addition (1h, 3h, 4h30 and 5h30).
Bacteria transformed with our biosensor used in each assays come from the same culture (OD = 0.1). This culture was well mixed and divided in 50 mL falcon. Toluene was then added in each Falcon.
Bioluminescence assays were realized quickly after substrate addition and the bioluminescence intensity was measured using Mithras² LB 943 Monochromator Multimode Reader. This machine was kindly lend by Berthold company for 3 days in order to let us realized the bioluminescence measurement. In addition to bioluminesence intensity, sample's OD was also measured. All tests were realized in triplicates.
Several negative controls were realized :
Measurement of LB bioluminescence intensity to determine the background noise
Measurement of LB + toluene bioluminescence intensity to determine toluene effect on the bioluminescent intensity
Measurement of the bioluminescence intensity of bacteria transformed with a genetic construction that do not contain the gaussia luciferase gene (Pr-XylR).
A positive control using Gluc codig device with Pr (BBa_K2023007) could have been made during the three days of measurement to test substrate efficiency and luminometer parameters. However, this BioBrick was created afterwards. We hoped that this BioBrick will serve as a future control for all bioluminescence assays in iGEM project to ensure the substrate efficiency.
Preliminary considerations
Toluene does not have auto-bioluminescence
We determine the effect of adding toluene to cell culture medium on bioluminescence intensity. As shown on the graph below the bioluminescence intensity of the LB + toluene at 10 mg/L (47.71 RLU) do not significantly differ from the bioluminescence intensity of the LB (48.93 RLU). The little bioluminescence found was due to the substrate added in our sample.
Therefore, toluene addition in our samples will not impact their bioluminescence intensity.
LB without toluene
LB with toluene at 10mg/L
Mean
48.93 RLU
47.71 RLU
Standard deviation (SD)
6.96
9.55
Std. error of mean(SEM)
1.87
2.55
Figure 4: Bioluminescence intensity expressed in RLU of LB and LB with toluene at 10mg/L. Mann whitney statistical test was performed to asses the effect of toluene on LB auto-bioluminescence (ns: non significant). Bacteria E.coli have little auto-bioluminescence
Bacteria transformed with a genetic construction that does not contain Gaussia luciferase gene were used in this study. The auto-bioluminescence of E.coli cells was assessed by comparate E.coli bacteria bioluminescence intensity to LB bioluminescence intensity. As shown on the figure 3, the bioluminescence intensity of E.coli cells (53.14 RLU) was a little superior to LB bioluminescence intensity (48.93 RLU) but not significantly superior. E.coli cells have little auto-bioluminescence. However, this bioluminescence in greatly inferior to the one produce by our biosensor in presence of pollutant (results presented after)
LB without toluene
BB12 without toluene
Mean
48.93 RLU
53.14 RLU
Standard deviation (SD)
6.96
11.34
Std. error of mean(SEM)
1.86
3.03
Figure 5: Bioluminescence intensity expressed in RLU of LB and LB + E.coli cells. Mann whitney statistical test was performed (ns: non significant). Little leakage in Gaussia luciferase expression
We investigate the inductility of the Pu promoter to ensure that the gaussia luciferase is not constitutively produce in our biosensor. To do so we compared the bioluminescence intensity of bacteria containing our biosensor to the bioluminescence intensity of bacteria transformed with a genetic construction that do not contain the Gaussia luciferase gene (BB12 : Pr-RBS-XylR).
The obtained results (figure 6) indicate that the gaussia luciferase gene is not produce in a constitutive manner in the cells. The bioluminescence intensity of our biosensor without toluene injection (158.86 RLU) is a little higher than the bioluminescence intensity of cells transformed with BB12 (53.14 RLU). However this bioluminescence intensity is significantly lower than the bioluminescence intensity of the cells transformed with our biosensor in presence of toluene (results presented in the next section)
BB12 without toluene
BB123 without toluene
Mean
53.14 RLU
158.86 RLU
Standard deviation (SD)
11.34
20.08
Std. error of mean(SEM)
3.03
3.10
Figure 6: Bioluminescence intensity expressed in RLU of E.coli cells transformed with a construction genetic that contain (BB123) or do not contain (BB12) the gaussia gene. A little leakage of the gaussia gene can be seen as the bioluminescence intensity of the sample that contain bacteria transformed with our biosensor is significantly superior to the one that contain bacteria transformed with Pr-RBS-XylR. Mann whitney statistical test was performed (*** : extremely significant).
Data processing
Due to the little LB background noise and leakage in gaussia luciferase synthesis, obtain bioluminescence intensity results were treated as followed. The background noise (LB+toluene) was subtracted to each sample’s bioluminescence intensity. Those latest were then expressed depending on the negative control (bacteria transformed with our biosensor in a medium without toluene, C0).
Results
Investigation of the best measurement time
It was first necessary to determine when, after toluene injection, bioluminescence results were the most relevant. To do so, we determine the bioluminescence intensity of our sample (bacteria transformed with our biosensor within LB medium containing different toluene concentration) several times after toluene addition.
The bioluminescence intensity of the negative control was also determine at each time after toluene addition and bioluminescent results for each assay were normalized on the corresponding negative control bioluminescence intensity. This normalization was necessary as we are dealing with several incubation time. This enable us to handle the bacteria OD increase within our samples and ensure that an increase in sample’s bioluminescence intensity is not correlated with an increase in cells concentration.
The graphs presented below represent the bioluminescence intensity of the sample depending on the time after a toluene addition for a fix concentration of toluene injected.
Figure 7: Evolution of bioluminescence intensity at different times after toluene addition (10ng/L). The bioluminescence intensity of each sample is normalized on the bioluminescence intensity of the corresponding negative control. Mann whitney statistical test was performed (ns : non significant, * : significant, ** : very significant, *** : extremely significant) Figure 8: Evolution of bioluminescence intensity at different times after toluene addition (100ng/L). The bioluminescence intensity of each sample is normalized on the bioluminescence intensity of the corresponding negative control. Mann whitney statistical test was performed (ns : non significant, * : significant, ** : very significant, *** : extremely significant) Figure 9: Evolution of bioluminescence intensity at different times after toluene addition (10µg/L). The bioluminescence intensity of each sample is normalized on the bioluminescence intensity of the corresponding negative control. Mann whitney statistical test was performed (ns : non significant, * : significant, ** : very significant, *** : extremely significant) Figure 10: Evolution of bioluminescence intensity at different times after toluene addition (10mg/L). The bioluminescence intensity of each sample is normalized on the bioluminescence intensity of the corresponding negative control. Mann whitney statistical test was performed (ns : non significant, * : significant, ** : very significant, *** : extremely significant)
The bioluminescence intensity of all our samples was compared to the bioluminescence intensity of the negative control for each time after toluene injection. Mann Whitney statistics test were realized to determine whether the bioluminescence intensity of the sample was significantly higher than the bioluminescence intensity of the negative control.
On the graphics we can notice that the bioluminescence intensity increase as the time after toluene injection increase. However, for the highest concentration (10 mg/L), a decrease in the bioluminescence intensity can be notice 5h30 after toluene injection. It is due to the accumulation of gaussia luciferase and the impact of toluene on the cell metabolism and especially protein synthesis (for more information click here ).
Therefore, it is essential to realize our bioluminescence test at a precise time after sampling. As we want to detect environmental toluene concentration (less than 20ng/L), it is to be suitable to wait 5h30 before proceeding to the bioluminescence test.
Investigation of detectable concentration 5h30 after toluene addition to the cells medium
As we determined that it was preferable to wait 5h30 before realizing the bioluminescence assay, we decided to focus on the results obtain for this time after toluene addition in the cells medium. We studied the evolution of the bioluminescence intensity according to the toluene concentration added in the cells medium 5h30 before. Those tests were realize in triplicates.
First of all, we wanted to ensure that the OD of the different samples is the same 5h30 after bacteria inoculation in presence of toluene. As shown on the Figure 11, the sample’s ODs are quite similar going from 1.464 to 1.61.
Figure 11: Sample's ODs depending on the toluene concentration added 5h30 before
The bioluminescence intensity of each samples was then determined and shown depending on the toluene concentration. As we are dealing with extremely different toluene concentration, it was preferable to give our results in function of the logarithm of the toluene concentration. The bioluminescence intensity data in RLU for each replicates (R1, R2, R3) are given in the table below and represented on the Figure 12.
Toluene concentration
Logarithm of toluene concentration
Mean - R1 (RLU)
SD - R1
Mean - R2 (RLU)
SD - R2
Mean - R3 (RLU)
SD - R3
0 ng/L
0
180.2
16.0
146.5
23.4
167.9
11.8
10 ng/L
1
1007.3
210.2
337.7
39.3
1041.9
139.4
100 ng/L
2
754.4
150.4
762.4
77.9
1823.1
243.1
10 µg/L
4
1110.4
150.0
1373.8
162.4
2073.7
202.9
10 mg/L
7
882.8
131.6
2485.4
259.5
1247.2
151.4
Figure 12: Evolution of bioluminescence intensity for each replicate depending on the logarithm of the toluene concentration injected 5h30 before
As shown on the graph, the obtained data differ from one replicates to another due to difference in the metabolism which is normal when working with living organism. However the curve profile stay is the same : the bioluminescence intensity of the sample increases until a toluene concentration of 10 µg/, then it reaches a plateau for two replicates and continues to increase for the other replicate.
Those results were pooled in order to draw a standard curve and being able to predict toluene concentration in a sample according to the bioluminescence intensity.
Quantification of environmentally relevant toluene concentration
The Figure 13 shows the evolution of the bioluminescence intensity depending on the logarithm of toluene concentration. For a toluene concentration inferior to 10 µg/L (10,000 ng/L : log(4)) the curve increases steadily, then the curve reach a plateau. This can be due to gaussia accumulation in the cells.
We were able to detect environmentally relevant toluene concentration and as the standard curve is linear for a toluene concentration inferior to 10µg/L, we are able to quantify the toluene concentration of a given sample.
0 ng/L
10 ng/L
100 ng/L
10 µg/L
10 mg/L
Mean (RLU)
164,84
795,63
1139,88
1519,28
1538,45
Standard deviation
17,62
129,73
146,68
171,75
180,84
Induction percentage
383%
592%
822%
833%
Figure 13: Evolution of bioluminescence intensity for pooled of the replicate depending on the logarithm of the toluene concentration injected 5h30 before. Correlation between toluene concentration in the cell culture medium and the bioluminescence intensity
Comparison with existing methods
Perspectives and improvement
Improve standard curve at low concentration
Determine the lowest concentration detectable
Figure 6: Bioluminescence intensity expressed in RLU of E.coli cells transformed with a construction genetic that contain (BB123) or do not contain (BB12) the gaussia gene. A little leakage of the gaussia gene can be seen as the bioluminescence intensity of the sample that contain bacteria transformed with our biosensor is significantly superior to the one that contain bacteria transformed with Pr-RBS-XylR. Mann whitney statistical test was performed (*** : extremely significant).
