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.

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 :

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

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.

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)

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)

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

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.

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.

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.

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.

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.

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.

Comparison with existing methods

Perspectives and improvement

Improve standard curve at low concentration
Determine the lowest concentration detectable