The Pr promoter is found in the toluene recognition system and is composed of 410 bp. It has a constitutive expression. This promoter is available on the iGEM registry at this ID access: BBa_I723018.

We chose to use this promoter because it is the specific promoter for the XylR gene. This promoter is naturally constitutive. It leads to the permanent production of the XylR protein. Indeed, the induced luminescence has to be proportional to the pollutant rate. Therefore, when pollutant molecules enter in the bacteria, the XylR protein has to be present in sufficient amount.

The XylR gene, a gene of 1795 bp, encodes for the XylR protein and is regulated by the Pr promoter in its natural organization. This gene is available on the iGEM registry at this ID access: BBa_K1834844.

The XylR protein, mined from Pseudomonas putida, is involved in the transcriptional activation of the toluene recognition system. This regulatory protein allows the detection of aromatic hydrocarbons that carry a methyl group, i.e. xylene, toluene and 1-chloro-3-methyl-benzene. The A domain of the XylR protein (sensing domain), reacts with the pollutant molecule by binding to its methyl group. This leads to the formation of a tetramer. The C domain is involved in the dimerization of XylR, which is ATP dependent. The made up tetramer acts as an activator transcriptional factor for the Pu promoter, through the DNA binding D domain.

Pu is a promoter found in the toluene recognition system and is composed of 320 bp. This promoter is available on the iGEM registry at this ID access: BBa_I723020.
We chose to use this promoter because of its sensibility to the tetramer formed by the transcriptional regulator XylR coupled with xylene, toluene or 1-chloro-3-methyl-benzene, in response to the detection of one of these environmental pollutants.

This gene is found in a well-known organism, the copepod Gaussia princeps. It encodes for a Luciferase enzyme, the Gaussia Luciferase, also known as GLuc, which is involved in a bioluminescence process. This enzyme degrades its substrate, the Coelenterazine, into a product, the Coelenteramide. With an optimal substrate level, this step produces energy in the form of protons that can be recovered with a fixed spectrophotometer at 475nm (green light).

We chose to use the GLuc-His part, a gene of 522 bp, available on the iGEM registry at the BBa_K1732027 ID access. It is an optimized version for E.Coli of its twin the GLUCCO-His part (BBa_K1732003), which codes for the Gaussia luciferase following by 6 histidines. In our plasmid, this gene is positioned after an induced promoter, known as Pu promoter to report the activation of the toluene recognition system. The Gaussia luciferase needs the addition of substrate to ensure its activity because this molecule is not synthetized by our biosensor. Due to its secreted form, lysing cells in order to assay GLuc activity is not necessary.

The Gaussia luciferase is an ideal reporter gene because of its stability at high temperature thanks to disulfide bonds and because it has extremely high activity in light production for very sensitive assays. When compared to Firefly and Renilla luciferase, GLuc generates over 1000-fold higher bioluminescent signal intensity. The NanoLuc has an activity a little higher but this luciferase is very recent and thus less characterized.

Advantages of luminescence, over fluorescence, include the absence of background noise, the amplification of signal and a high dynamic range that spans many orders of magnitude. Indeed, since light emission depends strictly on the chemical reaction between the substrate and the luciferase, there is no background noise originating from the sample. Furthermore, the turnover of the light reaction significantly amplifies the reporter signal.Even though bioluminescence is currently used mainly for transcription study and cell imaging, this method become increasingly popular for quantitative analysis.
Moreover, the fact that the luciferase substrate have to be added in the laboratory present an interesting way to control the reaction. Indeed the luciferase substrate can be added at the same time for each samples allowing a better compliance between the different samples.

The transcription of luciferase in the presence of specific pollutants and the use of its substrate as a manner to assess pollutant concentration is an innovative approach , yet poorly studied.

For more information about bioluminescence and the Gaussia luciferase, click here !

In our plasmid, all genes, XylR and GLuc, are preceded by a sequence that can easily affect the rate of translation: the Ribosome binding Sequence (RBS).

There are different types of RBS, depending on their binding strength:

Strong RBS:

The Elowitz RBS is the stronger RBS with an efficiency of 1.0. This RBS is a sequence of 12 bp and is available on the iGEM registry at this ID access: BBa_B0034.
An other strong RBS, based on Ron Weiss thesis, is available on the iGEM registry at this ID access: BBa_B0030. This RBS, a sequence of 15 bp, has an efficiency of 0.6.
This type of RBS allows a high translation rate that leads to a high protein production. However, a too high production can be harmful for the organism because a lot of proteins could be unfolded or misfolded.

Middle RBS:

An example of Middle RBS is available on the iGEM registry at this ID access: BBa_B0032. This RBS, a sequence of 13 bp derivative of BBa_B0030, has an efficiency of 0.3.

Weak RBS:

The weaker RBS is a sequence of 11 bp with an efficiency of 0.01, derivative of BBa_B0030. This RBS is available on the iGEM registry at this ID access: BBa_0033.
An other weak RBS is available on the iGEM registry at this ID access: BBa_B0031. This RBS, a sequence of 14 bp derivative of BBa_B0030, has an efficiency of 0.07.

In our project, we chose to use the stronger RBS, the Elowitz RBS (BBa_B0034), in order to have a maximal production rate. Indeed, as we already specified, the induced luminescence has to be proportional to the pollutant rate. Therefore, when pollutant molecules enter in the bacteria, the XylR protein has to be present in an enough amount and the Gaussia luciferase has to be produced rapidly in an amount that is proportional to the pollutant amount. This allows a luminescent response corresponding to the pollutant rate.

A terminator is a genetic part placed at the end of a gene, in order to end transcription thanks to a stop codon. It exists several types of terminators but the most used are forward terminators. In general, some RNA polymerases will continue the transcription after the terminator, thus the terminator efficiency is not 100% in the most cases.
In order to increase the terminator efficiency in our plasmid we chose to use a double terminator. We selected two different double terminators that we have to test.
The first one of 129 bp, consisting of BBa_B0010 and BBa_B0012 as shown in Figure 4, available on the iGEM registry at this ID access: BBa_B0015. It is the most used terminator for forward transcription with a forward-efficiency of 0,984 according to measurements done by Caitlin Conboy, and of 0,97 according to measurements done by Jason Kelly. Procedures used for these measurements are explained in the IGEM registry.