Team:Ionis Paris/Biobrick Design
In the context of iGEM, our genetic circuit, encoded as a plasmid, is defined as a BioBrick. The different elements of the genetic circuit contribute to the function of the BioBrick. In the iGEM registry, we can distinguish 2 components that compose a plasmid: the plasmid backbone and the BioBrick part (see Figure 2). Figure 1: General structure of a BioBrick (source here ) For the assembly of our biobrick we use the BioBrick RFC[10] assembly standard because it is the standard of the IGEM competition and most of the parts available on the registry match this standard. The BioBrick RFC[10] assembly standard is based on the use of a prefix and a suffix placed at the extremities of each part to be assembled in order to obtain standard BioBricks that are compatible and thus can be easily assembled. Prefix and suffix Prefix and suffix provide a method to obtain standard genetic construction. Their exact sequences are available in Figure 2. Each one includes two different restriction sites: - EcoRI and XbaI in the prefix - SpeI and PstI in the suffix For protein coding regions (that starts with ATG), there is an adapted prefix. Figure 2: Prefix and suffix sequences All components of the genetic circuit are added to the system with these prefix and suffix. Once added to the system, in order to be compatible with the RFC[10] standard, a part does not have to contain these four restriction sites, they have to be unique to the prefix and suffix. Assembling method Figure 3: Assembling method Scar The use of this assembling method leaves a scar between the 2 assembled parts, with the following DNA sequence: The only disadvantage of the RFC[10] assembly standard is the impossibility to make fusion proteins. The 6bp scar includes a stop codon and the 8bp scar includes a frame-shift and a stop codon. The alternative to this problem is to use a scar-less assembly method or DNA synthesis. The biobrick part is composed of the sequence located between the prefix and the suffix. This sequence includes the devices that will perform the given functions in the cell (i.e protein production). The plasmid backbone is the sequence that begins with the suffix and ends with the prefix. The plasmid backbone mainly serves as support for the propagation of the biobrick part. It includes the origin of replication and the antibiotic resistance marker. The biosensor cell contains a genetic circuit, located on a plasmid support, allowing the expression of genes involved in the detection of the pollutant.
The Pr promoter is a constitutive promoter driving the transcription of the XylR gene, coding for the XylR protein. The XylR protein, a transcriptional regulatory protein for the Pu promoter, is activated by aromatic hydrocarbons that carry a methyl group (like toluene and xylene). In our biosensor, the Pu promoter allows the transcription of the bioluminescent reporter gene GLuc, coding for the Gaussia luciferase. When this enzyme reacts with its substrate, a substance called the Coelenterazine, it emits luminescence. Figure 4: The biosensor mechanism For the construction of our plasmid, we selected parts that are RFC[10] compatible.
In Figure 4 below, the map of our plasmid is represented in a simplified way, and is composed of the pSB1C3 backbone and the biosensor part. Figure 4: Our plasmid map We chose to use Escherichia Coli strain DH5α as chassis for our plasmid. Indeed, this bacterium grows easily and has several mutations that make it an excellent choice for cloning procedures with a high efficiency transformation. All our parts are optimized for E. Coli. Moreover, E. Coli is a model organism, entirely sequenced. We selected the pSB1C3 plasmid, a high copy number assembly plasmid, as a backbone of 2070 pb, because it is the most used type of backbone to assemble BioBricks. This plasmid backbone includes a high copy replication origin that allows a high copy number per cell which facilitates DNA purification. All the plasmid backbone used for the competition are provided with a default insert: the ccdb gene. This gene can be used as a marker to establish whether the plasmid backbone have been successfully digested. If not, the cell death gene will remain in the plasmid backbone and kill the kill the transformed bacteria. The ccdb gene ensures to not keep cells transformed with the uncut plasmid during the assembly of two BioBrick parts. Figure 6: Standard structure of high copy number assembly plasmid backbone. (source here) The pSB1C3 plasmid backbone confers Chloramphenicol resistance and includes the pUC19-derived pMB1, replication origin with a copy number of 100-300 per cell. The map of pSB1C3 plasmid is available below in Figure 2. Figure 7: Map of the pSB1C3 plasmid (source here) The Pr promoter is found in the toluene recognition system and is composed of 410 bp. 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 should be present in sufficient amount. The 1704 bp XylR gene, encodes for the XylR protein and is regulated by the Pr promoter in its native context. This gene is available on the iGEM Registry of Standard Biological Parts (BBa_K1834844). The XylR that we designed for our biosensor is a bit different because it is optimized for E.Coli DH5-Alpha and for IDT synthesis. . 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), binds to the pollutant molecule. 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. Figure 8: The XylR CDS structure Pu promoter
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. This gene is found in a well-known organism, the copepod Gaussia princeps. It encodes for the Gaussia luciferase enzyme, also known as GLuc, which is involved in a bioluminescence process. This enzyme degrades its substrate, coelenterazine, into a product, celenteramide. With an optimal substrate level, this step produces energy in the form of light that can be detected with a fixed spectrophotometer at 488nm. We chose to use the GLuc-His part, a gene of 522 bp, available on the iGEM registry at the (BBa_K1732027) which codes for the Gaussia luciferase followed by 6 histidines and optimized it for E.coli. In our plasmid, this gene is positioned after an inducible promoter, the Pu promoter, to report the activation of the toluene recognition system. 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[1][2]. 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 [3]. 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. Even though bioluminescence is currently used mainly for transcription study and cell imaging, this method become increasingly popular for quantitative analysis. [3] For more information about bioluminescence and the Gaussia luciferase, click here ! [1] Inouye, S., Sahara-Miura, Y., Sato, J., Iimori, R., Yoshida, S., and Hosoya, T. (2013). Expression, purification and luminescence properties of coelenterazine-utilizing luciferases from Renilla, Oplophorus and Gaussia: Comparison of substrate specificity for C2-modified coelenterazines. Protein Expression and Purification 88, 150–156. 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. 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.
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. Several types of terminators exists 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. Figure 9: B0015 double terminator structure (Source here) The second one of 95 bp, consisting of BBa_B0011 and BBa_B0012 as shown in Figure 9, available on the iGEM registry at this ID access: BBa_B0014. It is a bidirectional terminator with a forward-efficiency of 0,604 according to measurements done by Caitlin Conboy and of 0,96 according to measurements done by Jason Kelly, and with a reverse-efficiency of 0,86 according to measurements done by Jason Kelly. Figure 10: B0014 double terminator structure (Source here)
Principle OF THE BIOBRICK DESIGN IN AN IGEM CONTEXT
Biobrick definition
Biobrick RFC[10] assembly standard
- 5' [part A] TACTAGAG [part B] 3’.
When using the alternate prefix in the case of the assembling of a RBS with a protein coding region, the scar DNA sequence would be:
- 5' [part A] TACTAG [part B, that starts with ATG] 3’.
Biobrick part
Each device is a composite part, which means it is composed of several basic parts assembled together to ensure a specific function. A basic part is a single functional unit coding for a basic biological function and cannot be split into smaller units. Promoters, coding sequences or RBSs are examples of basic parts. Two parts make up our device: one in charge of the XylR protein synthesis and one in charge of theGaussia luciferase synthesis.
Plasmid backbone
The presence of the antibiotic resistance marker in the backbone allows the selection of only clones that incorporate the plasmid thanks to an antibiotic in the medium. Clones that do not incorporate the plasmid do not have the antibiotic resistance and thus cannot survive in the selective medium.
In the iGEM competition, a set of 4 linearized plasmid backbones is sent to each team: pSB1A3, pSB1C3, pSB1K3.m1, and pSB1T3. They respectively include these 4 antibiotics: Ampicillin, Chloramphenicol, Kanamycin and Tetracycline.
PRINCIPLE AND FUNCTIONING OF OUR "BIOSENSOR" PLASMID
Plasmid map
Details on the used parts
Chassis
Plasmid Backbone
As already specified, the presence of the antibiotic resistance marker in the backbone allows the selection of clones that incorporated the plasmid.
This plasmid is also the designated plasmid backbone required for the registry shipping during the IGEM competition.
Constitutive Pr promoter
XylR gene
We chose to use this promoter because of its sensibility to the transcriptional regulator XylR bound to xylene, toluene or 1-chloro-3-methyl-benzene.
GLuc gene
The Gaussia luciferase needs the addition of substrate to ensure its activity because this molecule is not synthetized by our biosensor. Therefore, in the laboratory, luciferase substrate can be added at the same time in each sample ensuring that every measurement will be taken at the same time. This allow a better consistency between our different results. Also, due to its secreted form, lysing cells in order to assay GLuc activity is not necessary.
[2] S. Inouye, Y. Sahara, Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps, Biochem. Biophys. Res. Commun. 365 (2008) 96–101
[3]Wood, K (2011), The bioluminescence advantage, laboratorynews, available on: http://www.labnews.co.uk/features/the-bioluminescence-advantage-13-09-2011/Elowitz RBS
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.
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.
Double terminator
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.
