Experiments and Results
The biological component of GutLux is the biosensor that is designed to produce fluorescence upon the detection of the target metabolites: kynurenic acid and tryptamine. To get to the final biosensor design, we have had to work our way up from scratch, starting with designing the DNA inserts. We then set out to clone our DNA inserts into expression plasmids by employing techniques of Modular Cloning (MoClo), a Golden Gate-based method, to obtain our desired constructs.
We have designed our experiments to facilitate the construction of our Level 2 plasmids (containing the multigene cassette) and the reporter plasmid (containing the Dioxin Response Element (DRE) promoter upstream of the superfolder green fluorescent protein (sfGFP)).
The goals of our experiments were:
- To construct a plasmid containing a multigene cassette capable of expressing the proteins involved in our system: Aryl hydrocarbon Receptor (AhR), Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) and Aryl hydrocarbon Receptor Interacting Protein (AIP).
- To construct a plasmid that functions as the reporter by providing a binding site for the AhR-ARNT complex, the DRE, through the CYP1A1 promoter which can drive the expression of sfGFP based on metabolite detection.
- To validate if the constructed plasmids had the correct DNA inserts, in the desired order.
The AhR-ARNT mechanism is explained below in Figure 1.
Modular Cloning (MoClo)
We opted to work with MoClo because of its ability to create multigene assemblies efficiently and reliably by making use of the type IIS restriction enzymes (Weber et al., 2011). We based our cloning method on the RFC94 cloning system pioneered by the Boston U iGEM team. The type IIS enzymes used for the construction were BsaI and BbsI.
MoClo involves generating different levels of plasmids with increasing complexity to create a modular setup. The levels start with Level 0, where an individual genetic regulatory element such as the promoter will be in its own plasmid. Level 1 is the plasmid that is constructed using multiple Level 0 plasmids effectively creating a full transcription unit that can express a gene. Similarly, Level 2 plasmids are constructed using multiple transcription units from different Level 1 plasmids to generate a multigene cassette.
RFC94 makes use of BsaI and BbsI in an alternating fashion to go from a lower level to a higher level, thanks to its efficient way of designing the fusion sites. The orientation of BsaI and BbsI recognition sites in the fusion site will depend on the level of plasmid to be generated. We decided to use BsaI for Level 1 construction and BbsI for Level 2 construction. Irrespective of the enzyme used, the fusion sites were designed to always produce a four-nucleotide-long sticky end. This was to ensure the fragments digested from a lower level still retained the same fusion site for the construction of the next level. The design also ensured improved reaction efficiency by preventing ligated fragments from getting digested again. Since the MoClo construction involves using two plasmids, it is strongly recommended to use backbones containing two different antibiotic selection markers to facilitate efficient screening.
Construction of Plasmid Backbones Compatible for RFC94
We worked with pRSFDUET-1 and pC125 vectors to create our backbones for the Level 1 and Level 2 plasmids for Escherichia coli, respectively. pRSFDUET-1 was chosen due to its ability to express at a higher level in E. coli. pC125, provided kindly by Chris Jonkergouw of Aalto University, was chosen because it contained an origin of replication from the incompatibility group B, which is important to prevent incompatibility issues when transforming with other plasmids like our reporter plasmid (Thomas, 2014). For Saccharomyces cerevisiae, we used the yeast expression and integration vectors, pRS416 and pRS305K, respectively. Both vectors were kindly provided to us by Alexander Frey of Aalto University.
All the vectors were converted into RFC94 compatible backbones through restriction-ligation cloning (Figure 2). We designed DNA inserts called plugs that contained a transcription unit for sfGFP flanked by appropriate fusion sites. These plugs also contained restriction sites for the type II restriction enzymes EcoRI and XbaI at their 5’ and 3’ ends outside the fusion sites, respectively. The plug and the vectors were digested using EcoRI and XbaI. The digested fragments were then ligated to produce the RFC94 compatible backbones. To facilitate efficient screening, our Level 1 backbones contain a gene that confers resistance to the kanamycin antibiotic, and Level 0 and Level 2 backbones contain a gene that confers resistance to the ampicillin antibiotic.
We have successfully constructed 3 of our 4 E. coli backbones (Figure 3), but none of the 4 S. cerevisiae backbones were constructed as a result of time constraints. We are troubleshooting their construction as of the time of writing this wiki.
Construction of Level 0 Plasmids Through Blunt-End Cloning
We ordered our DNA inserts from IDT and TWIST as blunt-end fragments containing the required type IIS recognition and fusion sites. The inserts were then ligated to pJET1.2/Blunt, a blunt-end cloning vector, through standard ligation procedure. All Level 0 plasmids will have resistance to ampicillin (Figure 4).
We have constructed a total of twenty-three Level 0 plasmids for use in E. coli and S. cerevisiae. These twenty-three plasmids contain a DNA insert of a genetic regulatory element to be used for expression in either E. coli or S. cerevisiae. The breakdown of the plasmids is shown in Figures 5 and 6:
Construction of Level 1 Plasmids Using BsaI
Like mentioned above, the Level 1 plasmid will contain a full transcription unit capable of expressing one of the three proteins from our system (Figure 7). As such, we designed six Level 1 plasmids each for E. coli and S. cerevisiae - three with the Glutathione S-Transferase (GST) tag and three without it (Figure 8). As mentioned above, these plasmids were constructed through the MoClo method by using BsaI.
As of the time of writing of this wiki, five of the six Level 1 plasmids for E. coli and four of the six Level 1 plasmids for S. cerevisiae have been constructed successfully (Figure 9, Table 1). However, as no S. cerevisiae backbones have been yet constructed, the S. cerevisiae Level 1 transcription units have been constructed using the E. coli backbones. We are experiencing issues with the construction of the other three Level 1 plasmids, mainly with their MoClo reaction setup and transformation. We are currently working to resolve them.
Construction of Level 2 Plasmids Using BbsI
The Level 1 plasmids that are verified to contain full transcription units for the individual proteins will be chosen for the construction of Level 2 plasmids. Like mentioned above, the Level 2 plasmid will contain the desired multigene cassette capable of expressing all three proteins (AhR, ARNT, and AIP) of our system (Figure 10). We designed two different Level 2 plasmids - one containing the coding sequences of all individual proteins with GST solubility tags, and the other containing just the coding sequences of all individual proteins - for both E. coli and S. cerevisiae, respectively (Figure 11). BbsI was used to construct the Level 2 plasmids for both hosts.
Unfortunately, as of the time of writing of this wiki, none of the four Level 2 plasmids were constructed as a result of time constraints and the issues with our other pending Level 1 plasmids. We hope to troubleshoot the Level 2 construction and obtain the Level 2 plasmids before the iGEM judging session.
Construction of the Reporter Plasmid Containing the Dioxin Response Element Upstream of the sfGFP Sequence
Our reporter plasmid was designed using a combination of MoClo techniques and conventional restriction-ligation cloning. The Dioxin Response Element (DRE) is a sequence consisting of 5′-CACGCNA-3′ or 5′-TNGCGTG-3′, where N is a random nucleotide (Li et al., 2014). The DRE element is found in the coding sequences of the promoters that regulate the genes of human cytochrome proteins like CYP1A1, CYP1A2 and CYP1B1. We opted to use the CYP1A1 promoter because literature showed it had more DRE elements and thus a better induction of gene expression than the promoters of other cytochrome proteins (Xiao et al., 2015). To obtain the reporter backbone, we designed a plug similar to the one used for construction of RFC94 compatible backbones (Figure 12). The plug here contains the transcription unit for sfGFP, albeit without a promoter. In the space previously intended for the promoter, there are two BsaI restriction sites to insert the promoter.
The reporter plasmid will be constructed through restriction-digestion using the reporter plug and a Level 1 backbone. The CYP1A1 promoter sequence will be amplified and extracted from a commercially available plasmid through polymerase chain reaction (PCR) using primers that are designed to introduce BsaI sites to the termini of the promoter (Table 2). The primers were designed to amplify the region of 74725220-74727160 base pairs of chromosome 15 based on the GRCh38.p13 assembly (GenBank accession: NC_000015.10). These BsaI sites will be compatible to facilitate insertion into the reporter backbone.
Unfortunately, we could not amplify the promoter from the commercially available plasmid using the primers designed, as they were not complementary as expected and the company did not release a detailed CYP1A1 promoter sequence map for intellectual property reasons. We hope to obtain the promoter from the commercial plasmid using restriction-ligation and proceed with the construction of the reporter plasmid in the coming days.
We have compiled all our protocols for wet lab experiments as a single pdf file here.
Sequences and Resources
Please find the sequences to the DNA inserts that we have used for GutLux here.
1. Li, S., Pei, X., Zhang, W., Xie, H., & Zhao, B. (2014). Functional Analysis of the Dioxin Response Elements (DREs) of the Murine CYP1A1 Gene Promoter: Beyond the Core DRE Sequence. International Journal of Molecular Sciences, 15(4), 6475–6487. doi:10.3390/ijms15046475
2. Thomas, C. M. (2014). Plasmid Incompatibility. Molecular Life Sciences, 1–3. https://doi.org/10.1007/978-1-4614-6436-5_565-2
3. Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PloS one, 6(2), e16765. https://doi.org/10.1371/journal.pone.0016765
4. Xiao, W., Son, J., Vorrink, S. U., Domann, F. E., & Goswami, P. C. (2015). Ligand-independent activation of aryl hydrocarbon receptor signaling in PCB3-quinone treated HaCaT human keratinocytes. Toxicology Letters, 233(3), 258–266. doi:10.1016/j.toxlet.2015.02.005