Part:BBa_K5439004
FRET-based system for the detection of cadmium
FRET-based sensor system for the detection of cadmium and other heavy metals that consists of phytochelatin synthase from Thlaspi japnonicum (BBa_K5439001),an enzyme that catalyzes the biosynthesis of phytochelatins using as a co-substrate the heavy metal cadmium, flanked by two fluorescent proteins: ECFP (BBa_K1159302)as an energy donor and mVenus (BBa_K1907000)as an energy acceptor.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 895
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 745
Illegal BglII site found at 2154
Illegal XhoI site found at 2176 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2825
Contents
Usage and Biology
With advancements in synthetic biology, biosensors based on Förster resonance energy transfer (FRET) have become increasingly common for the analysis and study of biomolecules with high sensitivity and specificity. The FRET mechanism describes the energy transfer from a donor fluorophore to another nearby acceptor fluorophore, and in order for a noticeable fluorescence signal to occur, the fluorophores must be both close enough to each other and their emission spectra must overlap, which provides a condition for energy matching. Hence, onne of the most used applications of FRET is to reflect the distance information of two molecules. For exmaple, an increase in FRET signal is robust evidence of intermolecular interactions or conformational changes that cause the molecules to get closer together (Fang et. al, 2023).
In this composite part, we propose a new iteration of the biosensor developed by TecMonterrey_GDL in 2022 (BBa_K4447004), using their biosensor as a modular base for the development of 3 new biosensors for the detection of 3 different substrates: rifampicin (BBa_K5439003), cadmium (BBa_K5439004), and ibuprofen (BBa_K5439006). These new constructs are made up of the same general parts: an enzyme capable of recognizing a substrate of interest flanked by two fluorescent proteins (one donor fluorophore and one acceptor fluorophore). As with the original erythromycin biosensor, it bases its function on the conformational change the detector enzyme will undergo in the presence of the substrate of interest, bringing the fluorophores closer together and emitting a quantifiable FRET signal (Verma, 2023).
Since it builds upon the previous erythromycin biosensor (BBa_K4447004) and uses it as a base for assembly, it preserves its original design considerations: NcoI and XhoI restriction sites at the 5' and 3' ends, and a polyhistidine tag at the end of mVenus for purification. Due to the way it was assembled, there are no scars between genes.
Characterization
Gene Amplification, Assembly and Transformation
The basis for the assembly of this composite part was the previous iteration of the biosensor, ECFP_EryK_mVenus(BBa K4447004), which was made up of the 3 genes in a pET-28b backbone. In order to successfully assemble the construct through Gibson Assembly without scars between parts and ensure proper expression of the full fusion construct, we amplified the vector using primers that bind to the ends of both fluorescent proteins and exclude the center EryK gene, obtaining an empty FRET backbone with the homology regions corresponding to the gene of interest, in this case TjPCs. This was the basis for the construction of the other two versions of the biosensor: ECFP_RifMo_mVenus (BBa_K5439003), and ECFP_IpfF_mVenus (BBa_K5439006). This method of assembly effectively makes our system modular and customizable, as the detector gene can be switched out to cover a wider range of contaminants.
Along with amplifying the FRET backbone, we amplified TjPCs (BBa_K5439001)with primers that generate homology regions corresponding to those generated by the backbone amplification. After several optimization cycles, which included optimizing the annealing temperature, number of cycles, and elongation times, we obtained purified fragments to use in the assembly. Figure 2 displays the PCR gels for both the vector and the gene.
With both fragments amplified and purified, we proceeded to assemble the construct through Gibson Assembly (NEB Gibson Assembly Master Mix). Table 1 shows the components used for the assembly reaction. Assembly was done with 100 ng of vector and a 3-fold molar excess of insert, and the reaction was incubated at 50 °C for 1 hour.
| Reagent | Quantity |
|---|---|
| FRET backbone | 2.3 µL |
| TjPCs | 0.6 µL |
| Gibson Assembly Master Mix | 5 µL |
| Nuclease-free water | 2.1 µL |
After the assembly, the next step was to transform the assembled ECFP_TjPCs_mVenus product into E. coli BL21, an expression strain. This step required optimization as well, particularly regarding the efficiency of our competent cells. After optimization, we successfully obtained transformed colonies containing our construct (Figure 3.)
Confirmation of construct insertion through restriction digestion
As a confirmation step, we performed minipreps on transformed colonies and digested the resulting plasmid with Nco I and XhoI , in order to ensure the transformed colonies contained the plasmid with the full construct. Table 2 shows the components used for the restriction digest, while Figure 4 shows the resulting gel, showing bands that correspond to the approximate full length of the ECFP_TjPCs_mVenus construct and the rest of pET28b.
| Reagent | Quantity |
|---|---|
| Restriction Enzyme 10X Buffer | 5 µL |
| DNA (1 μg/μL) | 1 µL |
| NcoI restriction enzyme | 1 µL |
| XhoI restriction enzyme | 1 µL |
| BSA (10 μg/μL) | 0.2 µL |
| Nuclease-free water | To 20 µL |
| Total Volume | 20 µL |
Protein expression
Once we had the certainty of a successful assembly and transformation in BL21, selected colonies were induced with 0.4 mM IPTG to stimulate protein overexpression and obtain the full ECFP_TjPCs_mVenus fusion construct (Gomes, 2020). We attempted various temperatures and induction conditions, and the last trial at 16 °C for 16 hours yielded visible bands corresponding to the approximate molecular weight of the full construct (~106 kDa). As shown in Figure 5 , the bands corresponding to this weight were not visible in the negative controls, which were the same cells transformed with an empty pET28b backbone containing no insert. This shows evidence for the expression of our fusion construct. Further tests would include the purification of the protein and assays correlating the concentration of substrate to the fluorescence produced by the FRET system.
References
[1] Fang, C., Huang, Y., & Zhao, Y. (2023). Review of FRET biosensing and its application in biomolecular detection. American Journal of Translational Research, 15(2), 694–709. [2] Verma, A. K., Noumani, A., Yadav, A. K., & Solanki, P. R. (2023). FRET Based Biosensor: Principle Applications Recent Advances and Challenges. Diagnostics (Basel, Switzerland), 13(8). https://doi.org/10.3390/diagnostics13081375 [3] Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S., & Steinegger, M. (2022). ColabFold: making protein folding accessible to all. Nature Methods, 19(6), 679–682. https://doi.org/10.1038/s41592-022-01488-1 [4] Gomes, L., Monteiro, G., & Mergulhão, F. (2020). The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms. International Journal of Molecular Sciences, 21(2), 576. https://doi.org/10.3390/ijms21020576
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