Team:UParis BME/Proof Of Concept
Proof Of Concept
Introduction
When we first brainstormed about our possible project for iGEM 2021, one of the ideas was to focus on auto-testing for Marburg diseases. We then decided to look over a more severe and global problem, which was cancer. Particularly, we were interested in cancer diagnosis, since the most common method - tissue biopsy - is an invasive procedure, better suited for confirming than screening. We looked into the state-of-the-art regarding cancer diagnosis, and found out that cancer subtypes could be identified with the help of microRNA signatures (miRNA): coordinated dysregulation in specific miRNAs can indicate the onset of a specific cancer1. These marker miRNAs are often mediated by cancer cells via exosomes (small protecting vehicles), which makes them much more stable in blood (despite the presence of RNAse)2-3. With this, we had defined our topic of interest, but lacked the tool to detect and identify these markers. Further literature research and taking a look at the previous iGEM projects lead us to learning about the toehold switch, a riboswitch that, after binding with an RNA sequence of interest, translates a reporter gene. All of these findings have led us to an exciting idea that we have decided to carry out during iGEM 2021!
As described in implementation, our team aims to build a compact and accessible liquid biopsy tool for cancer diagnosis. We decided to use toehold switches to detect and quantify blood miRNAs that are abnormally expressed by cancer cells. These de-novo-designed riboregulators enable the control of transcription and translation in response to the target RNA, here miRNA.
As described in Figure 1, the toehold switch is composed of a “toehold” region that is complementary to a part of the miRNA at its 5' end. A repressed gene is located at the 3’ end of the riboregulator. The ribosome binding site (RBS) and the start codon (AUG) are restricted in the hairpin structure of the toehold switch. Once the trigger RNA binds to the toehold switch, the secondary structure is not favorable anymore and the hairpin is linearized1. The ribosome is then able to bind the RBS and start the translation of the repressed gene. Because we have more literature using fluorescent protein as a repressed gene, we decided to use EGFP4-6.
Toehold switch sensors have many advantages over conventional detection methods like PCR, benefiting from easy programming, rapidity, and low cost. Toehold switches can be combined with living cells or cell-free systems, offering a wide range of uses and applications, for in and out-lab, and do not require any huge expertise4. Our approach (Figure 2) starts with defining common miRNAs that are abnormally expressed in cancers and cancer subtypes8-10. SwitchMi Designer - the software we developed to design toehold switch candidates from target miRNAs - can generate banks of toehold switches targeting previously defined miRNAs, while taking into account the best possible amino acid sequence between the initiation codon and the EGFP.
Then, our proof-of-concept strategy includes 3 phases, described in Figure 3.
Phase 1 aims to select the toehold switch candidates that can detect target miRNAs from those that cannot. It consists of testing the candidates in Escherichia coli. The toehold switches are encoded by plasmids and expressed via T7 RNA polymerase. BL21 DE3 E. coli undergo a double transformation with plasmids encoding for the target miRNA and plasmids encoding for one of the toehold switch candidates. After overnight incubation, green fluorescence (from EGFP expression) can be measured by epifluorescence microscope. After this first phase, the toehold switch with the best sensitivity and specificity for its target is selected among the bank of candidates. We have found that toehold switches have a good detection efficiency for an RNA concentration of 170nM11 but previous iGEM teams, such as Evry Paris Saclay in 2020, were able to detect 2nM of trigger RNA using toehold switches.
Phase 2 aims to test the toehold switch on cancer cell-derived miRNAs and in a cell-free system. Cancer cell lines are cultured, then their exosomes are isolated. MiRNAs are extracted from the previously isolated exosomes after a lysis. MiRNAs and toehold switches are then put together in a cell-free system and green fluorescence is measured by epifluorescence. According to the toehold switch characterisation done on phase 1, the quantity detected by toehold switch is compared to the one obtained by the gold standard method, PCR. The miRNA quantification using toehold switch in the cell-free system should be optimized at the end of this second phase.
Phase 3 objective is to build the ExoSwitch prototype. A microfluidic chip is designed and produced to be loaded with blood samples in the first well, whereas the toehold switch is in the cell-free system on the last well. The microfluidic device allows exosome isolation from the blood sample and miRNA extraction with a lysis buffer. Several microfluidic chips are put on a 96-well plate, enabling the analysis of several miRNA detection at the same time using a plate reader.
At the end of the 3 phases, the ExoSwitch prototype is ready to be used!
Development
Our initial idea was to design a rapid, non invasive, easy-to-access low-cost cancer diagnosis tool. However, the limited time of the competition, and the difficulties encountered during our journey prevented our team from completing all three phases. Regarding Phase 1 (testing and selecting toehold switches targeting miRNA) we did not succeed in building functional toehold switches. We are close to getting the plasmid encoding for the toehold switch and we engineered BL21 DE3 E. coli expressing human miRNA. We have tried several methods to build the plasmid encoding for the toehold switch, which are described in the wetlab section. First, we tried to synthesize toehold switches using DNA Synthesis via Splicing for Overlap Extension Polymerase Chain Reaction (SOE PCR). However, this method requires much more expertise in the domain than what we have. Therefore, we changed our strategy and went to molecular cloning. We decided to assemble a plasmid containing the repressed gene, EGFP, and the T7 terminator. We ordered the T7 promoter followed by the variable part of the toehold switch (composed of the toehold region and the hairpin) from IDT, in order to insert this synthesized fragment into the receiver plasmid previously described. This strategy enabled us to save money when we ordered the fragment from IDT, because the sequence is more than 700bp shorter than the full toehold switch. We successfully produced a plasmid with the repressed gene, EGFP, and the T7 terminator (part BBa_K3878000). In the end, the molecular cloning method to assemble the synthesised fragment in the plasmid containing EGFP and the T7 terminator using restriction enzymes failed because our fragment had sticky ends after digestion. Finally, we tried the Golden Gate assembly (more details in wetlab), which benefits from sticky end fragments. However, our protocol was not optimized, which prevented us from getting our final construct.
For Phase 2, we completed half of the steps: we extracted exosomes from cancer cell cultures (MCF-7 and HeLa cell lines) and isolated miRNAs; we confirmed their presence thanks to a qRT-PCR method and quantified them. We had to stop right before the cell-free system step because of the troubles encountered in phase 1, that prevented us from moving forward within phase 2. Indeed, as previously described, all parts of the toehold switch were ready to be mixed (host plasmid and toehold Switch sequence), but we did not find the time to optimize the experiment in order to ligate them and progress through step 1 and step 2. Optimization of those digestion and ligation protocols were needed in order to have the best efficiency to build our final constructs.
Regarding Phase 3, we modeled the microfluidic chip using Clewin software. We decided to not produce it, even if we add protocols and materials to do it, because phase 1 and 2 were not achieved.
Perspectives
The immediate perspectives of the project are to achieve the final steps of phase 1 and phase 2.
For Phase 1, each of the toehold switch candidates has to be tested in the presence of a trigger miRNA, of a (theoretically) non-trigger miRNA, and in absence of miRNA. To do so, double transformation has to be performed on BL21 DE3 E. coli with plasmids encoding for the toehold switch, and with another plasmid either encoding for the trigger miRNA or for the non-trigger miRNA (or no other plasmid in the “absence of miRNA” condition). After overnight incubation, the fluorescence is measured using an epifluorescence microscope, allowing the toehold switches with the best specificity per trigger miRNA to be selected.
Each selected toehold switch has to be tested (in triplicate, for reproducibility) with an increasing quantity of trigger miRNA, in order to determine which concentration of toehold switch is required to quantify the smallest amount of miRNA. This experience enables the establishment of a calibration curve allowing us to link the fluorescence with the quantity of miRNA.
For Phase 2, exosomal miRNAs from HeLa and MCF-7 cell lines need to be quantified using the previously selected toehold switch in a cell-free system, with the results being compared to the gold standard method, PCR.
Then, the next step is the experimental part for Phase 3. The microfluidic chip can be produced following our guidelines and implemented in a 96-wells plate to be easily analysed by a plate reader.
In conclusion, we hope that future iGEM teams will continue to explore miRNA quantification using toehold switches and pursue similar projects to ExoSwitch. Throughout our journey, we have built various resources, such as the SwitchMi Designer, reviews about miRNAs, exosomes, toehold switches, and microfluidics. We hope these resources as well as our ambitious idea will inspire other students to resolve similar public health issues with the help of riboswitches and synthetic biology.
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- Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proceedings of the National Academy of Sciences. 2008;105(30):10513-10518. doi:10.1073/pnas.0804549105
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