Team:Tec-Monterrey/Design

DESIGN

Because we wanted to provide the whole method for the detection of Fusarium oxysporum on the field, we divided our project into different steps: Amplification, Detection and Cell-free.

Amplification

In order to have enough sample after DNA extraction using a technology with no laboratory requirements (such as the disposable polymeric microneedle patch used by the EPFL team in 2019 [1] and presented by the article Extraction of Plant DNA by Microneedle Patch for Rapid Detection of Plant Diseases [2]) an amplification step must be performed. Given the conditions in which we want our device to be used (on the field without specialized equipment) we need a portable, versatile and sensitive alternative to PCR. We found two alternatives commonly used for point-of-care biosensors: Recombinase Polymerase Amplification (RPA) and Nucleic Acid Sequence-Based Amplification (NASBA) [3][4]. NASBA is specifically designed for the amplification of RNA sequences and it works at 41°C, but for amplifying double stranded DNA it needs a denaturation step, which makes it no longer isothermal and represents a disadvantage [5]. RPA works at an optimal temperature of 37°C, but it has been reported to even perform amplification at the wide range of 25°C to 42°C [6], it uses nucleoprotein complexes conformed by oligonucleotide primers and recombinase proteins and does not require a denaturation step [5], this is why we decided to use RPA for our project.

Since we need the amplified sample in the form of RNA for its detection (see detection section) and we are using a cell-free system to do so (see cell-free section) we included a T7 promoter at the beginning of the forward RPA promoter for each sample, which will initiate a transcription step.

Detection

As explained in our Description page, the detection of Fusarium oxysporum is accomplished by the usage of toehold switches. These genetic tools are RNA sequences composed of four main parts (Figure 1). The first part is located at the beginning of the toehold and it is complementary to a given sequence of interest also called trigger (in this case, the fungus); next, there is a hairpin-like (loop) sequence, which hides an RBS and start codon; subsequently, at the end of the hairpin, a linker sequence is used as a spacer before the next component; finally, there is a coding sequence for a reporter gene [7].

Figure 1. Basic structure of a toehold switch. The reporter gene is represented here as RFP/AmilCP

The toehold itself, without the presence of the sequence of interest will remain with the RBS and start codon hiden, so there will be no expression of the reporter gene. If the sequence from Fusarium oxysporum is present with the toehold and since they are complementary, they will create a complex which will disrupt its original hairpin structure by a change in its conformation, leaving the RBS and start codon in the open [7], allowing the reporter gene to be synthesized and confirming that the plant is infected (Figure 2).

Figure 2. Functionality of toehold switches with and without the presence of a trigger sequence

Based on the literature reported by the 2019 EPFL team we decided to create not only toeholds for the detection of Fusarium, but also a positive control to make sure the toeholds are working, as they did. However, we too added two kinds of negative controls in order to prove the specificity of the genetic tools and correct functioning of the system as a whole.

  • Fusarium toehold: For the detection of Fusarium oxysporum we used the 28S-18S ribosomal RNA intergenic spacer sequence retrieved from NCBI [8] since it has been reported to be a consensus sequence and it has been used for PCR diagnosis [9].
  • Positive control toehold: We used a gene called SST1 (sucrose 1-fructosyltransferase) from Agave tequilana [10] as our positive control since it should always be present in the plant, so while using this toehold on the field it should as well always show expression of the reporter gene, proving the toeholds and cell-free are functioning adequately.
  • Negative control toehold (specificity): For the first type of negative control we decided to use the previous triggers, add single nucleotide mutations and test them, expecting that they should not express any quantity of reporter gene when the non-mutated triggers are present.
  • Negative control toehold (functioning of the system): This last control has a premature stop codon before the reporter gene, which should not let the process continue even in the presence of the correct trigger if the system is functioning correctly.

For the design of the toehold switches and to contribute to future works of fellow scientists, we decided to create our own software to standardize the process, since we realized that the current tools are not necessarily user friendly for those not specialized on the topic, and multiple platforms need to be utilized for the development of toeholds, especially if an amplification step is needed and thus the design of primers.

Read all about our software and its results here

We decided to use amilCP and RFP as options for reporter genes, since these proteins have a strong and highly visible color (blue and red respectively) when expressed, and are smaller than GFP which is typically used, not to mention that when being on the field the color of GFP can be mistaken with contamination on the sample. AmilCP and RFP are well characterized and RFP can be used for measurement of fluorescence protocols, as well as AmilCP in absorbance assays.

Our software gives as a result a library of toeholds based on several thermodynamic parameters. For the proof of concept of our detection system, we decided to test the best five different toeholds according to Toehold switch creator’s score both for Fusarium oxysporum and for the positive control (agave sequence). Furthermore, we only chose to test one toehold for each specificity control and one for the premature stop codon toehold since they are negative controls. This strategy was addressed in order to evaluate which toehold has a higher or better expression of the reporter genes.

In order to to take advantage of the sponsorships given in the form of gBlocks, we decided to synthesize the toeholds without the reporter genes and ligate them separately, however, to have a reference on the experimentation we selected the toeholds with the best scores for each trigger and ordered them with the reporter genes included: Fusarium toehold + amilCP, Fusarium toehold + RFP, positive control + amilCP and positive control + RFP.

We also wanted to test the advantages our software could provide to the functionality of the toehold itself and improve a previous genetic part from iGEM, so we agreed to design a set of toeholds for the bois noir disease, which was one of the targets of 2019 EPFL team, and compare our results, expecting an even better outcome. We also ordered two of these toeholds with the chosen reporter genes following the same criteria.

Finally, in order to have enough of each genetic part, instead of cloning in bacteria, we added sites for the primers M13 (-20) and M13 (-48) at each end so we can amplify them by PCR. In the case of the toeholds without the proteins, we added a prefix and a suffix to ligate them easily. Our final constructs looked as shown in Figure 3.

Figure 3. A) Final construct of our toeholds with reporter gene. B) Final construct of ur toehold without reporter gene. C) Representation of our toeholds for negative controls: specificity. D) Representation of our toeholds for negative controls: functioning of the system.

Cell-free

Since we expect our device to be used on the field by the farmers and don't want to rely on a living organism, we are using a cell-free method to carry on the detection of Fusarium oxysporum.

We decided to develop our own cell-free instead of using a commercial one for the device, with the goal of making the product more economically accessible. Given the characterization of E. coli as a source for the development of these kinds of systems and its high protein yield (up to 2.0 mg/mL in batch mode reactions) [11] we agreed on using it instead of other organisms with potential for cell-free’s, such as wheat germ and Vibrio natriegens.

Dopp et al provides a detailed protocol for using the E. coli strain BL21 Star™ (DE3), prefered due to its rapid doubling time, T7 transcription machinery and mutation in the RNaseE gene [12]. This method consists of a four day series of steps, as described on Figure 4.

Figure 4. workflow of the protocol for the development of an E. coli cell-free system

Finally, we planned on replacing the most expensive substrate used for E. coli cell-free system development: phosphoenolpyruvate (PEP), which is utilized as a source of energy. We found different articles addressing cost-effective alternatives such as glucose-6-phosphate (G6P) and starch. This last one has the advantage of maintaining the pH at a stable range (unlike G6P, due to the rapid formation of organic acids) while providing ATP [13][14].

Energy source Cost-effective Stable pH (prolonged reaction)
PEP No Yes
G6P Yes No
Starch Yes Yes

References

  1. iGEM EPFL 2019 team (2019). DNA extraction. https://2019.igem.org/Team:EPFL/Microneedles
  2. Paul, R., Saville, A. C., Hansel, J. C., Ye, Y., Ball, C., Williams, A., ... & Wei, Q. (2019). Extraction of plant DNA by microneedle patch for rapid detection of plant diseases. ACS nano, 13(6), 6540-6549.
  3. Daher, R. K., Stewart, G., Boissinot, M., & Bergeron, M. G. (2016). Recombinase polymerase amplification for diagnostic applications. Clinical chemistry, 62(7), 947-958.
  4. Chakravarthy A, Nandakumar A, George G, Ranganathan S, Umashankar S, Shettigar N, Palakodeti D, Gulyani A, Ramesh A. Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay. Life Sci Alliance. 2021 Sep 30;4(12):e202101213. https://doi.org/10.26508/lsa.202101213
  5. Zanoli, L. M., & Spoto, G. (2013). Isothermal amplification methods for the detection of nucleic acids in microfluidic devices. Biosensors, 3(1), 18-43. doi:10.3390/bios3010018
  6. TwistDx. Application Note 002: Evaluation of isothermal Recombinase Polymerase Amplification incubation temperature tolerance. https://www.twistdx.co.uk/docs/default-source/Application-notes/app-note-002---temp-tolerance-v1-3.pdf?sfvrsn=50570efc_16
  7. To, A. C. Y., Chu, D. H. T., Wang, A. R., Li, F. C. Y., Chiu, A. W. O., Gao, D. Y., ... & Yip, K. Y. (2018). A comprehensive web tool for toehold switch design. Bioinformatics, 34(16), 2862-2864.
  8. National Center for Biotechnology Information (NCBI). Nucleotide Database: Fusarium oxysporum isolate MCC2074 28S ribosomal RNA gene, partial sequence; 28S-18S ribosomal RNA intergenic spacer, complete sequence; and 18S ribosomal RNA gene, partial sequence. https://www.ncbi.nlm.nih.gov/nucleotide/MT254057.1?report=genbank&log$=nuclalign&blast_rank=1&RID=GV21GHZJ013
  9. Muhammad, A., Hussain, I., Khanzada, K. A., Kumar, L., Ali, M., Yasmin, T., & Hyder, M. Z. (2017). Molecular characterization of Fusarium oxysporum f. sp. Cubense (Foc) tropical race 4 causing Panama disease in Cavendish banana in Pakistan. Pakistan Journal of Agricultural Sciences, 54(1).
  10. National Center for Biotechnology Information (NCBI). Nucleotide Database: Agave tequilana sucrose:sucrose 1-fructosyltransferase (SST1) gene, complete cds https://www.ncbi.nlm.nih.gov/nuccore/JN790059.1
  11. Gregorio, N. E., Levine, M. Z., & Oza, J. P. (2019). A User’s Guide to Cell-Free Protein Synthesis. Methods and Protocols, 2(1), 24. doi:10.3390/mps2010024
  12. Dopp, J. L., Jo, Y. R., & Reuel, N. F. (2019). Methods to reduce variability in E. Coli-based cell-free protein expression experiments. Synthetic and systems biotechnology, 4(4), 204–211. https://doi.org/10.1016/j.synbio.2019.10.003
  13. Calhoun KA, Swartz JR. Energizing cell-free protein synthesis with glucose metabolism. Biotechnol Bioeng. 2005 Jun 5;90(5):606-13. doi: 10.1002/bit.20449. PMID: 15830344.
  14. Kim, H.-C., Kim, T.-W., & Kim, D.-M. (2011). Prolonged production of proteins in a cell-free protein synthesis system using polymeric carbohydrates as an energy source. Process Biochemistry, 46(6), 1366–1369. doi:10.1016/j.procbio.2011.03.008