Plasmid Construction

For proof of concept purposes, we constructed a pSB1C3-KPC-2-surrogate plasmid that carries a short snippet of the KPC-2 gene by assembling the KPC-2-surrogate gene fragment (BBa_K5041000) with pSB1C3 backbone using the RFC 10 BioBricks assembly standard [1]. Briefly, the partial sequence of the KPC-2-surrogate gene was amplified by PCR (TaKaRa Bio). Both the RFC 10-compatible amplicons and pSB1C3 (from Interlab 2018 Test Device 5 BBa_J364008, Distribution Kit 2024) were then double digested with EcoRI and PstI restriction enzymes (Anza, Thermo Fisher Scientific) and assembled with T4 ligation (New England Biolabs). Ligation product was used to transform chemically competent DH5α E. coli, and cloned plasmid was later harvested using DNA-spin™ Plasmid DNA Purification Kit (iNtRON Biotechnology) and verified by Sanger sequencing (BGI Genomics).

Rolling Circle Amplification Template Construction

To facilitate the RCA process, we have constructed a circularised third-generation (v3) RCA template (BBa_K5041004) with the sequence as shown in Figure 1.

Figure 1: BBa_K5041004 template sequence

The linear oligonucleotide strand was synthesised by IDT and assembled into circular ssDNA form using IDT-synthesised scaffold (5'-GCCACCGCGGTCTCACGGGT-3') and T4 DNA ligase (New England Biolabs), and thus, treated with Exonuclease I and III (Thermo Fisher Scientific and Takara Bio) to remove the remaining scaffold.

Cell Lysis

Prior to the detection system, the collected bacterial sample must be lysed to release its bacterial DNA. In this proof of concept stage, we choose to lyse the cells using SDS. Using SDS to lyse bacterial cells has been an established procedure, which is used in the extraction of plasmids from bacteria using mini-prep kits [2]. SDS lysis could serve as a benchmark for lysis efficiency and plasmid yield when compared to other lysis methods or buffer compositions. The proof of our methodology in lysing the cells using SDS can be viewed here. Figure 2 and Figure 3 illustrate the process of cell lysis and its significance in the downstream reactions.

Figure 2: Cell Lysis and Release of Genomic Material

Figure 3: Indication of Antibiotic Resistance Gene

Target Gene Extraction

After cell lysis, a snippet of the target gene, which is responsible for antibiotic resistance, is cut using a restriction enzyme and a nickase, as illustrated in Figure 4. The nickase is used to cut the 3' side of the double-stranded DNA (dsDNA), while the restriction enzyme cuts the 5' side of the gene snippet. The restriction enzyme and nickase used to digest the plasmid in the proof of concept stage are HhaI and Nt.BsmAI (New England Biolabs) respectively.

Figure 4: Restriction Digestion by Restriction Enzyme (HhaI) and Nickase (Nt.BsmAI)

Following the digestion, Φ29 DNA polymerase (New England Biolabs and Beyotime Biotechnology) releases a single-stranded DNA (ssDNA) through linear strand displacement amplification (LSDA) as illustrated in Figure 5. Φ-29 binds to the nicked 5' end of the gene, which is the pSB1C3-KPC-2-surrogate in this proof of concept, and then amplifies one strand of the plasmid up to the HhaI cutting site. This process is expected to amplify a 32nt ssDNA from the pSB1C3-KPC-2-surrogate plasmid, which will serve as the primer for the following steps, along with 724 and 534nt ssDNA strands as the side products of this LSDA process.

Figure 5: Working Mechanism of Φ29 DNA Polymerase

An experiment has also been conducted to test this process, and the results can be seen here.

Rolling Circle Amplification

The circularised RCA template consists of a primer binding site, where the 32nt ssDNA from the previous step will bind to. As presented in Figure 6, upon this binding, Φ29 DNA polymerase will start to add nucleotides following the primer 3'-end, according to sequences complementary to the RCA template [3]. During this step, we expect cells that do not carry KPC-2 gene will not produce complementary primer, thus, will not trigger the RCA process.

After completing its first round along the RCA template, Φ29 will continue to replicate the RCA template while displacing the complementary strands that it has formed during its previous round [4]. By including the complementary strand of our peroxidase-mimicking G-quadruplex DNAzyme into the RCA template, the RCA process is expected to generate repeats of the G-quadruplex DNAzyme upon the primer binding.

Figure 6: Step of Rolling Circle Amplification

The RCA template also includes the recognition site of the Nt.BsmAI nicking endonuclease. Without Nt.BsmAI, this RCA process will generate a long continuous strand that consists of repeats of the RCA template. However, with the addition of Nt.BsmAI, the Φ29-produced strands will be nicked by the nickase prior to its displacement, producing shorter products out of the amplification process, as shown in Figure 7. This aims to increase the detection sensitivity by mediating exponential RCA [5].

Figure 7: Rolling Circle Amplification Product

To test the RCA reactions and to eliminate interfering factors, we have also conducted the RCA experiments with synthetic RCA primer from IDT. We tested several conditions for the RCA process and the results can be viewed here.

Signal Expression

The previous step of the system generates G-quadruplex structures that act as peroxidase-mimicking DNAzymes. The products from RCA are then set up into a reaction consisting of haemin, TMB, and H₂O₂. If the RCA process was triggered, G-quadruplex DNAzyme will be formed, and thus produce an observable colour change of the reaction due to the oxidation of tetramethylbenzidine (TMB) to TMB⁺. The positive reaction is hence indicated by a blue colour while the negative reaction does not change colour and remains colourless. Figure 8 illustrates the colour change reaction in the presence of the G-quadruplex DNAzyme.

Figure 8: Colour Change of Reaction Mixture with G-Quadruplex DNAzyme

It is understood that the working mechanism of the DNAzyme involves the attachment of haemin to the G-quadruplex structure, a guanine-rich DNA sequence stabilised by hydrogen bonds, to produce a stable complex [6]. Haemin's iron has the ability to move electrons around to catalyse the conversion of TMB to TMB⁺ [6].

To confirm the process of the G-quadruplex DNAzyme reaction, we have also acquired an IDT-synthesised DNA (BBa_K5041001) that will fold into a G-quadruplex structure. All of the experiment results can be found here.

Remarks

While our detection system is designed to operate in isothermal conditions, particularly at room temperature, our experiments in the proof of concept stage were largely conducted at the suggested optimum temperatures of the enzyme. We have confirmed that most reactions can occur at 37°C or lower. However, the need for ongoing research is underscored by the requirement for further verification of the enzyme's performance at room temperature. 

References

1. "Help:Standards/Assembly/RFC10", Registry of Standard Biological Parts, https://parts.igem.org/Help:Standards/Assembly/RFC10 (accessed Sep. 24, 2024).
2. "DNA-spinTM Plasmid DNA Purification Kit", iNtRON Biotechnology, https://intronbio.com:6001/intronbioen/product/product_view.php?PRDT_ID=1 (accessed Sep. 24, 2024).
3. F. Li, Y. Zhou, H. Yin, and S. Ai, "Recent advances on signal amplification strategies in photoelectrochemical sensing of micrornas", Biosensors and Bioelectronics, vol. 166, p. 112476, Oct. 2020. doi: 10.1016/j.bios.2020.112476
4. R. Hull, "Assay, detection, and diagnosis of plant viruses", Plant Virology, pp. 755–808, 2014. doi: 10.1016/b978-0-12-384871-0.00013-3
5. H.-X. Jiang, Z.-Z. Liang, Y.-H. Ma, D.-M. Kong, and Z.-Y. Hong, "G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microrna detection", Analytica Chimica Acta, vol. 943, pp. 114–122, Nov. 2016. doi: 10.1016/j.aca.2016.09.019
6. N. Alizadeh, A. Salimi, and R. Hallaj, "Haemin/G-quadruplex horseradish peroxidase-mimicking dnazyme: Principle and biosensing application", Advances in Biochemical Engineering/Biotechnology, pp. 85–106, 2017. doi: 10.1007/10_2017_3