Cell-free protein expression systems, uses thereof and kits

Abstract

This invention relates to the field of biotechnology, specifically disclosing a cell-free protein expression system (CFS), its applications, and a kit. The CFS comprises: 35-50 vol% *E. coli* cell extract, 40-50 vol% lysis buffer, 8-12 vol% gene template, and 2-15 vol% nuclease-free water; wherein the *E. coli* is a recombinant bacterium obtained by modifying a recipient *E. coli*, the modification including: (1) knocking out the LacZ gene in the recipient *E. coli*; and (2) adding the LyseR gene to the recipient *E. coli*. The CFS of this invention features high expression activity and gene stability, making it suitable for expressing high molecular weight proteins and for in vitro diagnostic applications based on cell-free sensors.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a cell-free protein expression system, its applications, and a reagent kit. Background Technology

[0002] Cell-free protein expression systems (CFS) in *E. coli* are in vitro protein synthesis technologies based on cell extracts. These technologies are favored for their simple genetic background, ease of operation, rapid reaction speed, simplicity of process, and high-throughput processing capabilities. This technology has become a key tool in bioreaction system research. Recently, the advantages of cell-free protein synthesis systems in expressing complex proteins, toxic proteins, and signaling proteins have become increasingly apparent, and their application prospects in the biopharmaceutical industry are particularly broad. However, current cell-free protein expression systems (CFS) suffer from problems such as poor activity and low protection efficiency of linear templates.

[0003] On the other hand, citrus Huanglongbing (HLB) is a highly invasive and widely distributed disease of citrus plants caused by Asian phloem parasitic bacteria (CLas). In recent years, it has become a major disease seriously threatening the citrus industry. HLB has a long asymptomatic incubation period, making early detection challenging; and HLB with obvious symptoms is incurable. Currently, the best HLB management strategy is to control the vector with pesticides and eliminate affected trees, which hinders environmentally and economically sustainable citrus cultivation. Therefore, accurate and timely diagnosis of asymptomatic latent CLAs is crucial for the detection and control of citrus Huanglongbing. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a cell-free protein expression system that can achieve rapid protein expression and can be applied to cell-free sensor-based in vitro diagnostics.

[0005] Another technical problem to be solved by the present invention is to provide an application of a cell-free protein expression system in the preparation of a kit for detecting the pathogen of citrus Huanglongbing (HLB).

[0006] The technical problem that this invention also aims to solve is to provide a kit for detecting the pathogen of citrus Huanglongbing, which can achieve high specificity, high sensitivity and visual detection.

[0007] The technical problem that this invention also aims to solve is to provide a method for detecting the pathogen of citrus Huanglongbing, which can detect citrus Huanglongbing during the incubation period with high sensitivity and strong specificity.

[0008] To address the aforementioned technical problems, the present invention provides a cell-free protein expression system (CFS), comprising:

[0009] Escherichia coli cell extract 35-50 vol%, lysis buffer 40-50 vol%, gene template 8-12 vol%, nuclease-free water 2-15 vol%.

[0010] Exemplarily, in some embodiments, the amount of extract used is 37 vol%, 39 vol%, 41 vol%, 43 vol%, 45 vol%, or 47 vol%, but is not limited thereto. Preferably, it is 38 vol% to 42 vol%.

[0011] Exemplarily, in some embodiments, the amount of premix used is 42 vol%, 44 vol%, 45 vol%, 47 vol%, or 49 vol%, but is not limited thereto. Preferably, it is 42 vol% to 45 vol%.

[0012] Specifically, a premix includes, but is not limited to, amino acids, energy supply systems, buffers, and mixtures required for protein synthesis.

[0013] For example, in some embodiments, the amount of gene template used is 8.5 vol%, 9 vol%, 9.5 vol%, 10 vol%, 10.5 vol%, 11 vol%, or 11.5 vol%, but is not limited thereto.

[0014] Exemplary examples show that in some embodiments, the amount of nuclease-free water used is 3 vol%, 6 vol%, 9 vol%, 12 vol%, or 14 vol%, but is not limited thereto. Preferably, it is 2 to 5 vol%.

[0015] In addition, CFS also includes a gene template, which may be a linearized fragment of the gene to be expressed or a recombinant plasmid carrying the gene, preferably a recombinant plasmid carrying the gene.

[0016] It should be noted that, depending on the application field of CFS, other components may be introduced, and those skilled in the art can select them according to actual needs. For example, when applied in the field of virus detection, chromogenic agents for corresponding labeled proteins may be added, but this is not limited to these, and will not be listed here. The *E. coli* mentioned is a recombinant bacterium obtained by modifying the recipient *E. coli*, and the modification includes:

[0017] (1) Knock out the LacZ gene in the recipient Escherichia coli;

[0018] Specifically, the LacZ gene can be knocked out, or the Lac operon gene can be knocked out completely, but it is not limited to these two methods.

[0019] (2) Add the LyseR gene to the recipient Escherichia coli;

[0020] Based on the above-mentioned modified E. coli extract, proteins can be rapidly expressed. Furthermore, the LyseR gene product enzymatically cleaves the cell wall of E. coli during freeze-thaw cycles, thereby achieving autolysis and disruption (see...). Figure 1 ).

[0021] Specifically, the recipient Escherichia coli is BL21(DE3), BL21(a1), or C41(DE3), but is not limited thereto. Preferably, in some embodiments, the recipient Escherichia coli is BL21(DE3).

[0022] Preferably, in some embodiments, the modification further includes:

[0023] (3) The GamS gene was added to the recipient Escherichia coli. Specifically, the nucleotide sequence of the GamS gene is shown in SEQ ID NO: 1.

[0024] Specifically, the GamS gene can use either the T7 promoter or the AmpR promoter, but is not limited to these. Preferably, the GamS gene uses the AmpR promoter (SEQ ID NO: 2); the CFS expression level prepared using it is higher.

[0025] Based on the above modifications, the linear gene template in CFS can be protected from exonuclease degradation (see...). Figure 2 ).

[0026] Preferably, in some embodiments, the modification further includes:

[0027] (3) Knock out the RecBCD operon gene and ptrA gene of the recipient Escherichia coli and insert the LyseR gene at the knockout site.

[0028] Specifically, the RecBCD operon genes include the RecB gene, the RecC gene, and the RecD gene. The nucleotide sequence of the RecB gene is shown in SEQ ID NO: 3, the nucleotide sequence of the RecC gene is shown in SEQ ID NO: 4, the nucleotide sequence of the RecD gene is shown in SEQ ID NO: 5, and the nucleotide sequence of the ptrA gene is shown in SEQ ID NO: 6.

[0029] Preferably, in some embodiments, the RecBCD operon gene and ptrA gene of the *E. coli* are knocked out using the CRISPER / Cas combined λ-red dual plasmid gene editing system, and the LyseR gene is inserted at the knockout site;

[0030] The plasmids used include pUC19-LyseR and pCas12a-λred plasmids; specifically, the nucleotide sequence of pUC19-LyseR plasmid is shown in SEQ ID NO: 7, and the nucleotide sequence of pCas12a-λred plasmid is shown in SEQ ID NO: 8.

[0031] The gRNAs used include gRNA-Fw and gRNA-Rev, whose nucleotide sequences are shown in SEQ ID NO: 9-10, respectively;

[0032] The homologous arms used include TYB-Fw and TYB-Rev, whose nucleotide sequences are shown in SEQ ID NO: 13-14, respectively.

[0033] Preferably, in some embodiments, the lysis buffer comprises components at the following concentrations:

[0034] HEPES 40–60 mM, ATP 1–2 mM, GTP 1–2 mM, CTP 0.5–1.5 mM, UTP 0.5–1.5 mM, tRNA 0.1–0.5 mg / mL, CoA 0.1–0.5 mM, NAD 0.2–0.8 mM, cAMP 0.5–1 mM, leucovorin 0.01–0.1 mM, spermidine 0.5–1.5 mM, 3-PGA 10–50 mM, maltose 10–20 mM, amino acids 20–25 mM, PEG-8000 8–15 mg / mL, potassium glutamate 55–80 mM, magnesium glutamate 0.5–1.5 mM, DTT 0.5–1.5 mM. The solvent used in the lysis buffer (premix) is water, more specifically ddH2O or decpH2O.

[0035] Preferably, in some implementations, CFS includes:

[0036] Escherichia coli cell extract 40 vol%, lysis buffer 44.5 vol%, RNase inhibitor 0.5 vol%, gene template 10 vol%, nuclease-free water (depc H2O) 5 vol%.

[0037] The lysis solution comprises components at the following concentrations:

[0038] HEPES 50mM, ATP 1.5mM, GTP 1.5mM, CTP 0.9mM, UTP 0.9mM, tRNA 0.2mg / mL, CoA 0.26mM, NAD 0.33mM, cAMP 0.75mM, leucovorin 0.0678mM, spermidine 1mM, 3-PGA 30mM, maltose 12mM, amino acids 6mM, PEG-8000 11.2mg / mL, potassium glutamate 60.6mM, magnesium glutamate 0.815mM, DTT 1mM, Leucine 20mM, Glycine 24mM, Valine 24mM, Isoleucine 24mM, Methionine 24mM, Proline 24mM, Phenylalanine 24mM, Tyrosine 24mM, Serine 24mM, Threonine 24mM, Tryptophan 24mM, Asparagine 24mM, Glutamine 24mM, Lysine 24mM, Cysteine ​​24mM, Arginine 24mM, Aspartic Acid 24mM, Histidine 24mM, Glutamic Acid 24mM, Alanine 24mM.

[0039] Preferably, in some implementations, CFS includes:

[0040] Escherichia coli cell extract 40 vol%, lysate 44.5 vol%, RNase inhibitor 0.5 vol%, chlorophenol red-β-D-galactopyranoside (CPRG) 1 vol%, gene template 10 vol%, nuclease-free water (depc H2O) 4 vol.

[0041] Accordingly, the present invention also discloses the application of the above-mentioned cell-free protein expression system (CFS) in the preparation of a kit for detecting the pathogen of citrus Huanglongbing.

[0042] Accordingly, the present invention also discloses a kit for detecting the pathogen of citrus Huanglongbing, characterized in that it includes the above-mentioned cell-free protein expression system (CFS) and Toehold Switch element;

[0043] The nucleotide sequence of the Toehold Switch element is shown in SEQ ID NO: 15.

[0044] Preferably, in some embodiments, the kit further includes LAMP primers for amplifying a Trigger RNA sequence that is complementary to the Toehold sequence in the Toehold Switch element, and the Trigger RNA nucleotide sequence is shown in SEQ ID NO: 16;

[0045] The LAMP primers include L966-F3, L966-B3, L966-BIP, and L966-FIP, and their nucleotide sequences are shown in SEQ ID NO: 17-20, respectively.

[0046] Preferably, in some embodiments, the kit further includes: PCR buffer, dNTPs, MgCl2, Bst DNA / RNA polymerase and water, wherein the water is ddH2O or decpH2O, but is not limited thereto.

[0047] Preferably, in some embodiments, the cell-free protein expression system and the Toehold Switch element are freeze-dried on a paper-based material; the freeze-drying temperature is -60℃ to -40℃, and the freeze-drying time is 20h.

[0048] Accordingly, the present invention also discloses a method for detecting the pathogen of citrus Huanglongbing (HLB) for non-diagnostic purposes, comprising:

[0049] The genome of the sample to be tested is extracted and used as a gene template;

[0050] The gene template was amplified using LAMP primers, wherein the LAMP primers included L966-F3, L966-B3, L966-BIP, and L966-FIP, and their nucleotide sequences are shown in SEQ ID NO: 17-20, respectively.

[0051] The amplified products were reacted using the reaction system consisting of the cell-free protein expression system (CFS) and the Toehold Switch element described above; and the results were determined based on the color change.

[0052] Specifically, total RNA can be extracted using methods commonly used in the art, such as the Trizol method, guanidine isothiocyanate-phenol-chloroform extraction method, or other kits, but is not limited thereto. Preferably, in some embodiments, the step of extracting the genome of the sample to be tested as a gene template includes:

[0053] Take the midrib of the citrus leaf to be tested, add NaOH lysis buffer, grind, let stand, and then take the supernatant. Dilute with TE buffer to obtain the solution.

[0054] Specifically, the NaOH pyrolysis solution comprises: 0.2–1 M NaOH, 5–20 mM Na₂EDTA, and water. Preferably, the NaOH pyrolysis solution comprises: 0.5 M NaOH, 10 mM Na₂EDTA, and water.

[0055] Specifically, the amount of NaOH lysis buffer used is 500 μL for a 2 cm × 0.6 cm blade.

[0056] Specifically, the grinding time is 0.5 to 2 minutes, and the settling time is 1 to 5 minutes.

[0057] Specifically, the TE buffer is diluted 10 to 100 times.

[0058] Specifically, in the step of amplifying the gene template using LAMP primers, the amplification reaction system is as follows:

[0059] 10×buffer 1μL, Mg 2+ 0.6 μL, dNTP 1.2 μL, L966-F3 (2 μM) 1 μL, L966-B3 (2 μM) 1 μL, L966-BIP (16 μM) 1 μL, L966-FIP (16 μM) 1 μL, Bst4.2 DNA / RNA polymerase 0.4 μL, gene template 1 μL, decpH2O 1.8 μL.

[0060] The amplification reaction procedure was to react at 70℃ for 30 minutes.

[0061] Specifically, the step of reacting the amplification product using the reaction system composed of the cell-free protein expression system (CFS) and the ToeholdSwitch element, and determining the result based on the color change, includes:

[0062] The CFS and Toehold Switch components were freeze-dried on a paper-based material to obtain the reaction system;

[0063] Nuclease-free water and amplification products were added dropwise to the paper-based material, and after activation, the reaction was carried out at 20-35°C for 30-60 min.

[0064] The result is determined based on the color change.

[0065] Specifically, the reaction system was 10 μL, the amplification product was 0.4 μL, and the nuclease-free water was 9.6 μL.

[0066] Implementing this invention has the following beneficial effects:

[0067] 1. The CFS in one embodiment of the present invention has the advantages of high expression activity and gene stability, which enables the cell-free sensor to fully exert its signal amplification and rapid prototyping capabilities in in vitro diagnostic applications.

[0068] 2. In one embodiment of the present invention, the CFS can be freeze-dried on a paper-based material. The low cost, room temperature stability, ease of storage and distribution, and colorimetric results of this paper-based cell-free sensor reaction mean that these in vitro diagnostics have great application potential in resource-scarce areas and point-of-care testing (POCT) scenarios. Attached Figure Description

[0069] Figure 1 This is a schematic diagram illustrating the principle of LyseR gene product in CFS breaking down the cell wall of E. coli in this invention.

[0070] Figure 2 This is a schematic diagram illustrating the principle by which the GamS gene product in CFS protects the linear gene template from exonuclease degradation in this invention.

[0071] Figure 3 This is a diagram showing the results of LacZ protein expression in Example 1, prepared from the GamS gene using different promoters, based on autolysis procedures and exonuclease-inhibiting CFS expression.

[0072] Figure 4 This is a schematic diagram of the dual-plasmid gene editing principle in Example 2;

[0073] Figure 5 Composition diagram of plasmid pUC19-LyseR in Example 2;

[0074] Figure 6 Composition diagram of plasmid pCas12a-λred in Example 2;

[0075] Figure 7 This is a graph showing the results of expressing three CRISPR proteins based on the autolysis procedure and the exonuclease-deficient CFS in Example 2;

[0076] Figure 8 This is a graph showing the detection results of CLAs in leaves of citrus infected with Huanglongbing (HLB) using a paper-based Toehold Switch coupled with a LAMP system in Example 3. Detailed Implementation

[0077] To facilitate understanding of the present invention, it will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. Rather, these embodiments or examples are provided to make the disclosure of the present invention more thorough and complete.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments or examples only and is not intended to limit the invention. The optional range of the term "and / or" as used herein includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all related listed items.

[0079] The following embodiments are provided for the purpose of illustrating various embodiments of the present invention and are not intended to limit the scope of the invention in any way.

[0080] This invention. Those skilled in the art will understand that variations and other uses within the scope of the claims are included within the spirit and scope of this invention. Unless otherwise specified, the materials, reagents, etc., used in the following examples are commercially available. The promoter and terminator sequences mentioned in the examples can also be downloaded from NCBI, and the specific sequence start positions can be determined from the primers in the primer table. Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

[0081] In this invention, terms such as "first aspect" and "second aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features.

[0082] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0083] Unless otherwise specified, the percentage content involved in this invention refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0084] Unless otherwise specified, all percentage concentrations mentioned in this invention refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0085] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument.

[0086] Example 1: CFS Based on Autolysis Procedure and Exonuclease Activity Inhibition

[0087] 1. Preparation of extracts based on autolysis procedure and exonuclease activity inhibition

[0088] The plasmid pAD-AmpRPro-GamS (SEQ ID NO: 21) carrying the GamS gene with the AmpR promoter and the plasmid pAD-LyseR (addgene, Plasmid: #99245) carrying the LyseR gene were transformed into *E. coli* BL21(DE3)-Gold-dLac (Bacterial Strain #99247, addgene) cells. Cells were cultured in 2×YTPG medium containing the corresponding antibiotics at 37°C with shaking. When the cells reached OD600 = 0.6, 1 mM IPTG was added to induce T7 RNA polymerase expression. Cells were harvested when OD600 = 3.0, and the cell pellet was washed three times with S30A buffer (50 mM Tris, 60 mM potassium glutamate, 14 mM magnesium glutamate, final pH adjusted to 7.7, with 2 mM DTT added on the day of use). S30Abuffer was added at a buffer volume to bacterial precipitate weight ratio of 2:1. After freeze-thaw-vortex circulation, the extract was harvested by high-speed centrifugation (25000g / 1h / 4℃).

[0089] 2. Premix configuration

[0090] Prepare the premix according to the recipe in Table 1, mix thoroughly, and then dispense and store in a -80°C freezer.

[0091] Table 1 Premix Composition

[0092]

[0093] The table lists amino acids at the following concentrations:

[0094] Leucine 20mM, glycine 24mM, valine 24mM, isoleucine 24mM, methionine 24mM, proline 24mM, phenylalanine 24mM, tyrosine 24mM, serine 24mM, threonine 24mM, tryptophan 24mM, asparagine 24mM, glutamine 24mM, lysine 24mM, cysteine ​​24mM, arginine 24mM, aspartic acid 24mM, histidine 24mM, glutamic acid 24mM, alanine 24mM.

[0095] 3. Preparation of cell-free colorimetric reaction system

[0096] Prepare the cell-free colorimetric reaction system according to the specific formula in Table 2:

[0097] Table 2 Composition of the cell-free colorimetric reaction system

[0098]

[0099]

[0100] 4. Promoter optimization

[0101] Using the GamS gene driven by either the T7 promoter or the AmpR promoter, and the AmpR gene driven by the AmpR promoter...

[0102] The *E. coli* cells with the promoter-driven LyseR gene were prepared based on an autolysis procedure and an exonuclease-inhibiting CFS. Using plasmid pFN6A-(HQ)-LacZ (SEQ ID NO: 22) and the linear-LacZ gene as templates, and galactoside (CPRG) as a substrate, cell-free colorimetric reaction systems were prepared according to Table 2 for in vitro expression experiments. The system was incubated at 30℃ for 1 h. After the reaction, the color change of the reaction product was recorded, and the absorbance value of the reaction product at 570 nm was measured. The effect of different promoters driving GamS gene expression on CFS activity was compared by using the absorbance OD570. The results are as follows: Figure 3 As shown, the results indicate that, compared to using the T7 promoter, the GamS gene carrying the AmpR promoter showed a significant increase in the in vitro expression level of the corresponding CFS, and both the linear and plasmid gene reaction products changed to purple color.

[0103] Example 2: CFS Based on Autolysis Procedure and Exonuclease Activity Deficiency

[0104] 1. Preparation of engineered bacteria

[0105] The RecBCD operon gene and ptrA gene in *E. coli* BL21(DE3)-Gold-dLac (Bacterial Strain #99247) were knocked out using a CRISPR / Cas combined with λ-red dual plasmid gene editing system, and the LyseR gene was inserted at the knockout site to obtain the engineered strain BL21(DE3)-gold-dLac-dRecBCD:LyseR. The schematic diagram is shown below. Figure 4 As shown. The specific steps are as follows: The plasmid pCas12a-λred( Figure 6 (SEQ ID NO: 8) and pUC19-LyseR ( Figure 5The bacterial culture (SEQ ID NO: 7) was transformed into Escherichia coli BL21(DE3)-Gold-dLac (Bacterial Strain #99247, addgene) cells. The cells were cultured in LB medium containing the corresponding antibiotics at 37°C. When the cells grew to OD600 = 0.6, 40 mM arabinose and 0.25 mM IPTG were added to induce the expression of Cas12a and λred proteins, respectively. After culturing for another 5 h, the bacterial culture was diluted 1000 times and spread on agar plates containing 10% sucrose and AmpR resistance, and incubated upside down overnight. On the second day, single colonies were picked for colony PCR verification. Single colonies with the correct bands were selected and cultured and purified on sucrose plates until a single positive target band was shown in the colony PCR verification (using primers argA-F (SEQ ID NO: 10) and ppdB-R (SEQ ID NO: 11)).

[0106] 2. Preparation of extracts based on autolysis procedure and exonuclease activity deficiency

[0107] The verified cells were cultured in 2×YTPG medium containing the corresponding antibiotic at 37°C with shaking. When the cells grew to OD600 = 0.6, 1 mM IPTG was added to induce T7 RNA polymerase expression. When the cells grew to OD600 = 3.0, the cells were harvested, and the cell pellet was washed three times with S30A buffer. Finally, S30A buffer was added at a ratio of 2:1 (S30A buffer volume to cell pellet weight). After freeze-thaw-vortex cycling, the extract was harvested by high-speed centrifugation (25000 g / 1 h / 4°C).

[0108] 3. Premix configuration

[0109] Prepare the premix according to the recipe in Table 1, mix thoroughly, and then dispense and store in a -80°C freezer.

[0110] 4. Preparation of cell-free reaction system

[0111] Prepare the cell-free reaction system according to the specific formula in Table 3:

[0112] Table 3 Composition of the cell-free reaction system

[0113]

[0114] 5. CFS expresses three CRISPR proteins and LacZ protein in vitro.

[0115] Using plasmids pMBP-LbCas12a(addgene, 113431) and pET-28b-T7-henAsCas12a(E174R / S542R / K548R)-NLS(nucleoplasmin-6xHis), respectively...

[0116] In vitro expression experiments were conducted using (AAS1885)(addgene, 114072), pC013-Twinstrep-SUMO-huLwCas13a(addgene, 90097), pFN6A-(HQ)-LacZ(SEQ ID NO:22), and linear gene templates: Linear-LbCas12a, Linear-henAsCas12a, Linear-LwaCas13a, and Linear-LacZ. The linear gene structure was: 5'-T7 promoter-target gene-T7 terminator-3', with 10bp non-coding sequences retained at both ends of the T7 promoter and T7 terminator. Cell-free reaction systems were prepared according to Table 3, and after uniform mixing, the mixtures were incubated at 30℃ for 1 h. After cell-free expression, 10 μL of sample was mixed with 30 μL of TBS, and then 40 μL of 2×LD and 4 μL of DTT (1M) were added. After thorough mixing, the mixture was heated at 95℃ for 3 min. 16.8 μL of the mixture was then loaded onto an SDS gel. After protein electrophoresis, the expression effects of plasmid gene templates and linear gene templates in CFS were identified by Coomassie Brilliant Blue staining and Western blot. The results are as follows: Figure 7 As shown in the figure. The results showed that both plasmid genes and linear genes had high expression levels in CFS, with the plasmid gene template showing higher expression levels compared to the linear gene template.

[0117] Example 3 uses a paper-based Toehold Switch coupled with a LAMP detection system for rapid lysis and visual detection of citrus Huanglongbing pathogen.

[0118] 1. Design of Toehold Switch Elements Based on CLas Genome Sequences

[0119] Genomic DNA of *Bacillus citrus* (NCBI: 537021) was downloaded from the NCBI website. Using the phage DNA polymerase I, β-operon, and outer membrane protein *omp* gene sequences as source sequences, Toehold Switch elements were designed using software developed by iGEM in 2017. The source sequences were divided into 1000bp segments. The segmented gene sequences were then input into the software for screening Switch RNA and Trigger RNA. Based on the software's efficiency ranking, Toehold Switch elements with higher efficiency rankings were prioritized for further in vitro validation experiments.

[0120] The LacZ gene was used as the reporter gene for the switch RNA element. The 5' and 3' ends of the element include the T7 promoter and T7 terminator, respectively. A linear switch gene was obtained by PCR. The corresponding ssDNA of the trigger RNA was synthesized, and in vitro validation experiments were performed using the linear switch gene and ssDNA. In the in vitro validation experiments, the combination with the highest fold change value and the most significant color difference between the positive and negative groups was selected as the effective toehold switch element, namely S7-2 (SEQ ID NO: 15) and T7-2 (SEQ ID NO: 16).

[0121] 2. Design LAMP amplification primers targeting effective toehold switch element sequences.

[0122] Based on the genomic RNA sequence of CLas, highly conserved fragments were selected from the upstream and downstream regions of the trigger RNA. Primers were designed using PrimerExplorerV5 software, following LAMP primer design principles. The selected LAMP primers were L966-F3 (SEQ ID NO: 17), L966-B3 (SEQ ID NO: 18), L966-BIP (SEQ ID NO: 19), and L966-FIP (SEQ ID NO: 20).

[0123] 4. Rapid lysis of citrus leaves

[0124] Take a midrib (2cm × 0.6cm) from a citrus leaf to be tested, cut the midrib into small pieces and place it in a mortar. Add 500μL of 0.5M NaOH lysis buffer, grind immediately for 1 min, and then let stand at room temperature for 2 min. Take the clear supernatant and dilute it 50 times with TE buffer. The diluted sample is the DNA solution.

[0125] The formulation of 0.5M NaOH lysis buffer is: 0.5M NaOH, 10mM Na2EDTA, with the balance being ddH2O. The formulation of TE buffer is: 10mM Tris pH=8.0, 1mM EDTA, with the balance being ddH2O.

[0126] 5. Construction of the Toehold Switch Reaction System

[0127] (1) Construction of the Switch plasmid

[0128] Two rounds of repeat PCR were performed using designed long primers to amplify the Switch element into the LacZ gene, which includes a T7 promoter sequence and a T7 terminator sequence at each end. Based on candidate Switch RNA gene sequences, long primers were designed using snapgene, specifically S7-2-F1 (SEQ ID NO: 24), S7-2-F2 (SEQ ID NO: 25), Switch-T7Ter-R-55℃ (SEQ ID NO: 26), Switch-T7Ter-R-58℃ (SEQ ID NO: 27), and Switch-T7Ter-R-61℃ (SEQ ID NO: 28).

[0129] Primers S7-2-F1 and S7-2-F2 contain the Switch sequence. The Switch sequence was ligated to the LacZ gene by PCR amplification. The PCR amplification system for the Switch fragment is shown in Table 4 below:

[0130] Table 4. First-round PCR amplification system and conditions

[0131]

[0132]

[0133] As shown in the table above, PCR amplification was performed using recombinant plasmid pFN6A-S4-3-LacZ (SEQ ID NO: 23) as a template. After the reaction, the product was used as the template for the second round of PCR. The second round of PCR amplification system is shown in Table 5 below:

[0134] Table 5. Second-round PCR amplification system and conditions

[0135]

[0136] After the second round of PCR reaction, the amplification product was digested with DpnI enzyme and then the DNA was purified. The purified linear DNA was used as the switch element to be verified in the next in vitro verification experiment.

[0137] (2) Configuration of the reaction system

[0138] Prepare the reaction system according to the specific formula in Table 6:

[0139] Table 6. Composition of the Toehold Switch reaction system

[0140]

[0141] The Toehold Switch reaction system was freeze-dried, and the specific steps are as follows:

[0142] (1) Use a handheld puncher of different shapes with a diameter of 0.9cm to punch holes in cellulose quantitative filter paper (Whatman, 1442-042), collect the small filter paper pieces cut from the puncher, and place them at the bottom of a 96-well plate;

[0143] (2) The Toehold Switch reaction system was added dropwise at a volume of 9.6 μL per well to the bottom of the wells of a 96-well plate containing filter paper.

[0144] (3) The paper-based material containing the Toehold Switch reaction system was freeze-dried in a freeze dryer. The freeze-drying program was set to freeze at -50℃ for 5 hours and dry at -50℃ for 20 hours.

[0145] 6. LAMP reaction

[0146] Prepare the reaction system according to the specific formula in Table 7:

[0147] Table 7 Composition of the LAMP reaction system

[0148]

[0149] The extracted DNA solution was used as a gene template for amplification. The specific amplification reaction procedure was 70℃ for 30 minutes. After amplification, the amplification products were collected.

[0150] 7. Color reaction

[0151] 9.6 μL of nuclease-free water was thoroughly mixed with 0.4 μL of the amplification product and then added dropwise to a paper substrate loaded with the Toehold Switch reaction system to activate the lyophilized system. The system was incubated at 30°C for 30–60 min, and the color change of the reaction product and its absorbance at 570 nm were recorded. The detection results are as follows: Figure 8As shown in the figure, NC represents healthy citrus leaves, while the others represent leaves from citrus trees susceptible to Huanglongbing (HLB). The test results indicate that samples prepared from infected citrus leaves through rapid lysis with alkaline lysis buffer and a 50-fold dilution with TE buffer can be detected in a paper-based Toehold Switch coupled LAMP system, resulting in a purple color change in the positive group.

[0152] The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. The above embodiments only illustrate several implementation methods of the present invention to facilitate a specific and detailed understanding of the technical solution of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A cell-free protein expression system, characterized in that, include: Escherichia coli cell extract 35-50 vol%, lysis buffer 40-50 vol%, gene template 8-12 vol%, nuclease-free water 2-15 vol%; Wherein, the *E. coli* is a recombinant bacterium obtained by modifying the recipient *E. coli*, and the modification includes: (1) Knock out the LacZ gene in the recipient Escherichia coli; (2) Add the LyseR gene to the recipient Escherichia coli.

2. The cell-free protein expression system of claim 1, wherein, The modifications also include: (3) Add the GamS gene to the recipient Escherichia coli, wherein the GamS gene uses the AmpR promoter.

3. The cell-free protein expression system of claim 1, wherein, The modifications also include: (4) Knock out the RecBCD operon gene and ptrA gene of the recipient Escherichia coli and insert the LyseR gene at the knockout site.

4. The cell-free protein expression system of claim 3, wherein, The RecBCD operon gene and ptrA gene of the *E. coli* were knocked out using a CRISPR / Cas combined with λ-red dual plasmid gene editing system, and the LyseR gene was inserted at the knockout site. The plasmids used include pUC19-LyseR plasmid and pCas12a-λred plasmid; the nucleotide sequence of the pUC19-LyseR plasmid is shown in SEQ ID NO: 7, and the nucleotide sequence of the pCas12a-λred plasmid is shown in SEQ ID NO:

8. The gRNAs used include gRNA-Fw and gRNA-Rev, whose nucleotide sequences are shown in SEQ ID NO: 9-10, respectively; The homologous arms used include TYB-Fw and TYB-Rev, whose nucleotide sequences are shown in SEQ ID NO: 13-14, respectively.

5. The cell-free protein expression system of claim 1, wherein, The lysis buffer comprises the following components at the following concentrations: HEPES 40–60 mM, ATP 1–2 mM, GTP 1–2 mM, CTP 0.5–1.5 mM, UTP 0.5–1.5 mM, tRNA 0.1–0.5 mg / mL, CoA 0.1–0.5 mM, NAD 0.2–0.8 mM, cAMP 0.5–1 mM, leucovorin 0.01–0.1 mM, spermidine 0.5–1.5 mM, 3-PGA 10–50 mM, maltose 10–20 mM, PEG-8000 8–15 mg / mL, potassium glutamate 55–80 mM, magnesium glutamate 0.5–1.5 mM, DTT 0.5–1.5 mM, amino acids 20–25 mM.

6. The cell-free protein expression system of claim 1 or 5, wherein, include: Escherichia coli cell extract 40 vol%, lysis buffer 44.5 vol%, RNase inhibitor 0.5 vol%, gene template 10 vol%, nuclease-free water 5 vol%. The lysis solution comprises components at the following concentrations: HEPES 50mM, ATP 1.5mM, GTP 1.5mM, CTP 0.9mM, UTP 0.9mM, tRNA 0.2mg / mL, CoA 0.26mM, NAD 0.33mM, cAMP 0.75mM, leucovorin 0.0678mM, spermidine 1mM, 3-PGA 30mM, maltose 12mM, PEG-8000 11.2mg / mL, potassium glutamate 60.6mM, magnesium glutamate 0.815mM, DTT 1mM, Leucine 20mM, Glycine 24mM, Valine 24mM, Isoleucine 24mM, Methionine 24mM, Proline 24mM, Phenylalanine 24mM, Tyrosine 24mM, Serine 24mM, Threonine 24mM, Tryptophan 24mM, Asparagine 24mM, Glutamine 24mM, Lysine 24mM, Cysteine ​​24mM, Arginine 24mM, Aspartic Acid 24mM, Histidine 24mM, Glutamic Acid 24mM, Alanine 24mM.

7. The use of the cell-free protein expression system according to any one of claims 1 to 6 in the preparation of a kit for detecting the pathogen of citrus Huanglongbing.

8. A kit for detecting Candidatus Liberibacter asiaticum, characterized by, Includes the cell-free protein expression system and the Toehold Switch element as described in any one of claims 1 to 6; The nucleotide sequence of the Toehold Switch element is shown in SEQ ID NO:

15.

9. The kit for detecting citrus huanglongbing pathogen according to claim 8, wherein, It also includes LAMP primers for amplifying a Trigger RNA sequence complementary to the Toehold sequence in the ToeholdSwitch element, the nucleotide sequence of which is shown in SEQ ID NO: 16; the LAMP primers include L966-F3, L966-B3, L966-BIP, and L966-FIP, the nucleotide sequences of which are shown in SEQ ID NO: 17-20, respectively; and / or The cell-free protein expression system and the Toehold Switch element were freeze-dried on a paper-based material at a freeze-drying temperature of -60°C to -40°C.

10. A method for detecting the pathogen of citrus Huanglongbing (HLB) for non-diagnostic purposes, characterized in that, include: The genome of the sample to be tested is extracted and used as a gene template; The gene template was amplified using LAMP primers, wherein the LAMP primers included L966-F3, L966-B3, L966-BIP, and L966-FIP, and their nucleotide sequences are shown in SEQ ID NO: 17-20, respectively. The amplified products were reacted using a reaction system consisting of the cell-free protein expression system and the ToeholdSwitch element as described in any one of claims 1 to 6; and the results were determined based on the color change.

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