Preparation method of high-purity TIR2 and T1R3 recombinant plasmids
High-purity T1R2/T1R3 recombinant plasmids were prepared by combining thermal shock and alkaline lysis with column purification technology, which solved the problems of low plasmid preparation efficiency and low purity in the existing technology, and realized efficient and stable sweet taste perception research.
Patent Information
- Application Number
- CN202511177898.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-21
- Publication Date
- 2025-11-14
AI Technical Summary
In existing technologies, plasmid preparation is inefficient, has low purity, unstable transfection effect, and complex process, making it difficult to meet the high standards required for sweetness perception research.
The T1R2 and T1R3 recombinant plasmids were transformed into modified Tran1-T1 Escherichia coli competent cells using the heat shock method. The cells were then amplified using their high-efficiency cloning characteristics, and the recombinant plasmids were extracted and purified using an optimized alkaline lysis combined with column purification technique.
It significantly improved the yield and purity of T1R2/T1R3 recombinant plasmids, meeting the requirements of transfection experiments, ensuring consistent expression of receptor proteins, simplifying the operation process, and improving the sensitivity and stability of experiments.
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Figure CN120944928A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological cell technology, and more specifically, to a method for preparing high-purity TIR2 and T1R3 recombinant plasmids. Background Technology
[0002] Sweetness, as a core element of basic human taste perception, plays a crucial guiding role in dietary choices. While traditional sweeteners (such as sucrose and fructose) provide energy, excessive intake is significantly positively correlated with the occurrence of metabolic diseases such as obesity and type II diabetes. The inappropriate use of sweeteners is a significant contributing factor to overweight. Therefore, there is a need to develop novel, low-calorie sweetener alternatives.
[0003] At the molecular level, sweetness perception relies on the T1R2 / T1R3 heterodimer receptor—a specific molecular sensor belonging to the Class C G protein-coupled receptor (Class C GPCRs) family. The receptor's unique structural features include: an extracellular N-terminal domain (ATD) responsible for ligand recognition and binding; a transmembrane domain (TMD) mediating signal transduction; and an intracellular circular structure involved in G protein coupling.
[0004] In existing technologies, sweet taste receptor cell models are typically constructed in vitro for sweet taste perception evaluation and mechanism analysis. However, the following key bottlenecks are currently faced: i) Low plasmid preparation efficiency: Traditional methods cannot simultaneously guarantee the yield and purity of T1R2 / T1R3 recombinant plasmids; ii) Unstable transfection effect: Fluctuations in receptor expression levels lead to differences in detection sensitivity; iii) Complex process: Multi-step operations introduce uncontrollable variables.
[0005] Therefore, there is an urgent need for a method to prepare high-purity TIR2 and T1R3 recombinant plasmids. Summary of the Invention
[0006] The purpose of this invention is to provide a method for preparing high-purity TIR2 and T1R3 recombinant plasmids to solve the problems in the prior art. By improving the transformation, amplification and purification processes, the yield and purity of T1R2 / T1R3 recombinant plasmids can be significantly improved, meeting the high standard requirements of transfection experiments.
[0007] This invention provides a method for preparing high-purity TIR2 and T1R3 recombinant plasmids, comprising:
[0008] The T1R2 and T1R3 recombinant plasmids were transformed into modified Tran1-T1 Escherichia coli competent cells by heat shock.
[0009] Large-scale amplification of plasmids was carried out using the high-efficiency cloning characteristics of Tran1-T1 Escherichia coli.
[0010] Recombinant plasmids were extracted and purified from bacterial cells using an optimized alkaline lysis combined with column purification technique.
[0011] The method for preparing high-purity TIR2 and T1R3 recombinant plasmids as described above, preferably, involves transforming the T1R2 and T1R3 recombinant plasmids into modified Tran1-T1 Escherichia coli competent cells via heat shock, comprising:
[0012] Obtain the target recombinant plasmids T1R2 and T1R3;
[0013] Take 50 μL of frozen Tran1-T1 competent cells and thaw them on ice;
[0014] Add 1 μL of the target plasmid to Tran1-T1 competent cells, incubate on ice for 30 min, heat shock in a 42℃ water bath for 30 s, and immediately incubate on ice for 2 min.
[0015] Add 700 μL of preheated antibiotic-free LB medium and mix well;
[0016] The cells were cultured at 37°C and 200 rpm for 1 h with shaking to restore cell activity.
[0017] The method for preparing high-purity TIR2 and T1R3 recombinant plasmids as described above, preferably, involves large-scale amplification of the plasmids utilizing the high-efficiency cloning characteristics of Tran1-T1 Escherichia coli, including:
[0018] The transformed bacterial culture was spread on LB agar plates containing 100 μg / mL ampicillin and incubated upside down at 37°C for 12–16 h.
[0019] Pick a single colony and inoculate it into 3 mL of antibiotic-free LB medium, and incubate at 37°C and 250 rpm for 12-14 h with shaking.
[0020] The method for preparing high-purity TIR2 and T1R3 recombinant plasmids as described above, preferably, involves extracting and purifying the recombinant plasmids from bacterial cells using an optimized alkaline lysis combined with column purification technique, comprising:
[0021] Centrifuge to collect 5 mL of bacterial cells, and discard the supernatant;
[0022] Add Buffer P1, Buffer P2 and Buffer N3 sequentially to the bacterial cells after discarding the supernatant, and centrifuge to remove the precipitate;
[0023] Pass the supernatant through an adsorption column and collect the plasmid by centrifugation;
[0024] Wash twice with 700 μL of Buffer PW;
[0025] Elution of plasmids using 50 μL of elution buffer.
[0026] The method for preparing high-purity TIR2 and T1R3 recombinant plasmids as described above, preferably, involves centrifuging to collect 5 mL of bacterial cells and discarding the supernatant, comprising:
[0027] Take 5.0 mL of overnight cultured bacterial solution into a centrifuge tube, centrifuge at 12000-13000 rpm for 30-60 seconds, collect the bacteria, and discard the supernatant.
[0028] The method for preparing high-purity TIR2 and T1R3 recombinant plasmids as described above, preferably, involves sequentially adding Buffer P1, Buffer P2, and Buffer N3 to the bacterial cells after discarding the supernatant, followed by centrifugation to remove the precipitate, comprising:
[0029] Add 250 μL of Buffer P1 containing RNase A to the bacterial cells after discarding the supernatant, vortex to mix, and then transfer to a 1.5 mL centrifuge tube;
[0030] Add 250 μL of Buffer P2 to lyse, and gently invert and mix 6 times.
[0031] Add 350 μL of Buffer N3, immediately invert and mix 7 times, and centrifuge at 12,000 r / min for 10 min.
[0032] The method for preparing high-purity TIR2 and T1R3 recombinant plasmids as described above, preferably, involves passing the supernatant through an adsorption column and collecting the plasmids by centrifugation, comprising:
[0033] Take the supernatant and inject it into an EP tube containing an adsorption column. Centrifuge at 12,000 r / min for 1 min and discard the liquid.
[0034] The method for preparing high-purity TIR2 and T1R3 recombinant plasmids as described above, preferably, involves eluting plasmids using 50 μL of elution buffer, comprising:
[0035] The EP tube was evacuated for 2 minutes at a rotation speed of 12,000 r / min. The adsorption column was then transferred to a new EP tube, the EP tube cap was opened, and the tube was allowed to air at room temperature for 15 minutes.
[0036] Add 50 μL of elution buffer to the center of the membrane, let it stand at room temperature for 2 min, centrifuge at 12,000 r / min for 1 min, and then store at -20°C for later use.
[0037] The method for preparing high-purity TIR2 and T1R3 recombinant plasmids as described above, preferably, includes the extraction and purification of the recombinant plasmids from bacterial cells using an optimized alkaline lysis combined with column purification technique, further comprising:
[0038] The concentration and purity of the recombinant plasmid were determined using a nucleic acid concentration analyzer.
[0039] This invention provides a method for preparing high-purity TIR2 and T1R3 recombinant plasmids. Based on an optimized competent cell transformation system, the method involves introducing the target DNA fragment or its constructed recombinant DNA plasmid into competent cells. The genetic material is amplified using the host cell's replication and expression system. Finally, high-purity DNA plasmids are obtained through extraction and purification. By improving the transformation, amplification, and purification processes, the yield and purity of T1R2 / T1R3 recombinant plasmids are significantly increased, meeting the high standards required for transfection experiments. Its breakthrough advantages are: a highly efficient transformation system: by precisely controlling parameters such as heat shock and culture conditions, and optimizing heat shock conditions, the plasmid introduction efficiency is improved, resulting in a significant increase in transformation efficiency; and an intelligent purification process: using an optimized alkaline lysis binding column. The purification technique employs a specific buffer system, gradient centrifugation, and adsorption column to obtain a high proportion of T1R2 and T1R3 recombinant plasmids, reducing endotoxin and host DNA residues. Stable expression is ensured: plasmid purity (A260 / A280 = 1.98 / 2.01) meets transfection-grade standards, ensuring consistent expression of the receptor protein, and can be directly used for eukaryotic cell transfection experiments. This provides a key material basis for establishing a highly sensitive and stable sweetness evaluation cell model, significantly accelerating the development of novel sweeteners. The integrated transformation-amplification-purification technology established in this invention is simple to operate, highly reproducible, and yields high results. Plasmid yield and purity are significantly improved compared to traditional methods, providing crucial material support for downstream experiments such as gene function research and protein expression. Attached Figure Description
[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to the accompanying drawings, wherein:
[0041] Figure 1 A flowchart illustrating an embodiment of the method for preparing high-purity TIR2 and T1R3 recombinant plasmids provided by the present invention;
[0042] Figure 2 This is a diagram showing the transformation results of the recombinant plasmid.
[0043] Figure 3 The results are the sequencing results of the transformed T1R2 and T1R3 recombinant DNA. Detailed Implementation
[0044] Various exemplary embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings. The descriptions of the exemplary embodiments are merely illustrative and are in no way intended to limit the present disclosure or its application or use. The present disclosure may be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided so that the present disclosure will be thorough and complete, and will fully express the scope of the disclosure to those skilled in the art. It should be noted that, unless specifically stated otherwise, the relative arrangement of components and steps, the composition of materials, numerical expressions, and values set forth in these embodiments should be interpreted as exemplary only and not as limiting.
[0045] The terms “first,” “second,” and similar terms used in this disclosure do not indicate any order, quantity, or importance, but are merely used to distinguish different parts. Terms such as “including” or “contains” mean that the element preceding the term encompasses the element listed after it, and do not exclude the possibility of encompassing other elements as well. Terms such as “above” and “below” are used only to indicate relative positional relationships; when the absolute position of the described object changes, this relative positional relationship may also change accordingly.
[0046] In this disclosure, when a specific component is described as being located between a first component and a second component, an intermediary component may or may not be present between the specific component and the first or second component. When a specific component is described as connecting to other components, the specific component may be directly connected to the other components without having an intermediary component, or it may not be directly connected to the other components but may have an intermediary component.
[0047] All terms used in this disclosure (including technical or scientific terms) have the same meaning as understood by one of ordinary skill in the art to which this disclosure pertains, unless otherwise specifically defined. It should also be understood that terms defined in a general dictionary, such as a dictionary, should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and not as having an idealized or highly formalized meaning, unless expressly defined herein.
[0048] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, they should be considered part of the specification.
[0049] like Figure 1 and Figure 2 As shown, the method for preparing high-purity TIR2 and T1R3 recombinant plasmids provided in this embodiment includes the following steps in actual implementation:
[0050] Step S1: Transform the T1R2 and T1R3 recombinant plasmids into the modified Tran1-T1 Escherichia coli competent cells by heat shock.
[0051] In one embodiment of the method for preparing high-purity TIR2 and T1R3 recombinant plasmids of the present invention, step S1 may specifically include:
[0052] Step S11: Obtain the target recombinant plasmids T1R2 and T1R3.
[0053] Step S12: Take 50 μL of frozen Tran1-T1 competent cells and thaw them on ice.
[0054] Specifically, 50 μL of Tran1-T1 competent cells were taken from a -80°C freezer and thawed on ice.
[0055] Step S13: Add 1 μL of the target plasmid to Tran1-T1 competent cells, incubate on ice for 30 min, heat shock in a 42℃ water bath for 30 s, and immediately incubate on ice for 2 min.
[0056] Specifically, after the competent cells thaw, add 1 μL each of the constructed recombinant plasmids (pcDNA3-His-T1R2, pcDNA3-Flag-T1R3) or ligation products to the competent cells, gently stirring with a pipette tip or tapping the tube to mix. Incubate on ice for 30 min; then, heat shock at 42°C for 30 s, immediately followed by an ice bath for 2 min to improve transformation efficiency. Specifically, after heat shock at 42°C for 30 s, immediately place on ice for 2 min without shaking.
[0057] Step S14: Add 700 μL of preheated antibiotic-free LB medium and mix well.
[0058] Specifically, 700 μL of preheated antibiotic-free LB liquid medium was added to the Tran1-T1 competent cells obtained in step S13 and gently pipetted to mix.
[0059] Step S15: Incubate the cells at 37°C and 200 rpm for 1 h with shaking to restore cell activity.
[0060] Bacteria can be restored to their normal growth state through shaking culture.
[0061] Step S2: Utilize the high-efficiency cloning characteristics of Tran1-T1 Escherichia coli to perform large-scale amplification of plasmids.
[0062] In one embodiment of the method for preparing high-purity TIR2 and T1R3 recombinant plasmids of the present invention, step S2 may specifically include:
[0063] Step S21: Spread the transformed bacterial culture onto LB agar plates containing 100 μg / mL ampicillin and incubate upside down at 37°C for 12-16 h.
[0064] Specifically, a small amount of bacterial culture obtained in step S1 was spread onto an LB agar plate containing a final concentration of 100 μg / mL ampicillin. The plate was placed at 37°C until the liquid was absorbed. The plate was then inverted and incubated overnight at 37°C (12-16 h).
[0065] Step S22: Pick a single colony and inoculate it into 3 mL of antibiotic-free LB culture medium. Incubate at 37℃ and 250 rpm for 12-14 h with shaking to amplify the plasmid.
[0066] Specifically, pick one independent, round, and appropriately sized colony from each of the LB plates used for plating culture, inoculate it into 3 mL of antibiotic-free LB culture medium, and incubate at 37°C and 250 rpm for 12–14 h.
[0067] Step S3: Extract and purify recombinant plasmids from bacterial cells using optimized alkaline lysis combined with column purification technology.
[0068] In one embodiment of the method for preparing high-purity TIR2 and T1R3 recombinant plasmids of the present invention, step S3 may specifically include:
[0069] Step S31: Centrifuge to collect 5 mL of bacterial cells and discard the supernatant.
[0070] Specifically, take 5.0 mL of overnight cultured bacterial solution into a centrifuge tube, centrifuge at 12000-13000 rpm for 30-60 seconds, collect the bacteria, and discard the supernatant.
[0071] Step S32: Add Buffer P1 (to resuspend the cells), Buffer P2 (to lyse the cells), and Buffer N3 (to neutralize the lysis buffer) to the bacterial cells after discarding the supernatant, and centrifuge to remove the precipitate.
[0072] In one embodiment of the method for preparing high-purity TIR2 and T1R3 recombinant plasmids of the present invention, step S32 may specifically include:
[0073] Step S321: Add 250 μL of Buffer P1 containing RNase A to the bacterial cells after discarding the supernatant, vortex to mix, and then transfer to a 1.5 mL centrifuge tube.
[0074] Step S322: Add 250 μL of Buffer P2 for lysis, and gently invert and mix 6 times.
[0075] Step S323: Add 350 μL of Buffer N3, immediately invert and mix 7 times, and centrifuge at 12,000 r / min for 10 min.
[0076] Step S33: Pass the supernatant through an adsorption column and collect the plasmid by centrifugation.
[0077] Specifically, the supernatant was taken and injected into an EP tube containing an adsorption column, centrifuged at 12,000 r / min for 1 min, and the liquid was discarded.
[0078] Step S34: Wash twice with 700 μL of Buffer PW to remove impurities.
[0079] Step S35: Elute plasmids with 50 μL of elution buffer.
[0080] In one embodiment of the method for preparing high-purity TIR2 and T1R3 recombinant plasmids of the present invention, step S35 may specifically include:
[0081] Step S351: Isolate the EP tube for 2 min at a rotation speed of 12,000 r / min, transfer the adsorption column to a new EP tube, open the EP tube cap, and let it air dry at room temperature for 15 min.
[0082] Step S352: Add 50 μL of elution buffer to the center of the membrane, let it stand at room temperature for 2 min, centrifuge at 12,000 r / min for 1 min, and then store it at -20°C for later use.
[0083] Figure 2 The diagram shows the transformation results of the recombinant plasmid. Figure 3 The sequencing results of the transformed T1R2 and T1R3 recombinant DNA are shown.
[0084] Furthermore, in one embodiment of the method for preparing high-purity TIR2 and T1R3 recombinant plasmids of the present invention, step S3 may further include:
[0085] Step S36: Use a nucleic acid concentration analyzer to determine the concentration and purity of the recombinant plasmid.
[0086] The test results of the concentration and purity of T1R2 and T1R3 recombinant plasmids are shown in Table 1. The plasmid purity (A260 / A280=1.98 / 2.01) meets the transfection grade standard. Therefore, high-purity plasmids can be obtained by the method of the present invention.
[0087] Table 1. Concentration and purity of T1R2 and T1R3 recombinant plasmids
[0088]
[0089] The method for preparing high-purity TIR2 and T1R3 recombinant plasmids provided in this invention is based on an optimized competent cell transformation system. Specifically, it involves introducing the target DNA fragment or its constructed recombinant DNA plasmid into competent cells, utilizing the host cell's replication and expression system to amplify the genetic material, and finally extracting and purifying the high-purity DNA plasmid. By improving the transformation, amplification, and purification processes, the yield and purity of T1R2 / T1R3 recombinant plasmids are significantly improved, meeting the high standards required for transfection experiments. Its breakthrough advantages are reflected in: a highly efficient transformation system: by precisely controlling parameters such as heat shock and culture conditions, optimizing heat shock conditions, and improving plasmid introduction efficiency, the transformation efficiency is significantly enhanced; and an intelligent purification process: employing an optimized alkaline lysis combination... The column purification technology, employing a specific buffer system, gradient centrifugation, and adsorption column, achieves a high proportion of T1R2 and T1R3 recombinant plasmids, reducing endotoxin and host DNA residues. Stable expression is ensured: plasmid purity (A260 / A280 = 1.98 / 2.01) meets transfection-grade standards, ensuring consistent expression of the receptor protein, and can be directly used in eukaryotic cell transfection experiments. This provides a key material basis for establishing a highly sensitive and stable sweetness evaluation cell model, significantly accelerating the development of novel sweeteners. The integrated transformation-amplification-purification technology established in this invention is simple to operate, highly reproducible, and yields high output. Plasmid yield and purity are significantly improved compared to traditional methods, providing crucial material support for downstream experiments such as gene function research and protein expression.
[0090] The embodiments of this disclosure have now been described in detail. To avoid obscuring the concept of this disclosure, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.
[0091] While specific embodiments of this disclosure have been described in detail by way of examples, those skilled in the art should understand that the examples are for illustrative purposes only and not intended to limit the scope of this disclosure. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this disclosure. The scope of this disclosure is defined by the appended claims.
Claims
1. A method for preparing high-purity TIR2 and T1R3 recombinant plasmids, characterized in that, include: The T1R2 and T1R3 recombinant plasmids were transformed into modified Tran1-T1 Escherichia coli competent cells by heat shock. Large-scale amplification of plasmids was carried out using the high-efficiency cloning characteristics of Tran1-T1 Escherichia coli. Recombinant plasmids were extracted and purified from bacterial cells using an optimized alkaline lysis combined with column purification technique.
2. The method for preparing high-purity TIR2 and T1R3 recombinant plasmids according to claim 1, characterized in that, The process of transforming the T1R2 and T1R3 recombinant plasmids into modified Tran1-T1 E. coli competent cells via heat shock includes: Obtain the target recombinant plasmids T1R2 and T1R3; Take 50 μL of frozen Tran1-T1 competent cells and thaw them on ice; Add 1 μL of the target plasmid to Tran1-T1 competent cells, incubate on ice for 30 min, heat shock in a 42℃ water bath for 30 s, and immediately incubate on ice for 2 min. Add 700 μL of preheated antibiotic-free LB medium and mix well; The cells were cultured at 37°C and 200 rpm for 1 h with shaking to restore cell activity.
3. The method for preparing high-purity TIR2 and T1R3 recombinant plasmids according to claim 1, characterized in that, The large-scale amplification of plasmids utilizing the high-efficiency cloning characteristics of Tran1-T1 Escherichia coli includes: The transformed bacterial culture was spread on LB agar plates containing 100 μg / mL ampicillin and incubated upside down at 37°C for 12-16 h. Pick a single colony and inoculate it into 3 mL of antibiotic-free LB medium, and incubate at 37°C and 250 rpm for 12-14 h with shaking.
4. The method for preparing high-purity TIR2 and T1R3 recombinant plasmids according to claim 1, characterized in that, The method of extracting and purifying recombinant plasmids from bacterial cells using optimized alkaline lysis combined with column purification technology includes: Centrifuge to collect 5 mL of bacterial cells, and discard the supernatant; Add Buffer P1, Buffer P2 and Buffer N3 sequentially to the bacterial cells after discarding the supernatant, and centrifuge to remove the precipitate; Pass the supernatant through an adsorption column and collect the plasmid by centrifugation; Wash twice with 700 μL of Buffer PW; Elution of plasmids using 50 μL of elution buffer.
5. The method for preparing high-purity TIR2 and T1R3 recombinant plasmids according to claim 4, characterized in that, The centrifugation process collects 5 mL of bacterial cells, discarding the supernatant, including: Take 5.0 mL of overnight cultured bacterial solution into a centrifuge tube, centrifuge at 12000-13000 rpm for 30-60 seconds, collect the bacteria, and discard the supernatant.
6. The method for preparing high-purity TIR2 and T1R3 recombinant plasmids according to claim 4, characterized in that, The step of adding Buffer P1, Buffer P2, and Buffer N3 sequentially to the bacterial cells after discarding the supernatant, and centrifuging to remove the precipitate, includes: Add 250 μL of Buffer P1 containing RNase A to the bacterial cells after discarding the supernatant, vortex to mix, and then transfer to a 1.5 mL centrifuge tube; Add 250 μL of Buffer P2 to lyse, and gently invert and mix 6 times. Add 350 μL of Buffer N3, immediately invert and mix 7 times, and centrifuge at 12,000 r / min for 10 min.
7. The method for preparing high-purity TIR2 and T1R3 recombinant plasmids according to claim 4, characterized in that, The supernatant is passed through an adsorption column and centrifuged to collect the plasmid, including: Take the supernatant and inject it into an EP tube containing an adsorption column. Centrifuge at 12,000 r / min for 1 min and discard the liquid.
8. The method for preparing high-purity TIR2 and T1R3 recombinant plasmids according to claim 4, characterized in that, The eluted plasmids using 50 μL of elution buffer comprise: The EP tube was evacuated for 2 minutes at a rotation speed of 12,000 r / min. The adsorption column was then transferred to a new EP tube, the EP tube cap was opened, and the tube was allowed to air at room temperature for 15 minutes. Add 50 μL of elution buffer to the center of the membrane, let it stand at room temperature for 2 min, centrifuge at 12,000 r / min for 1 min, and then store at -20°C for later use.
9. The method for preparing high-purity TIR2 and T1R3 recombinant plasmids according to claim 4, characterized in that, The method of extracting and purifying recombinant plasmids from bacterial cells using optimized alkaline lysis combined with column purification technology also includes: The concentration and purity of the recombinant plasmid were determined using a nucleic acid concentration analyzer.