Construction and activity detection method of semisynthetic chassis cell for genetic material expansion based on vibrio natrieus

Abstract

The invention discloses construction and activity detection methods of semisynthetic chassis cells for genetic material expansion based on vibrio natrieti. The VnDX / pET23b-PtNTT2 (66-575) cell has a relatively wide nucleoside triphosphate transport active substrate spectrum, and has recognition and transport activities on natural nucleoside triphosphate (dNTPs and NTPs), pentose modified nucleoside triphosphate (2 '-OMe-NTPs and 2'-F-NTPs), and nucleoside triphosphate (dTPT3TP, dNaMTP, dCNMOTP, TPT3TP and NaMTP) containing non-natural basic groups, and the VnDX / pET23b-PtNTT2 (66-575) cell has the recognition and transport activities on the natural nucleoside triphosphate (dNTPs and NTPs) and the pentose modified nucleoside triphosphate (2 '-OMe-NTPs and 2'-F-NTPs). By means of the transport characteristic of the nucleoside triphosphate transporter PtNTT2 (66-575) and by detecting the content of ATP transported out of cells within the same time, the relative strength of the transport activity of various nucleoside triphosphate of the PtNTT2 (66-575) is simply, conveniently and rapidly determined.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology, and specifically relates to the construction of a cell based on Vibrio natriuresis VnDX capable of transporting non-natural nucleoside triphosphates mediated by a truncated mutant strain of nucleoside triphosphate transporter PtNTT2 (66-575), as a new chassis cell for realizing the expansion of the genetic alphabet of organisms and the types of genetic information carriers. Background Art

[0002] Natural nucleic acids DNA and RNA are the carriers of all life genetic information and the basis for the storage, use and transmission of genetic information in all cells. They have rich structures, properties and functions, making them widely used in biotechnology and biomedicine. However, the limited number of bases in natural nucleic acids greatly limits the genetic information capacity and chemical diversity of DNA and RNA. At the same time, the poor biochemical stability of DNA and RNA has great limitations in their application. In recent years, many DNA and RNA analogs have been developed, which not only retain the ability of bases to encode genetic information, but also have richer sequences and structural units and better physical and chemical properties, and provide a material basis for new semi-synthetic artificial life. These molecules are collectively referred to as non-natural nucleic acids. The development of non-natural nucleic acids mainly includes the substitution of a molecule on the base, pentose and phosphate group or the replacement of the entire group. Among them, the most widely studied non-natural nucleic acids XNA produced by the substitution or modification of pentose, such as: 2'-F-DNA, 2'-OMe-DNA, FANA, TNA, LNA, etc. XNA is widely used in the development of nucleic acid aptamers and biomaterials with stronger biological stability. In addition, the development of non-natural base pairs is also an important research direction of non-natural nucleic acids. Benner et al. developed non-natural base pairs based on hydrogen bond rearrangement, including isoG-isoC, VJ, KX, ZP, and SB. Romesberg et al. developed non-natural base pairs based on hydrophobic interaction and base stacking force, including MMO2-5SICS, NaM-5SICS, NaM-TPT3, TPT3-CNMO, PTMO-TPT3, CNMO-TPT3, and NaM-TAT1. Hirao et al. developed non-natural base pairs based on hydrophobic interactions, such as Ds-Pa and Ds-Px, and non-natural base pairs based on hydrogen bonds and steric hindrance, such as xy, sy, and Q-Pa. Among them, only NaM-5SICS and NaM-TPT3 and their analogs have achieved replication, transcription, and translation in vivo.

[0003] In 2014, Romesberg et al. constructed the first hexabasic semisynthetic organism containing non-natural base pairs that can stably proliferate. This semisynthetic organism is an artificial semisynthetic organism that can stably replicate and inherit the non-natural base pair d5SICS-dNaM based on Escherichia coli. The replication and maintenance of non-natural base pairs in this semisynthetic organism depend on the heterologous expression and function of the nucleoside triphosphate transporter PtNTT2. The nucleoside triphosphate transporter PtNTT2 is derived from Phaeodactylum tricornutum and is a membrane protein. Its function is to transport nucleoside triphosphates across the cell membrane, providing the semisynthetic organism with non-natural nucleoside triphosphates required for the replication and maintenance of non-natural base pairs, and at the same time releasing adenosine triphosphate (ATP) across the membrane. Its transport activity is usually detected using isotope-labeled nucleoside triphosphates. This semi-synthetic organism containing non-natural base pairs that are orthogonally paired with natural base pairs provides an important platform for synthetic biology to expand genetic letters and codes, and can be used for site-specific labeling of nucleic acids in living cells and for the production and evolution of proteins containing a variety of non-natural amino acids. At the same time, the transporter PtNTT2 heterologously expressed in Escherichia coli has also been shown to be able to transport nucleoside triphosphates containing non-natural pentose sugars into Escherichia coli cells, which lays the foundation for the synthesis and function of new genetic information carriers in living cells.

[0004] Vibrio natriegens is a Gram-negative bacterium and the fastest growing non-pathogenic bacterium known so far. Under optimal growth conditions, its generation time is less than 10 minutes. In contrast, the generation time of Escherichia coli and most bacteria is 20 minutes or even longer. This means that the protein synthesis rate of Vibrio natriegens is also relatively high, so Vibrio natriegens has great potential as a chassis cell for genetic engineering and a host for the expression of foreign genes. At present, the T7 RNA polymerase gene is integrated into the genome of Vibrio natriegens through genetic engineering to obtain a genetically engineered strain of Vibrio natriegens VnDX carrying a T7 RNA polymerase expression cassette, realizing the regulatory role of the T7 promoter in VnDX, enabling Vibrio natriegens to be directly applied to the mature pET-T7 foreign gene expression system developed in Escherichia coli, and realizing the controllable and efficient expression of foreign genes in VnDX.

[0005] In summary, heterologous expression of the nucleoside triphosphate transporter PtNTT2 in Vibrio natriuresis VnDX and exploration of its transport activity for various non-natural nucleoside triphosphates is expected to establish a new type of semi-synthetic life chassis cell that can be used to efficiently expand the genetic alphabet and the types of genetic information carriers, which is of great significance for the efficient expression of proteins containing a variety of non-natural amino acids and the efficient production of non-natural nucleic acid molecules. Summary of the invention

[0006] The primary purpose of the present invention is to develop a new type of high-quality chassis cell for semi-synthetic life construction.

[0007] Another object of the present invention is to provide a method for detecting the non-natural nucleoside triphosphate transport activity in the semi-synthetic life form Vibrio natriuresis VnDx chassis cells.

[0008] The construction of chassis cells in the present invention is achieved by the following steps:

[0009] (1) The extracted pET23b(+) plasmid was treated with restriction endonucleases Nde I and Xho I to obtain a cut vector.

[0010] (2) The transport protein PtNTT2 (66-575) was obtained by PCR amplification, and restriction sites Nde I and Xho I were introduced at both ends to obtain the inserted fragment after restriction digestion.

[0011] (3) The insert fragment obtained in step (2) and the cut vector obtained in step (1) were mixed in a ratio of 3:1, and the ligation product pET23b-PtNTT2 (66-575) was obtained under the action of T4 DNA polymerase.

[0012] (4) The ligation product obtained in step (3) is transformed into VnDX cells by electroporation to obtain VnDX / pET23b-PtNTT2(66-575) cells.

[0013] Preferably, the VnDX novel chassis cells use the pET-T7 exogenous gene expression system matured in Escherichia coli.

[0014] Preferably, the exogenous gene vector is transformed into VnDX cells by electroporation.

[0015] The transport activity determination of non-natural nucleoside triphosphates in the present invention is achieved by the following technical scheme:

[0016] ATP detection reagent was used to detect the ATP content transported out of the cell of VnDx by the transporter, and the activity of the nucleoside triphosphate transporter was indirectly determined.

[0017] (1) Pick the constructed VnDX / pET23b-PtNTT2(66-575) monoclonal colony and culture it in liquid culture medium until OD 600 = about 0.4, and then induced with IPTG at a final concentration of 1 mM for 1 hour to obtain induced expression cells.

[0018] (2) replacing the culture medium of the induced expression cells with fresh culture medium to eliminate the influence of metabolites on the detection, and diluting the induced expression cells obtained in step (1) to obtain a cell resuspension.

[0019] (3) The resuspension obtained in step (2) is transferred to a 96-well plate with a black bottom, and the nucleoside triphosphate substrate to be tested is added. After 30 minutes of transfer, ATP detection solution is added for detection.

[0020] Preferably, the final concentration of the transported nucleoside triphosphate is 10-500 μM, and the transport time is 10-60 minutes.

[0021] Preferably, the total detection volume is 80-200 μL, the volume of bacterial solution and nucleoside triphosphate substrate is 75-190 μL, and 2-10 μL ATP detection solution is added after the transfer is completed.

[0022] Preferably, the transport time of nucleoside triphosphates in the bacterial solution is 10-60 minutes, and after the transport is completed, ATP detection solution is added to react for 2-10 minutes.

[0023] A VnDX cell capable of transporting various natural or non-natural nucleoside triphosphates mediated by the expression of an exogenous gene PtNTT2 (66-575), having a broad substrate spectrum for the transport of various nucleoside triphosphates and significant transport activity;

[0024] The transport activity of the transporter PtNTT2(66-575) for natural deoxyribonucleoside triphosphate substrates in VnDX cells includes:

[0025] (1) The transporter PtNTT2 (66-575) has dATP transport activity in VnDX, and the transport activity is strong even without induced expression;

[0026] (2) The transport protein PtNTT2 (66-575) has transport activity for dTTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression;

[0027] (3) The transporter PtNTT2 (66-575) has transport activity for dCTP in VnDX, and the transport activity is already strong even without induced expression;

[0028] (4) The transporter protein PtNTT2 (66-575) has dGTP transport activity in VnDX, and the transport activity is more significant after induced expression.

[0029] The transport activity of the transporter PtNTT2(66-575) for natural ribonucleoside triphosphate substrates in VnDX cells includes:

[0030] (1) The transport protein PtNTT2 (66-575) has UTP transport activity in VnDX, and the transport activity is more significant after induced expression;

[0031] (2) The transport protein PtNTT2 (66-575) has transport activity for CTP in VnDX, and the transport activity is more significant after induced expression;

[0032] (3) The transporter PtNTT2 (66-575) has GTP transport activity in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression.

[0033] The transport activity of the transporter PtNTT2(66-575) for 2'-OMe-modified ribonucleoside triphosphate substrates in VnDX cells includes:

[0034] (1) The transporter PtNTT2 (66-575) has transport activity for 2'-OMe-ATP in VnDX, and the uninduced transport activity is already strong;

[0035] (2) The transport protein PtNTT2 (66-575) has transport activity for 2'-OMe-UTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression;

[0036] (3) The transport protein PtNTT2 (66-575) has transport activity for 2'-OMe-CTP in VnDX, and the transport activity is more significant after induced expression;

[0037] (4) The transporter protein PtNTT2 (66-575) has transport activity for 2'-OMe-GTP in VnDX, and the transport activity is more significant after induced expression.

[0038] The transport activity of the transporter PtNTT2(66-575) for 2'-F modified ribonucleoside triphosphate substrates in VnDX cells includes:

[0039] (1) The transport protein PtNTT2 (66-575) has transport activity for 2'-F-UTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression;

[0040] (2) The transport protein PtNTT2 (66-575) has transport activity for 2'-F-CTP in VnDX, and the transport activity is more significant after induced expression;

[0041] (3) The transporter protein PtNTT2(66-575) has transport activity for 2'-F-GTP in VnDX, and the transport activity is more significant after induced expression.

[0042] The transport activity of the transporter PtNTT2 (66-575) for deoxyribonucleoside triphosphate substrates containing unnatural bases in VnDX cells includes:

[0043] (1) The transport protein PtNTT2 (66-575) has transport activity for dTPT3TP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression;

[0044] (2) The transport protein PtNTT2 (66-575) has transport activity for dNaMTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression;

[0045] (3) The transporter PtNTT2(66-575) has transport activity for dCNMOTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression.

[0046] The transport activity of the transporter PtNTT2(66-575) for ribonucleoside triphosphate substrates containing unnatural bases in VnDX cells includes:

[0047] (1) The transport protein PtNTT2 (66-575) has transport activity for TPT3TP in VnDX, and the transport activity is already strong even without induced expression;

[0048] (2) The transporter PtNTT2 (66-575) has transport activity for NaMTP in VnDX, and the transport activity is already strong even without induced expression.

[0049] Compared with the prior art, the present invention has the following advantages and effects:

[0050] (1) The present invention obtains a novel, high-quality chassis cell of semi-synthetic artificial life based on Vibrio natriuresis VnDX.

[0051] (2) The present invention can rapidly, conveniently and at low cost detect the strength of the activity of nucleoside triphosphate transporters in the chassis cells in transporting various nucleoside triphosphates by an indirect determination method.

[0052] (3) The present invention uses a simple and easily available ATP detection kit to replace the original detection method of using radioactive elements to detect the activity of nucleoside triphosphate transporters, which is safer, more efficient and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is the molecular structure of rNTPs, dNTPs, 2'-F-NTPs, and 2'-OMe-NTPs.

[0054] Figure 2 It is the molecular structure of nucleoside triphosphates containing unnatural bases.

[0055] Figure 3 This is a schematic diagram of the nucleoside triphosphate transporter PtNTT2 (66-575) transporting various nucleoside triphosphate substrates from the extracellular space into the cell while releasing ATP to the extracellular space.

[0056] Figure 4 These are the results of an activity test of the nucleoside triphosphate transporter PtNTT2 (66-575) in transporting various natural deoxyribonucleoside triphosphates.

[0057] Figure 5 These are the activity test results of the nucleoside triphosphate transporter PtNTT2 (66-575) in transporting various natural ribonucleoside triphosphates.

[0058] Figure 6 These are the results of an activity test of the nucleoside triphosphate transporter PtNTT2(66-575) in transporting various 2'-OMe-modified nucleoside triphosphates.

[0059] Figure 7 These are the results of an activity test of the nucleoside triphosphate transporter PtNTT2 (66-575) in transporting various 2'-F modified nucleoside triphosphates.

[0060] Figure 8 These are the results of an activity test of the nucleoside triphosphate transporter PtNTT2 (66-575) in transporting deoxyribonucleoside triphosphates (dUBTP) containing various unnatural bases.

[0061] Fig. 9 These are the results of an activity test of the nucleoside triphosphate transporter PtNTT2 (66-575) in transporting ribonucleoside triphosphates (UBTPs) containing various unnatural bases. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. It should be noted that if there are any details not particularly described below, they can be realized or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, it is regarded as a conventional product that can be purchased commercially.

[0063] The natural ribonucleoside triphosphates and natural deoxyribonucleoside triphosphates used in the following examples were purchased from Shanghai Bioengineering Co., Ltd.; the 2'-modified ribonucleoside triphosphates used were purchased from Wuhu Huaren Technology Co., Ltd.; the ribonucleoside triphosphates containing non-natural bases dTPT3TP, dNaMTP, dCNMOTP, NaMTP, and TPT3TP used were purchased from Wuxi AppTec Pharmaceuticals Co., Ltd.; the VnDX strain used was donated by Yang Sheng's research group at the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences.

[0064] Example 1. Construction of the expression strain of the transport protein PtNTT2 (66-575)

[0065] The strain used in this example is a modified strain VnDX of Vibrio natrii, which was donated by the Yang Sheng research group of the Center for Excellence in Molecular Plant Sciences of the Chinese Academy of Sciences. The plasmid used is pET23b(+) which can be obtained through conventional commercial channels. The gene fragment of PtNTT2 cloned on the pLysS plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0066] Using the gene of PtNTT2 as a template, a mutant strain of PtNTT2 (66-575) was constructed, retaining the sequence of codon 66 to codon 575. Specifically, the primer sequences used to amplify the PtNTT2 (66-575) gene are shown in Table 1, and the specific steps are as follows:

[0067] (1) Using PtNTT2 as template and high-fidelity enzyme PtNTT2 (66-575) was amplified by HSDNA polymerase. The reaction system was shown in Table 2 and the PCR program was shown in Table 3. After the PCR reaction, an agarose gel DNA recovery kit (Guangzhou Meiji Biotechnology Co., Ltd.) was used to cut the gel and recover the DNA fragment to obtain the gene fragment of PtNTT2 (66-575).

[0068] (2) The DNA fragment of PtNTT2(66-575) and the pET23b(+) plasmid were double-digested with restriction endonucleases Nde I and Xho I. The enzyme digestion system is shown in Table 4. After incubation at 37°C for 3 hours, the DNA fragment and the linearized pET23b(+) plasmid were respectively recovered using an agarose gel DNA recovery kit.

[0069] The PtNTT2 (66-575) gene fragment with the same sticky ends and the linearized plasmid pET23b (+) were ligated at 16°C for 12 hours using T4 DNA ligase. The reaction system is shown in Table 5. The ligated product was purified using an agarose gel DNA recovery kit to obtain pET23b-PtNTT2 (66-575).

[0070] (3) Preparation of VnDX competent cells: Pick a single VnDX colony and place it in 2×YT3 medium (16% peptone, 10% yeast extract powder, 15% sodium chloride) at 37°C. Shake at 220 rpm until OD 600 =0.4-0.6, incubate the culture liquid on ice for more than 30 minutes, collect the cells by centrifugation at 4°C and 6000rpm, discard the supernatant and resuspend the cells with 680mM sucrose solution, repeat this step twice, and finally resuspend the cells with 680mM sucrose solution to OD 600 =1.8-2.5.

[0071] (4) The obtained pET23b-PtNTT2 (66-575) enzyme-linked product was transformed into VnDX competent cells by electroporation (electroporation conditions were 1.8 kV, 3 milliseconds) to obtain a monoclonal strain. Colony PCR and sequencing were performed on the monoclonal strain to verify its correctness. The PCR amplification system is shown in Table 6, and the strain VnDX / pET23b-PtNTT2 (66-575) was finally obtained. The protein sequence of PtNTT2 (66-575) is shown in SEQ ID NO.1, and the gene sequence of PtNTT2 (66-575) is shown in SEQ ID NO.2.

[0072] Table 1. Primer sequences for PCR amplification of PtNTT2 (66-575)

[0073]

[0074] Table 2. PCR reaction system of PtNTT2 (66-575)

[0075]

[0076] Table 3. PCR reaction program for PtNTT2 (66-575)

[0077]

[0078] Table 4. Enzyme digestion system of PtNTT2 (66-575) and pET23b (+)

[0079]

[0080] Table 5. Enzyme ligation reaction system of PtNTT2 (66-575) and pET23b (+)

[0081]

[0082] Table 6. Colony PCR reaction system of VnDX / pET23b-PtNTT2 (66-575)

[0083]

[0084] Example 2. Detection of the transport activity of nucleoside triphosphate transporter PtNTT2 (66-575) for natural deoxyribonucleoside triphosphates in Vibrio natriuresis VnDX

[0085] A single colony of VnDX / pET23b-PtNTT2(66-575) was picked and inoculated into 20 mL of 2×YT3 medium (1.6% peptone, 1% yeast extract powder, 1.5% sodium chloride) containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and cultured at 37°C with shaking at 220 rpm until OD 600 = 0.4, the culture medium was divided into two tubes, each with 10 mL, one of which was used as cells for inducing the expression of the transport protein PtNTT2 (66-57) without adding IPTG (isopropylthiogalactoside); the other tube was added with IPTG at a final concentration of 1 mM, and cultured at 220 rpm for 1 hour at 37 ° C to induce the expression of the transport protein. The induced cells and the uninduced cells were washed, and the cells were collected by centrifugation at 4 ° C and 4600 rpm. After discarding the supernatant, the cells were resuspended in pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and the above steps were repeated twice. The last collected cells were resuspended in 50 mL of pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin for detection.

[0086] Four orthogonal experiments were set up for induction and addition of nucleoside triphosphate substrates. The components of each experimental group and the control group are shown in Table 7. dATP, dTTP, dCTP or dGTP with a final concentration of 100 μM and 97 μL of induced bacterial solution or control bacterial solution were added to a black 96-well ELISA plate, and the volume was made up to 100 μL. After standing at 37°C for 30 minutes, 2 μL of ATP detection liquid (Beyotime Biotechnology) was added to each well. The transport activity of the transporter to transport the target nucleoside triphosphate into the cell was detected by indirectly measuring the content of ATP transported out of the nucleoside triphosphate transporter to the extracellular space. After standing for 5 minutes, the chemiluminescence intensity of each group was read using an ELISA instrument.

[0087] Table 7. VnDX / pET23b-PtNTT2(66-575) bacterial solution transport dNTP system

[0088]

[0089] like Figure 4 As shown: Comparison of the chemiluminescence values ​​after 30 minutes of transport with dATP as the transport substrate revealed that the transport protein PtNTT2 (66-575) has transport activity for dATP in VnDX, and the strain without induced expression also has a higher chemiluminescence intensity, which should be due to the background expression of the transport protein. Comparison of the chemiluminescence values ​​after 30 minutes of transport with dTTP as the transport substrate revealed that the transport protein PtNTT2 (66-575) has transport activity for dTTP in VnDX, and the transport activity after induced expression is slightly more significant than the transport activity without induced expression. The results after 30 minutes of transport with dCTP as the transport substrate showed that the transport protein PtNTT2 (66-575) has transport activity for dCTP in VnDX, and the strain without induced expression also has a higher chemiluminescence intensity, which should be due to the background expression of the transport protein. The results after 30 minutes of transport with dGTP as the transport substrate showed that the transport protein PtNTT2 (66-575) had transport activity for dGTP in VnDX, and the transport activity of induced expression was more significant.

[0090] Example 3. Detection of the transport activity of nucleoside triphosphate transporter PtNTT2 (66-575) for natural ribonucleoside triphosphates in Vibrio natriuresis VnDX

[0091] A single colony of VnDX / pET23b-PtNTT2(66-575) was picked and inoculated into 20 mL of 2×YT3 medium (1.6% peptone, 1% yeast extract powder, 1.5% sodium chloride) containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and cultured at 37°C with shaking at 220 rpm until OD 600= 0.4, the culture medium was divided into two tubes, each with 10 mL, one of which was used as cells for inducing the expression of the transport protein PtNTT2 (66-57) without adding IPTG; the other tube was added with IPTG at a final concentration of 1 mM, and cultured at 220 rpm for 1 hour at 37 ° C to induce the expression of the transport protein. The induced cells and the uninduced cells were washed, and the cells were collected by centrifugation at 4 ° C and 4600 rpm. After discarding the supernatant, the cells were resuspended in pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and the above steps were repeated twice. The last collected cells were resuspended in 50 mL of pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin for detection.

[0092] Four orthogonal experiments were set up for induction and addition of nucleoside triphosphate substrates. The components of each experimental group and the control group are shown in Table 8. Since ATP itself is the reaction substrate of the detection solution, only UTP, CTP or GTP with a final concentration of 100 μM and 97 μL of induced bacterial solution or control bacterial solution were added to a black 96-well ELISA plate, and the volume was made up to 100 μL. After standing at 37°C for 30 minutes, 2 μL of ATP detection liquid was added to each well. The transport activity of the transporter to transport the target nucleoside triphosphate into the cell was detected by indirectly measuring the content of ATP transported out of the nucleoside triphosphate transporter to the extracellular space. After standing for 5 minutes, the chemiluminescence intensity of each group was read using an ELISA instrument.

[0093] Table 8. VnDX / pET23b-PtNTT2(66-575) bacterial liquid transport NTP system

[0094]

[0095] like Figure 5 As shown: Comparison of the chemiluminescence values ​​after 30 minutes of transport with UTP as the transport substrate revealed that the transport protein PtNTT2 (66-575) has transport activity for UTP in VnDX, and the transport activity is more significant after induced expression. Comparison of the chemiluminescence values ​​after 30 minutes of transport with CTP as the transport substrate revealed that the transport protein PtNTT2 (66-575) has transport activity for CTP in VnDX, and the transport activity is more significant after induced expression. The results after 30 minutes of transport with GTP as the transport substrate showed that the transport protein PtNTT2 (66-575) has transport activity for GTP in VnDX, and the transport activity after induced expression is slightly more significant than the transport activity without induced expression.

[0096] Example 4. Detection of the transport activity of nucleoside triphosphate transporter PtNTT2 (66-575) for 2'-OMe-modified ribonucleoside triphosphate in Vibrio natriuresis VnDX

[0097] A single colony of VnDX / pET23b-PtNTT2(66-575) was picked and inoculated into 20 mL of 2×YT3 medium (1.6% peptone, 1% yeast extract powder, 1.5% sodium chloride) containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and cultured at 37°C with shaking at 220 rpm until OD 600 = 0.4, the culture medium was divided into two tubes, each with 10 mL, one of which was used as cells for inducing the expression of the transport protein PtNTT2 (66-57) without adding IPTG; the other tube was added with IPTG at a final concentration of 1 mM, and cultured at 220 rpm for 1 hour at 37 ° C to induce the expression of the transport protein. The induced cells and the uninduced cells were washed, and the cells were collected by centrifugation at 4 ° C and 4600 rpm. After discarding the supernatant, the cells were resuspended in pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and the above steps were repeated twice. The last collected cells were resuspended in 50 mL of pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin for detection.

[0098] Four orthogonal experiments were set up for whether to induce and whether to add nucleoside triphosphate substrates. The components of each experimental group and the control group are shown in Table 9. 2'-OMe-ATP, 2'-OMe-UTP, 2'-OMe-CTP or 2'-OMe-GTP with a final concentration of 100 μM and 97 μL of induced bacterial solution or control bacterial solution were added to a black 96-well ELISA plate, and the volume was made up to 100 μL. After standing at 37°C for 30 minutes, 2 μL of ATP detection liquid was added to each well. The transport activity of the transporter to transport the target nucleoside triphosphate into the cell was detected by indirectly measuring the content of ATP transported out of the nucleoside triphosphate transporter to the extracellular space. After standing for 5 minutes, the chemiluminescence intensity of each group was read using an ELISA instrument.

[0099] Table 9. VnDX / pET23b-PtNTT2(66-575) bacterial solution transport 2'-OMe-NTP system

[0100]

[0101]

[0102] like Figure 6As shown: Comparison of the chemiluminescence values ​​after 30 minutes of transport with 2'-OMe-ATP as the transport substrate revealed that the transport protein PtNTT2 (66-575) has transport activity for 2'-OMe-ATP in VnDX, and the strain without induced expression also has a high chemiluminescence intensity, which should be due to the background expression of the transport protein. Comparison of the chemiluminescence values ​​after 30 minutes of transport with 2'-OMe-UTP as the transport substrate revealed that the transport protein PtNTT2 (66-575) has transport activity for 2'-OMe-UTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression. The results after 30 minutes of transport with 2'-OMe-CTP as the transport substrate showed that the transport protein PtNTT2 (66-575) has transport activity for 2'-OMe-CTP in VnDX, and the transport activity of induced expression is more significant. The results after 30 minutes of transport with 2'-OMe-GTP as the transport substrate showed that the transport protein PtNTT2(66-575) had transport activity for 2'-OMe-GTP in VnDX, and the transport activity of induced expression was more significant.

[0103] Example 5. Detection of the transport activity of nucleoside triphosphate transporter PtNTT2 (66-575) for 2'-F modified ribonucleoside triphosphate in Vibrio natriuresis VnDX

[0104] A single colony of VnDX / pET23b-PtNTT2(66-575) was picked and inoculated into 20 mL of 2×YT3 medium (1.6% peptone, 1% yeast extract powder, 1.5% sodium chloride) containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and cultured at 37°C with shaking at 220 rpm until OD 600 = 0.4, the culture medium was divided into two tubes, each with 10 mL, one of which was used as cells for inducing the expression of the transport protein PtNTT2 (66-57) without adding IPTG; the other tube was added with IPTG at a final concentration of 1 mM, and cultured at 220 rpm for 1 hour at 37 ° C to induce the expression of the transport protein. The induced cells and the uninduced cells were washed, and the cells were collected by centrifugation at 4 ° C and 4600 rpm. After discarding the supernatant, the cells were resuspended in pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and the above steps were repeated twice. The last collected cells were resuspended in 50 mL of pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin for detection.

[0105] Four groups of orthogonal experiments were set up for induction and addition of nucleoside triphosphate substrates. The components of each experimental group and the control group are shown in Table 10. Since 2'-F-ATP is similar in structure to ATP itself, it was verified through preliminary experiments that the ATP detection solution 2'-F-ATP also had a strong reaction. Therefore, only 2'-F-UTP, 2'-F-CTP or 2'-F-GTP with a final concentration of 100 μM and 97 μL of induced bacterial solution or control group bacterial solution were added to a black 96-well ELISA plate, and the volume was made up to 100 μL. After standing at 37°C for 30 minutes, 2 μL of ATP detection liquid was added to each well. The transport activity of the transporter to transport the target nucleoside triphosphate into the cell was detected by indirectly measuring the content of ATP transported out of the nucleoside triphosphate transporter to the extracellular space. After standing for 5 minutes, the chemiluminescence intensity of each group was read using an ELISA instrument.

[0106] Table 10. VnDX / pET23b-PtNTT2(66-575) bacterial liquid transport 2'-F-NTP system

[0107]

[0108] like Figure 7 As shown: Comparison of the chemiluminescence values ​​after 30 minutes of transport with 2'-F-UTP as the transport substrate revealed that the transport protein PtNTT2 (66-575) has transport activity for 2'-F-UTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression. Comparison of the chemiluminescence values ​​after 30 minutes of transport with 2'-F-CTP as the transport substrate revealed that the transport protein PtNTT2 (66-575) has transport activity for 2'-F-CTP in VnDX, and the transport activity is more significant after induced expression. The results after 30 minutes of transport with 2'-F-GTP as the transport substrate showed that the transport protein PtNTT2 (66-575) has transport activity for 2'-F-GTP in VnDX, and the transport activity is more significant after induced expression.

[0109] Example 6. Detection of the transport activity of nucleoside triphosphate transporter PtNTT2 (66-575) for deoxyribonucleoside triphosphates containing non-natural bases in Vibrio natriuresis VnDX

[0110] A single colony of VnDX / pET23b-PtNTT2(66-575) was picked and inoculated into 20 mL of 2×YT3 medium (1.6% peptone, 1% yeast extract powder, 1.5% sodium chloride) containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and cultured at 37°C with shaking at 220 rpm until OD 600= 0.4, the culture medium was divided into two tubes, each with 10 mL, one of which was used as cells for inducing the expression of the transport protein PtNTT2 (66-57) without adding IPTG; the other tube was added with IPTG at a final concentration of 1 mM, and cultured at 220 rpm for 1 hour at 37 ° C to induce the expression of the transport protein. The induced cells and the uninduced cells were washed, and the cells were collected by centrifugation at 4 ° C and 4600 rpm. After discarding the supernatant, the cells were resuspended in pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and the above steps were repeated twice. The last collected cells were resuspended in 50 mL of pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin for detection.

[0111] Four orthogonal experiments were set up for induction and addition of nucleoside triphosphate substrates. The components of each experimental group and the control group are shown in Table 11. dTPT3TP, dNaMTP or dCNMOTP with a final concentration of 100 μM and 97 μL of induced bacterial solution or control bacterial solution were added to a black 96-well ELISA plate, and the volume was made up to 100 μL. After standing at 37°C for 30 minutes, 2 μL of ATP detection liquid was added to each well. The transport activity of the transporter to transport the target nucleoside triphosphate into the cell was detected by indirectly measuring the content of ATP transported out of the nucleoside triphosphate transporter to the extracellular space. After standing for 5 minutes, the chemiluminescence intensity of each group was scanned using an ELISA instrument.

[0112] Table 11. VnDX / pET23b-PtNTT2(66-575) bacterial liquid transport dUBTP* system

[0113]

[0114] *dUBTP means: dTPT3TP, dNaMTP or dCNMOTP

[0115] like Figure 8As shown: Comparison of the chemiluminescence values ​​after 30 minutes of transport with dTPT3TP as the transport substrate revealed that the transport protein PtNTT2 (66-575) has transport activity for dTPT3TP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression. Comparison of the chemiluminescence values ​​after 30 minutes of transport with dNaMTP as the transport substrate revealed that the transport protein PtNTT2 (66-575) has transport activity for dNaMTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression. The results after 30 minutes of transport with dCNMOTP as the transport substrate revealed that the transport protein PtNTT2 (66-575) has transport activity for dCNMOTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression.

[0116] Example 7. Detection of the transport activity of nucleoside triphosphate transporter PtNTT2 (66-575) for ribonucleoside triphosphates containing non-natural bases in Vibrio natriuresis VnDX

[0117] A single colony of VnDX / pET23b-PtNTT2(66-575) was picked and inoculated into 20 mL of 2×YT3 medium (1.6% peptone, 1% yeast extract powder, 1.5% sodium chloride) containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and cultured at 37°C with shaking at 220 rpm until OD 600 = 0.4, the culture medium was divided into two tubes, each with 10 mL, one of which was used as cells for inducing the expression of the transport protein PtNTT2 (66-57) without adding IPTG; the other tube was added with IPTG at a final concentration of 1 mM, and cultured at 220 rpm for 1 hour at 37 ° C to induce the expression of the transport protein. The induced cells and the uninduced cells were washed, and the cells were collected by centrifugation at 4 ° C and 4600 rpm. After discarding the supernatant, the cells were resuspended in pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin, and the above steps were repeated twice. The last collected cells were resuspended in 50 mL of pre-cooled 2×YT3 medium containing 50 μg / mL spectinomycin and 50 μg / mL carbenicillin for detection.

[0118] Four orthogonal experiments were set up for induction and addition of nucleoside triphosphate substrates. The components of each experimental group and the control group are shown in Table 12. TPT3TP or NaMTP with a final concentration of 100 μM and 97 μL of induced bacterial solution or control bacterial solution were added to a black 96-well ELISA plate, and the volume was made up to 100 μL. After standing at 37°C for 30 minutes, 2 μL of ATP detection liquid was added to each well. The transport activity of the transporter to transport the target nucleoside triphosphate into the cell was detected by indirectly measuring the content of ATP transported out of the cell by the nucleotide transporter. After standing for 5 minutes, the chemiluminescence intensity of each group was scanned using an ELISA instrument.

[0119] Table 12. VnDX / pET23b-PtNTT2(66-575) bacterial liquid transport UBTP* system

[0120]

[0121] *UBT means: TPT3TP or NaMTP.

[0122] like Fig. 9 As shown in the figure: By comparing the chemiluminescence values ​​after 30 minutes of transport with TPT3TP as the transport substrate, it was found that the transport protein PtNTT2 (66-575) has transport activity for TPT3TP in VnDX, and the strain without induced expression also has a higher chemiluminescence intensity, which should be due to the background expression of the transport protein. By comparing the chemiluminescence values ​​after 30 minutes of transport with NaMTP as the transport substrate, it was found that the transport protein PtNTT2 (66-575) has transport activity for NaMTP in VnDX, and the strain without induced expression also has a higher chemiluminescence intensity, which should be due to the background expression of the transport protein.

[0123] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for constructing a novel VnDX chassis cell capable of stably maintaining and expressing the exogenous gene PtNTT2 (66-575), characterized in that: Using the pET-T7 exogenous gene expression system, pET23b(+) was used as a vector to carry the exogenous gene PtNTT2(66-575), including the following steps: (1) using the pET23b(+) plasmid extracted by treating with restriction endonucleases Nde I and Xho I as a vector; (2) The transport protein PtNTT2 (66-575) gene fragment was obtained by PCR amplification, and restriction sites NdeI and Xho I were introduced at both ends for restriction digestion; (3) The fragment obtained in step (2) and the vector obtained in step (1) were mixed in a ratio of 3:1, and the enzyme-linked product pET23b-PtNTT2 (66-575) was obtained under the action of T4 DNA polymerase; (4) The enzyme-linked product pET23b-PtNTT2 (66-575) obtained in step (3) is transformed into VnDX cells by electroporation to obtain VnDX / pET23b-PtNTT2 (66-575) cells.

2. The method for constructing a VnDX novel chassis cell according to claim 1, characterized in that: The VnDX novel chassis cells use the pET-T7 exogenous gene expression system matured in Escherichia coli.

3. A method for determining the nucleoside triphosphate transport activity of a nucleoside triphosphate transporter heterologously expressed in Vibrio natriuresis VnDx, characterized in that: ATP detection reagent was used to detect the ATP content transported out of the cell of VnDx by the transporter, and the activity of the nucleoside triphosphate transporter was indirectly determined. The following steps are involved: (1) Pick the constructed VnDX / pET23b-PtNTT2(66-575), transfer the single clone to 20 mL of 2×YT3 medium, and culture in shaking medium at 220 rpm at 37°C until OD 600 =0.4-0.8, divided equally into two tubes A and B; (2) adding IPTG at a final concentration of 0.5-1 mM to tube A obtained in step (1), incubating the cells at 37° C. and shaking at 220 rpm to induce protein expression for 0.5-3 hours to obtain induced expression cells; tube B contains uninduced expression cells; (3) Collect the cells by centrifugation at 4°C and 3000-6000 rpm, resuspend the cells in fresh 2×YT3 medium to eliminate the influence of metabolites on the detection, and dilute the cells to an appropriate concentration; (4) The diluted cells obtained in step (3) were transferred to a black-bottom 96-well plate, and nucleoside triphosphate substrates with a final concentration of 10-500 μM were added. After 10-60 minutes of transport, 2-10 μL of ATP detection solution was added, and the cells were allowed to stand at room temperature for 2-10 minutes before detection.

4. The method for measuring nucleoside triphosphate transport activity according to claim 3, characterized in that: The total detection volume is 80-200 μL, the volume of bacterial solution and nucleoside triphosphate substrate is 75-190 μL, and 2-10 μL ATP detection solution is added after the transfer is completed.

5. A VnDX cell capable of transporting various natural or non-natural nucleoside triphosphates mediated by the expression of an exogenous gene PtNTT2 (66-575), characterized in that: It has a broad substrate spectrum for the transport of various nucleoside triphosphates and has significant transport activity; The transport activity of the transporter PtNTT2(66-575) for natural deoxyribonucleoside triphosphate substrates in VnDX cells includes: (1) The transporter PtNTT2 (66-575) has dATP transport activity in VnDX, and the transport activity is strong even without induced expression; (2) The transport protein PtNTT2 (66-575) has transport activity for dTTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression; (3) The transporter PtNTT2 (66-575) has transport activity for dCTP in VnDX, and the transport activity is already strong even without induced expression; (4) The transporter protein PtNTT2 (66-575) has dGTP transport activity in VnDX, and the transport activity is more significant after induced expression.

6. The VnDX cell having the ability to transport various natural or non-natural nucleoside triphosphates mediated by the expression of the exogenous gene PtNTT2 (66-575) according to claim 5, characterized in that: The transport activity of the transporter PtNTT2(66-575) for natural ribonucleoside triphosphate substrates in VnDX cells includes: (1) The transport protein PtNTT2 (66-575) has UTP transport activity in VnDX, and the transport activity is more significant after induced expression; (2) The transport protein PtNTT2 (66-575) has transport activity for CTP in VnDX, and the transport activity is more significant after induced expression; (3) The transporter PtNTT2 (66-575) has GTP transport activity in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression.

7. The VnDX cell having the ability to transport various natural or non-natural nucleoside triphosphates mediated by the expression of the exogenous gene PtNTT2 (66-575) according to claim 5, characterized in that: The transport activity of the transporter PtNTT2(66-575) for 2'-OMe-modified ribonucleoside triphosphate substrates in VnDX cells includes: (1) The transporter PtNTT2 (66-575) has transport activity for 2'-OMe-ATP in VnDX, and the uninduced transport activity is already strong; (2) The transport protein PtNTT2 (66-575) has transport activity for 2'-OMe-UTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression; (3) The transport protein PtNTT2 (66-575) has transport activity for 2'-OMe-CTP in VnDX, and the transport activity is more significant after induced expression; (4) The transporter protein PtNTT2 (66-575) has transport activity for 2'-OMe-GTP in VnDX, and the transport activity is more significant after induced expression.

8. The VnDX cell having the ability to transport various natural or non-natural nucleoside triphosphates mediated by the expression of the exogenous gene PtNTT2 (66-575) according to claim 5, characterized in that: The transport activity of the transporter PtNTT2(66-575) for 2'-F modified ribonucleoside triphosphate substrates in VnDX cells includes: (1) The transport protein PtNTT2 (66-575) has transport activity for 2'-F-UTP in VnDX, and the transport activity after induced expression is slightly more significant than that after uninduced expression; (2) The transport protein PtNTT2 (66-575) has transport activity for 2'-F-CTP in VnDX, and the transport activity is more significant after induced expression; (3) The transporter protein PtNTT2(66-575) has transport activity for 2'-F-GTP in VnDX, and the transport activity is more significant after induced expression.

9. The VnDX cell having the ability to transport various natural or non-natural nucleoside triphosphates mediated by the expression of the exogenous gene PtNTT2 (66-575) according to claim 5, characterized in that: The transport activity of the transporter PtNTT2 (66-575) for deoxyribonucleoside triphosphate substrates containing unnatural bases in VnDX cells includes: (1) The transport protein PtNTT2 (66-575) has transport activity for dTPT3TP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression; (2) The transport protein PtNTT2 (66-575) has transport activity for dNaMTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression; (3) The transporter PtNTT2(66-575) has transport activity for dCNMOTP in VnDX, and the transport activity after induced expression is slightly more significant than that without induced expression.

10. The VnDX cell having the ability to transport various natural or non-natural nucleoside triphosphates mediated by the expression of the exogenous gene PtNTT2 (66-575) according to claim 5, characterized in that: The transport activity of the transporter PtNTT2(66-575) for ribonucleoside triphosphate substrates containing unnatural bases in VnDX cells includes: (1) The transport protein PtNTT2 (66-575) has transport activity for TPT3TP in VnDX, and the transport activity is already strong even without induced expression; (2) The transporter PtNTT2 (66-575) has transport activity for NaMTP in VnDX, and the transport activity is already strong even without induced expression.

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