AUTOINDUCER-2 (AI-2) MOLECULAR RESPONSE-BASED STARTING ELEMENT AND ESCHERICHIA COLI (E. COLI) DYNAMIC REGULATION SYSTEM AND METHOD CONSTRUCTED THEREBY

Abstract

An autoinducer-2 (AI-2) molecular response-based starting element and an Escherichia coli (E. coli) dynamic regulation system and method constructed thereby are provided. A cell density-dependent starting element P.sub.J23119-LsrR-P.sub.lsrA based on an AI-2 molecular response is constructed. The element can be used to self-induce the expression of dCpf1, and crRNAs of different target genes are further assembled, such that the self-inducible element can be used for dCpf1-CRP to achieve the dynamic regulation of genes in a synthesis pathway. In the present disclosure, vectors pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-dCpf1-CRP, pRSFDuet-GFP-mCherry, and pETDuet-crRNA can be constructed to simultaneously achieve the transcriptional activation and inhibition of different genes. The construction method of recombinant E. coli in the present disclosure is simple and has promising application prospects.

Claims

1. An autoinducer-2 (AI-2) molecular response-based starting element, wherein the AI-2 molecular response-based starting element is a cell density-dependent starting element P.sub.J23119-LsrR-P.sub.lsrA; the cell density-dependent starting element P.sub.J23119-LsrR-P.sub.lsrA is constructed as follows: optimizing a native cell density-dependent promoter P.sub.lsrA in an AI-2 signaling molecular response-based wild-type (WT) starting element gene fragment P.sub.lsrR-LsrR-P.sub.lsrA, using a constitutive promoter P.sub.J23119 to express a transcriptional regulation factor LsrR, and constructing the cell density-dependent starting element P.sub.J23119-LsrR-P.sub.lsrA based on an AI-2 molecular response; and the cell density-dependent starting element P.sub.J23119-LsrR-P.sub.lsrA has a nucleotide sequence shown in SEQ ID NO: 1, and the AI-2 signaling molecular response-based WT starting element gene fragment P.sub.lsrR-LsrR-P.sub.lsrA has a nucleotide sequence shown in SEQ ID NO: 2.

2. A method of a use of a cell density-dependent starting element P.sub.J23119-LsrR-P.sub.lsrA based on an AI-2 molecular response for automatically triggering a CRISPR-dCpf1 regulation system.

3. An AI-2 molecular response-based Escherichia coli (E. coli) CRISPR-dCpf1 dynamic regulation system, comprising a dCpf1 protein expression vector pACYDuet-dCpf1, a reporter fluorescent protein GFP, an mCherry expression vector pRSFDuet-GFP-mCherry, and a crRNA expression vector pETDuet-crRNA of a reporter protein, wherein the dCpf1 protein expression vector pACYDuet-dCpf1 comprises a cell density-dependent starting element P.sub.J23119-LsrR-P.sub.lsrA.

4. The AI-2 molecular response-based E. coli CRISPR-dCpf1 dynamic regulation system according to claim 3, wherein a transcriptional activator CRP in E. coli is selected and fused to a C terminus of a dCpf1 protein.

5. A construction method of an AI-2 molecular response-based E. coli CRISPR-dCpf1 dynamic regulation system, comprising the following steps: (1) selecting plasmids pACYDuet, pRSFDuet, and pETDuet as starting vectors; recombinantly ligating FndCpf1 and P.sub.J23119-LsrR-P.sub.lsrA gene fragments with the plasmid pACYDuet to construct a vector pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-dCpf1, recombinantly ligating a GFP gene with a linearized vector pRSFDuet to construct a vector pRSFDuet-GFP, and recombinantly ligating a crRNA fragment with a linearized vector pETDuet to construct a crRNA gene-containing vector pETDuet-crRNA; (2) with the vector pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-dCpf1 as a template, fusing a transcriptional activator CRP to a C terminus of a dCpf1 protein to construct a dCpf1 activation plasmid pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-dCpf1 -CRP; (3) with the vector pRSFDuet-GFP obtained in step (1) as a template, recombinantly ligating a gene fragment of mCherry with the vector pRSFDuet-GFP to construct a reporter vector pRSFDuet-GFP-mCherry for fluorescence activation and inhibition; and (4) co-transforming the dCpf1 activation plasmid pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-dCpf1 -CRP into an E. coli MG1655 competent cell with the reporter vector pRSFDuet-GFP-mCherry and the crRNA gene-containing vector pETDuet-crRNA.

6. A method of a use of an AI-2 molecular response-based E. coli CRISPR-dCpf1 dynamic regulation system in a regulation of an expression of a synthesis pathway gene in E. coli.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 shows an AI-2 signaling molecular response-based E. coli QS system;

[0023] FIG. 2 is a schematic diagram illustrating the construction of a CRISPR-dCpf1 regulation system;

[0024] FIG. 3 shows the activation intensities of transcription factors CRP and RpoZ on a green fluorescent protein (GFP);

[0025] FIG. 4 shows the regulatory effects of arabinose induction-based CRISPR-dCpf1 on the activation and inhibition of genes;

[0026] FIG. 5 shows the fluorescence intensity of a cell density-dependent promoter P.sub.lsrA based on an AI-2 signaling molecule; and

[0027] FIG. 6 shows the regulatory effects of cell density-dependent CRISPR-dCpf1 on the activation and inhibition of genes.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present disclosure provides a cell density-dependent element P.sub.J23119-LsrR-P.sub.lsrA based on an AI-2 signaling molecular response, and the element can be used to automatically trigger a CRISPR-dCpf1 regulation system, which provides a tool for dynamic regulation of a gene in a metabolic synthesis pathway. In the present disclosure, the element P.sub.J23119-LsrR-P.sub.lsrA has a nucleotide sequence shown in SEQ ID NO: 1.

[0029] In the present disclosure, to construct a CRISPR-dCpf1 system with a regulatory effect for activation and inhibition, 3 compatible plasmids are first constructed, including a dCpf1 protein expression vector pACYDuet-dCpf1, a reporter fluorescent protein GFP, an mCherry expression vector pRSFDuet-GFP-mCherry, and a crRNA expression vector pETDuet-crRNA of a reporter protein. Transcriptional activators (CRP and RpoZ) in E. coli are selected and then each fused to a C terminus of a dCpf1 protein, and the activation intensities of different transcription factors are detected.

[0030] In the present disclosure, since a native cell density-dependent promoter P.sub.lsrA based on an AI-2 signaling molecular response has a low fluorescence intensity, the promoter needs to be optimized, including replacement of a core region of the promoter, random mutation of a ribosome binding site (RBS) sequence, and expression of a transcription factor LsrR. Subsequently, the arabinose induction-based promoter P.sub.araB in the CRISPR-dCpf1 system is replaced with the cell density-dependent promoter P.sub.lsrA to construct a CRISPR-dCpf1 dynamic regulation system with both activation and inhibition effects. With the AI-2 molecular response-based CRISPR-dCpf1 dynamic regulation system constructed in the present disclosure, the expression of synthesis pathway genes in E. coli can be dynamically regulated by simply designing and assembling crRNAs of metabolic synthesis pathway-associated genes.

[0031] The present disclosure also provides a construction method of an AI-2 signaling molecule response-based E. coli CRISPR-dCpf1 dynamic regulation system, including the following steps:

(1) Construction of a CRISPR-dCpf1 Activation System

[0032] The construction of the CRISPR-dCpf1 regulation system is shown in FIG. 2. The plasmids pACYDuet, pRSFDuet, and pETDuet commonly used for E. coli are selected as starting vectors. The gene fragments of FndCpf1 and araB inducing elements (araC-P.sub.araB) are recombinantly ligated with pACYDuet using a seamless cloning kit to construct a vector pACYDuet-P.sub.araB-dCpf1 with arabinose to induce the expression of dCpf1. The GFP gene is recombinantly ligated with a linearized vector pRSFDuet, and a high-copy replicon pRSF ori on the initial vector pRSFDuet is replaced with a low-copy replicon SC101 ori to construct a vector pRSFDuet-GFP. A crRNA fragment is recombinantly ligated with a linearized vector pETDuet to construct a vector pETDuet-crRNA with P.sub.J23119 to express crRNA. With pACYDuet-P.sub.araB-dCpf1 as a template, the transcriptional activators CRP and RpoZ are each fused to a C terminus of a dCpf1 protein to construct two dCpf1 activation plasmids pACYDuet-P.sub.araB-dCpf1-CRP and pACYDuet-P.sub.araB-dCpf1-RpoZ. The dCpf1 expression plasmids pACYDuet-P.sub.araB-dCpf1, pACYDuet-Parma-dCpf1-CRP, and pACYDuet-P.sub.araB-dCpf1-RpoZ are each co-transformed into an E. coli MG1655 competent cell with the vectors pRSFDuet-GFP and pETDuet-crRNA, and resulting single colonies are picked and cultivated in a 96-well plate to investigate the activation effects of these two transcription factors.

(2) Construction of a CRISPR-dCpf1-CRP Activation and Inhibition System

[0033] With the vector pRSFDuet-GFP obtained in step (1) as a template, a gene fragment of mCherry is recombinantly ligated with pRSFDuet-GFP using a seamless cloning kit to construct a reporter vector pRSFDuet-GFP-mCherry for fluorescence activation and inhibition. The transcription factor CRP vector pACYDuet-P.sub.araB-dCpf1-CRP with high fluorescence intensity in step (1) is co-transformed into an E. coli MG1655 competent cell with the vectors pRSFDuet-GFP-mCherry and pETDuet-crRNA, and resulting single colonies are picked and cultivated in a 96-well plate to verify that the CRISPR-P.sub.araB-dCpf1-CRP system can achieve both activation and inhibition effects.

(3) Construction of a Cell Density-Dependent Element Based on an AI-2 Signaling Molecular Response

[0034] The P.sub.araB promoter gene fragment and P.sub.lsrA ID promoter gene fragment (SEQ NO: 2) on the E. coli MG1655 genome are each recombinantly ligated with the vector pACYDuet using a seamless cloning kit to construct control vectors pACYDuet-P.sub.araB-gfp and pACYDuet-P.sub.lsrR-LsrR-P.sub.lsrA-gfp, respectively. Since a native cell density-dependent promoter P.sub.lsrA based on an AI-2 signaling molecular response has a low fluorescence intensity, the transcription factor LsrR is further expressed using a constitutive promoter P.sub.J23119 to construct a vector pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-gfp. The pACYDuet-P.sub.araB-gfp pACYDuet-P.sub.lsrR-LsrR-P.sub.lsrA-gfp, and pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-gfp plasmids are each transformed into an E. coli MG1655 competent cell, and resulting single colonies are picked and cultivated in a 96-well plate to compare the fluorescence intensities of the cell density-dependent promoter P.sub.lsrA and the inducible promoter P.sub.araB.

(4) Construction of a CRISPR-dCpf1-CRP Dynamic Regulation System

[0035] The arabinose-induced promoter P.sub.araB in the CRISPR-dCpf1 system is replaced with each of the gene sequences of P.sub.lsrR-LsrR-P.sub.lsrA and P.sub.J23119-LSIR-P.sub.lsrA to construct plasmids pACYDuet-P.sub.lsrR-LsrR-P.sub.lsrA-dCpf1 -CRP and pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-dCpf1 -CRP, respectively. The two plasmids are each co-transformed into an E. coli MG1655 competent cell with the vectors pRSFDuet-GFP-mCherry and pETDuet-crRNA, and resulting single colonies are picked and cultivated in a 96-well plate to verify that CRISPR-P.sub.lsrA-dCpf1-CRP can spontaneously initiate the effects of activating and inhibiting a gene according to a change in cell density.

[0036] To make the objectives, technical solutions, and advantages of the present disclosure clear, the present disclosure will be described in detail below in conjunction with examples, but the examples should not be construed as limiting the protection scope of the present disclosure.

Example 1

(1) Construction of a CRISPR-dCpf1 Activation System

[0037] The plasmids pACYDuet, pRSFDuet, and pETDuet commonly used by E. coli were selected as starting vectors, each was linearized by polymerase chain reaction (PCR), and the template DNA was digested with a DpnI enzyme. An FndCpf1 gene was amplified with the pLCg6-dCpf1 plasmid in Chinese Patent CN201911387447.8 as a template. The arabinose promoter P.sub.araB was amplified with the E. coli genome as a template; purified and recovered fragments of FndCpf1 and P.sub.araB were ligated to pACYDuet using a seamless cloning kit to construct a vector pACYDuet-P.sub.araB-dCpf1. The gfp gene in Chinese Patent CN201911387447.8 was amplified. A promoter P.sub.J23117 and an upstream sequence thereof (from the literature: Bikard, D.; Jiang, W.; Samai, P.; Hochschild, A.; Zhang, F.; Marraffini, L. A., Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013, 41 (15), 7429-7437.) were acquired through gene synthesis, and the two fragments were recombinantly ligated with a linearized vector pRSFDuet. The replicon pSC101 ori was amplified with a plasmid pSC101-Donor (Addgene #140630) as a template to construct a low-copy reporter protein plasmid pRSFDuet-GFP. A crRNA fragment was amplified with the perF11 vector in Chinese Patent CN201911387447.8 as a template and then recombinantly ligated with pETDuet through seamless cloning, then crRNA of an upstream sequence of the gfp gene promoter was designed on the CRISPR-DT web site (http://bioinfolab.miamioh.edu/CRISPR-DT/interface/Cpf1_design.php), and a vector pETDuet-crRNA for activating the gfp gene was constructed. The vector pACYDuet-P.sub.araB-dCpf1 was then linearized, gene fragments of the transcriptional activators CRP and RpoZ were each amplified with the E. coli genome as a template, and the transcription factors were each fused to a C terminus of a dCpf1 protein through a (GGGGS).sub.2 flexible Linker to construct dCpf1 activation plasmids pACYDuet-P.sub.araB-dCpf1-CRP and pACYDuet-P.sub.araB-dCpf1-RpoZ, respectively. The dCpf1 expression plasmids pACYDuet-P.sub.araB-dCpf1, pACYDuet-Parma-dCpf1-CRP, and pACYDuet-P.sub.araB-dCpf1-RpoZ were each co-transformed into an E. coli MG1655 competent cell with the vectors pRSFDuet-GFP and pETDuet-crRNA, then a resulting competent cell was coated on a chloramphenicol, kanamycin, and ampicillinum-resistant plate and cultivated at 30°C. for 16 h to 18 h. Positive single colonies were picked and cultivated in a 96-well plate, and activation intensities of the two transcription factors (CRP and RpoZ) on a GFP were detected by a Tecan microplate reader. An excitation wavelength of the GFP was set as 490 mm, and an emission wavelength was set as 530 mm. As shown in FIG. 3, an activation effect of the transcription factor RpoZ on GFP was not significant, while an activation effect of the transcription factor CRP on GFP was significant. The green fluorescence intensity was increased by 1.8 times compared with the case without the fusion of a transcription factor.

(2) Construction of a CRISPR-dCpf1-CRP Activation and Inhibition System

[0038] The plasmid pRSFDuet-GFP was linearized by PCR, the template DNA was digested with a DpnI enzyme, the red fluorescent protein (RFP) gene mCherry in Chinese Patent CN201911387447.8 was amplified, and the gene fragment of mCherry was recombinantly ligated with a linearized vector pRSFDuet-GFP using a seamless cloning kit to construct a reporter vector pRSFDuet-GFP-mCherry for fluorescence activation and inhibition. The vector pACYDuet-P.sub.araB-dCpf1-CRP with high activation intensity for the transcription factor CRP (FIG. 3) was selected and co-transformed into an E. coli MG1655 competent cell with the reporter protein plasmids pRSFDuet-GFP-mCherry and pETDuet-crRNA. The competent cell was coated on a chloramphenicol, kanamycin, and ampicillinum-resistant plate and cultivated at 30°C. for 16 h to 18 h. Positive single colonies were picked and cultivated in a 96-well plate for 12 h to 15 h, and arabinose was added at a final concentration of 40 mM to 60 mM to induce the expression of GFP and mCherry. The expression intensities of GFP and RFP were detected using a Tecan microplate reader. An excitation wavelength of GFP was set as 490 mm and an emission wavelength of GFP was set as 530 mm; an excitation wavelength of RFP was set as 580 mm and an emission wavelength of RFP was set as 615 mm. As shown in FIG. 4, after arabinose was added to induce the expression of dCpf1, starting from 15 h, the expression intensity of GFP was significantly increased, while the expression intensity of RFP was gradually decreased. It indicates that the constructed CRISPR-dCpf1 regulation system can simultaneously activate and inhibit the expression intensities of different genes under the action of arabinose induction.

Example 2

Construction of a Vector pACYDuet-P.SUB.lsrR.-LsrR-P.SUB.lsrA.-gfP

[0039] The plasmid pACYDuet was selected as a starting plasmid and linearized by PCR, and the template DNA was digested with a DpnI enzyme. A WT arabinose promoter P.sub.araB and a cell density-dependent promoter P.sub.lsrA were each amplified with an E. coli genome as a template, and purified and recovered fragments of P.sub.araB and P.sub.lsrA were ligated to a linearized vector pACYDuet using a seamless cloning kit to construct GFP reporter vectors pACYDuet-P.sub.araB-gfp and pACYDuet-P.sub.lsrR-LsrR-P.sub.lsrA-gfp, respectively. The constructed plasmids pACYDuet-P.sub.araB-gfp and pACYDuet-P.sub.lsrR-LsrR-P.sub.lsrA-gfp were each transformed into an E. coli MG1655 competent cell, and resulting single colonies were picked and cultivated in a 96-well plate to compare the fluorescence intensities of the cell density-dependent promoter P.sub.lsrA and the inducible promoter P.sub.araB. The expression intensities of GFP were detected using a Tecan microplate reader. An excitation wavelength of GFP was set as 490 mm, and an emission wavelength of GFP was set as 530 mm.

Example 3

Construction of a Vector pACYDuet-P.SUB.J23119.-LsrR-P.SUB.lsrA.-gfp

[0040] Since the pACYDuet-P.sub.lsrR-LsrR-P.sub.lsrA-gfp had a weak fluorescence intensity when the transcription factor LsrR was expressed using a WT promoter P.sub.lsrR, a constitutive promoter P.sub.J23119 was selected to express the transcriptional regulation factor LsrR. With the plasmid pACYDuet-P.sub.lsrR-LsrR-P.sub.lsrA-gfp as a template, the WT P.sub.lsrR promoter was replaced with a P.sub.J23119 promoter through cyclic PCR, and the template DNA was digested with a DpnI enzyme. Purified and recovered fragments were transformed into an E. coli MG1655 competent cell. The competent cell was coated on a chloramphenicol-resistant plate and cultivated overnight at 37°C., and positive single colonies were picked for testing. The plasmid pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-gfp sequenced as positive and the plasmids pACYDuet-P.sub.araB-gfp and pACYDuet-P.sub.lsrR-LsrR-P.sub.lsrA-gfp successfully constructed in Example 2 were each transformed into an E. coli MG1655 competent cell once again. The competent cell was coated on a chloramphenicol-resistant plate, and single colonies were picked and cultivated in a 96-well plate. An excitation wavelength of GFP was set as 490 mm, and an emission wavelength of GFP was set as 530 mm. These three plasmids were compared in terms of the expression intensity of GFP. As shown in FIG. 5, the WT P.sub.lsrA promoter had the weakest intensity and was barely expressed. After the transcription factor LsrR was expressed using the constitutive promoter P.sub.J23119, a GFP fluorescence intensity was 2.2 times higher than a GFP fluorescence intensity of the inducible promoter P.sub.araB.

Example 4

Construction of a CRISPR-dCpf1-CRP Dynamic Regulation System

[0041] The plasmid pACYDuet-P.sub.araB-dCpf1-CRP constructed in Example 1 was linearized by PCR, and the template DNA was digested with a DpnI enzyme. A gene fragment P.sub.J23119-LsrR-P.sub.lsrA was amplified with the plasmid pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-gfp constructed in Example 3 as a template. Purified and recovered fragments of P.sub.J23119-LsrR-P.sub.lsrA were ligated to a linearized vector pACYDuet-P.sub.araB-dCpf1-CRP using a seamless cloning kit, and the arabinose-induced promoter P.sub.araB in the CRISPR-dCpf1 system was replaced with the cell density-dependent starting element P.sub.J23119-LsrR-P.sub.lsrA constructed in Example 3 to construct a plasmid pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-dCpf1-CRP. The plasmid pACYDuet-P.sub.J23119-LsrR-P.sub.lsrA-dCpf1-CRP was co-transformed into an E. coli MG1655 competent cell with the reporter protein plasmids pRSFDuet-GFP-mCherry and pETDuet-crRNA. The competent cell was coated on a chloramphenicol-resistant plate, and single colonies were picked and cultivated in a 96-well plate. An excitation wavelength of GFP was set as 490 mm, and an emission wavelength of GFP was set as 530 mm; an excitation wavelength of RFP was set as 580 mm, and an emission wavelength of RFP was set as 615 mm. It was verified that CRISPR-P.sub.lsrA-dCpf1-CRP could spontaneously activate GFP and inhibit RFP according to a change in cell density. As shown in FIG. 6, after the cell was cultivated for 22 h to 26 h, the CRISPR-dCpf1 dynamic regulation system exhibited significant activation (1.3 times) and inhibition (1.5 times) effects on GFP and RFP.

[0042] The above are merely preferred examples of the present disclosure and are not intended to limit the present disclosure, and various changes and modifications can be made to the present disclosure by those skilled in the art. Any modifications, equivalent substitutions, improvements, and the like made within the spirit and principle of the present disclosure should be included within the protection scope of the present disclosure.