Patent Application
20230167435
This application is based upon and claims priority to Chinese Patent Application No. 202110968581.8, filed on Aug. 23, 2021, the entire contents of which are incorporated herein by reference.
The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy is named GBDZ045 Sequence Listing.xml, created on Oct. 24, 2022, and is 5,208 bytes in size.
The present disclosure belongs to the technical field of genetic engineering and particularly relates to an autoinducer-2 (AI-2) molecular response-based starting element and an Escherichia coli (E. coli) dynamic regulation system and method constructed thereby.
E. coli has long been widely used as a laboratory strain for molecular biology to facilitate the discovery of many important mechanisms in DNA replication, transcription, translation, and expression. In addition, E. coli is a preferred prokaryote for laboratory and industrial applications due to its rapid growth and development, simple cultivation conditions, strong metabolic plasticity, and rich biochemical and physiological functions. The relatively clear biochemical, physiological, and genetic properties of E. coli allow rapid progress in the research of metabolic engineering and synthetic biology, and E. coli has been widely used as an industrial host for the metabolic synthesis of a compound such as a lysine, 1,3-propanediol, and 1,4-butanediol. E. coli strains are generally considered to be harmless. For example, the laboratory strain E. coli MG1655 is considered to lack virulence factors associated with human diseases and has been widely used in the production of various biochemicals, food, and biofuels.
Programmable site-specific gene regulatory elements play an important role in genome-wide gene function research, metabolic pathway regulation, and artificial gene circuit design. Gene regulation tools based on clustered regularly interspaced short palindromic repeats (CRISPRs) are currently one of the most popular gene regulation techniques, and CRISPR technology can be used for the polygenic regulation of microorganisms. When a CRISPR gene regulation system guides the location of a defective nuclease (such as dCas9 and dCpf1) to a target gene site through a guide RNA (such as sgRNA and crRNA), DNA is not cleaved but is fused to a transcriptional repressor domain (TRD) to form an RNA-protein complex, and the RNA-protein complex is guided to a transcription start site of a target gene to inhibit the transcription. When a C terminus of the dCas protein is fused to a transcriptional activator, the dCas protein and the transcriptional activator can activate the transcription of the target gene at the transcription start site. In recent years, with the advent of programmable transcription factors for the CRISPR-dCpf1 system, the synthetic control of gene expression becomes simple. Compared with the CRISPR-dCas9 system, CRISPR-dCpf1 has significant advantages such as low off-target efficiency. In addition, the guide of dCpf1 only requires a single RNA, while the guide of dCas9 requires the joint action of sgRNA and tracrRNA. Therefore, the CRISPR-dCpf1 system is suitable for the regulation of multiple gene sites.
The dynamic regulation of gene expression has become a common regulatory strategy, and microorganisms can autonomously regulate their metabolic fluxes according to a microbial community or a cellular metabolism status. Quorum sensing (QS) is an information exchange mechanism by which bacteria monitor a population density and coordinate a microbial function by secreting signaling molecules, which has been widely used for automatic regulation of the expression of key genes in metabolic synthesis pathways or self-induction of the expression of recombinant proteins. An AI-2 signaling molecular response-based QS system (AI-2 QS) is an endogenous QS system in E. coli MG1655. In the AI-2 QS system, when a cell density is low, the transcription factor LsrR binds to an lsrA box sequence on a PlsrA promoter, thereby inhibiting the transcription of the PlsrA promoter. As bacterial cell density increases, a signaling molecule AI-2 synthesized in cells is continuously transported out of the cells through a TqsA protein. When the extracellular AI-2 signaling molecule accumulates to a specified threshold, the AI-2 molecule is transported into cells by the LsrABCD transport system and converted into a phosphorylated AI-2 molecule under the action of an LsrK protein, and the phosphorylated AI-2 molecule and the lsrA box sequence located on the PlsrA promoter compete with each other to bind to the LsrR molecule, thereby releasing the transcriptional inhibition of LsrR on P and PlsrA activating the expression of the promoter PlsrA (
The CRISPR-dCpf1 system is currently a popular gene editing and regulation system, which requires a shorter guide RNA (crRNA) sequence than the CRISPR-dCas9 system and can achieve the polygenic regulation by constructing an array of different crRNAs. Miao Chengsi et al. investigated the influence of a target sequence position, lengths of repeat and spacer sequences in crRNA, a protospacer adjacent motif (PAM) sequence, and the like in the CRISPR-dCpf1 system on the regulation performance of the CRISPR-dCpf1 system in E. coli and discovered an efficient CRISPR-dCpf1 gene regulation tool based on isopropyl-β-D-thiogalactoside (IPTG) induction. Wu Yaokang et al. also constructed a xylose induction-based CRISPR/Cpf1 polygenic editing and transcriptional regulation system (CAMERS-B) in Bacillus subtilis (B. subtilis) by designing a crRNA array, which can not only achieve the double-gene knockout, multi-point mutation, or single-gene insertion in B. subtilis but also achieve the transcriptional inhibition and activation of multiple genes. The CAMERS-B system can be further used to inhibit the genes (bdhA and acoA) of an acetoin catabolism pathway and the genes (ldh and pta) of the synthesized byproducts of lactic acid and acetic acid and to activate the pathway gene alsSD in the acetoin synthesis pathway and the gene alsR of a transcriptional activator. A crRNA array for bdhA, acoA, ldh, pta, and alsR is constructed to achieve the transcriptional inhibition and activation of the CAMERS-B system in acetoin synthesis-associated pathways, thereby increasing a yield of the acetoin by 44.8% and to 25.8 g/L.
Although a CRISPR-dCpf1 regulation system based on IPTG induction has been developed, the induction of overexpression of dCpf1 by IPTG may cause a metabolic burden on cells, thereby negatively affecting metabolic synthesis. Therefore, if a self-inducible signaling molecule is used to automatically trigger the CRISPR-dCpf1 regulation system, the metabolic burden of the recombinant protein on cells and the potential toxic effect of IPTG on cells can be avoided. When some exogenous metabolic synthesis pathways are introduced, it is difficult to develop a self-inducible element that can respond to a specific signaling molecule for dynamic regulation of the expression of key genes in a synthesis pathway due to a specified threshold concentration range for responding to an intermediate metabolite or due to the lack of research on regulation mechanisms for responding to an intermediate metabolite. The dynamic regulation based on cell QS is currently a regulation system with the potential for metabolic synthesis. Without responding to a specific intermediate metabolite, the system can secrete a relevant signaling molecule through a change in a cell population density to activate or inhibit the expression of a related gene. Therefore, an endogenous cell density-dependent AI-2 signaling molecule of E. coli can be used to develop a self-inducible dCpf1 expression element, and crRNA of a specific gene can be further constructed to achieve the dynamic activation or inhibition of the expression of related genes in the metabolic synthesis pathway.
To solve the problem that the IPTG induction-based CRISPR-dCpf1 regulation system may have a potential toxic effect on cells, the present disclosure constructs a cell density-dependent response element PJ23119-LsrR-PlsrA (SEQ ID NO: 1) based on an AI-2 signaling molecule. The element can self-induce the expression of dCpf1, and crRNA of a specific gene can be further constructed to dynamically activate or inhibit the expression of related genes in a metabolic synthesis pathway, thereby avoiding the potential toxic effect of an inducer such as IPTG on cells. The present disclosure mainly adopts the following technical solutions to solve the above technical problem:
The present disclosure provides an AI-2 molecular response-based starting element, where the AI-2 molecular response-based starting element is a cell density-dependent starting element PJ23119-LsrR-PlsrA. The cell density-dependent starting element PJ23119-LsrR-PlsrA is constructed as follows: optimizing a native cell density-dependent promoter PlsrA in an AI-2 signaling molecular response-based wild-type (WT) starting element gene fragment PlsrR-LsrR-PlsrA, using a constitutive promoter PJ23119 to express a transcriptional regulation factor LsrR, and constructing the cell density-dependent starting element PJ23119-LsrR-PlsrA based on an AI-2 molecular response. Preferably, the starting element PJ23119-LsrR-PlsrA has a nucleotide sequence shown in SEQ ID NO: 1, and the WT starting element gene fragment PlsrR-LsrR-PlsrA has a nucleotide sequence shown in SEQ ID NO: 2.
The present disclosure also provides a use of a cell density-dependent starting element PJ23119-LsrR-PlsrA based on an AI-2 molecular response for automatically triggering a CRISPR-dCpf1 regulation system.
The present disclosure also provides an AI-2 molecular response-based E. coli CRISPR-dCpf1 dynamic regulation system, 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. Preferably, the expression vector pACYDuet-dCpf1 includes a cell density-dependent starting element PJ23119-LsrR-PlsrA.
Preferably, a transcriptional activator CRP in E. coli is selected and fused to a C terminus of a dCpf1 protein.
The present disclosure also provides a construction method of an AI-2 molecular response-based E. coli CRISPR-dCpf1 dynamic regulation system, including the following steps:
(1) selecting plasmids pACYDuet, pRSFDuet, and pETDuet as starting vectors, recombinantly ligating FndCpf1 and PJ23119-LsrR-PlsrA gene fragments with pACYDuet to construct a vector pACYDuet-PJ23119-LsrR-PlsrA-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 pACYDuet-PJ23119-LsrR-PlsrA-dCpf1 as a template, fusing a transcriptional activator CRP to a C terminus of a dCpf1 protein to construct a dCpf1 activation plasmid pACYDuet-PJ23119-LsrR-PlsrA-dCpf1-CRP;
(3) with the vector pRSFDuet-GFP obtained in step (1) as a template, recombinantly ligating a gene fragment of mCherry with pRSFDuet-GFP to construct a reporter vector pRSFDuet-GFP-mCherry for fluorescence activation and inhibition; and
(4) co-transforming the plasmid pACYDuet-PJ23119-LsrR-PlsrA-dCpf1-CRP into an E. coli MG1655 competent cell with the vectors pRSFDuet-GFP-mCherry and pETDuet-crRNA.
The present disclosure also provides a use of an AI-2 molecular response-based E. coli CRISPR-dCpf1 dynamic regulation system in the regulation of expression of a synthesis pathway gene in E. coli.
Compared with the prior art, the present disclosure has the following positive progressive effects:
The present disclosure constructs a cell density-dependent starting element PJ23119-LsrR-PlsrA based on an AI-2 molecular response. A strong promoter PJ23119 is used to express a transcription factor LsrR, which can effectively enhance a fluorescence intensity of a reporter protein. By assembling crRNAs of different target genes, 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-PJ23119-LsrR-PlsrA-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.
The present disclosure provides a cell density-dependent element PJ23119-LsrR-PlsrA 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 PJ23119-LsrR-PlsrA has a nucleotide sequence shown in SEQ ID NO: 1.
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.
In the present disclosure, since a native cell density-dependent promoter PlsrA 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 ParaB in the CRISPR-dCpf1 system is replaced with the cell density-dependent promoter PlsrA 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.
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:
The construction of the CRISPR-dCpf1 regulation system is shown in
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-ParaB-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-ParaB-dCpf1-CRP system can achieve both activation and inhibition effects.
The ParaB promoter gene fragment and PlsrA 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-ParaB-gfp and pACYDuet-PlsrR-LsrR-PlsrA-gfp, respectively. Since a native cell density-dependent promoter PlsrA based on an AI-2 signaling molecular response has a low fluorescence intensity, the transcription factor LsrR is further expressed using a constitutive promoter PJ23119 to construct a vector pACYDuet-PJ23119-LsrR-PlsrA-gfp. The pACYDuet-ParaB-gfp pACYDuet-PlsrR-LsrR-PlsrA-gfp, and pACYDuet-PJ23119-LsrR-PlsrA-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 PlsrA and the inducible promoter ParaB.
The arabinose-induced promoter ParaB in the CRISPR-dCpf1 system is replaced with each of the gene sequences of PlsrR-LsrR-PlsrA and PJ23119-LSIR-PlsrA to construct plasmids pACYDuet-PlsrR-LsrR-PlsrA-dCpf1 -CRP and pACYDuet-PJ23119-LsrR-PlsrA-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-PlsrA-dCpf1-CRP can spontaneously initiate the effects of activating and inhibiting a gene according to a change in cell density.
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.
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 ParaB was amplified with the E. coli genome as a template; purified and recovered fragments of FndCpf1 and ParaB were ligated to pACYDuet using a seamless cloning kit to construct a vector pACYDuet-ParaB-dCpf1. The gfp gene in Chinese Patent CN201911387447.8 was amplified. A promoter PJ23117 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-ParaB-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)2 flexible Linker to construct dCpf1 activation plasmids pACYDuet-ParaB-dCpf1-CRP and pACYDuet-ParaB-dCpf1-RpoZ, respectively. The dCpf1 expression plasmids pACYDuet-ParaB-dCpf1, pACYDuet-Parma-dCpf1-CRP, and pACYDuet-ParaB-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
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-ParaB-dCpf1-CRP with high activation intensity for the transcription factor CRP (
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 ParaB and a cell density-dependent promoter PlsrA were each amplified with an E. coli genome as a template, and purified and recovered fragments of ParaB and PlsrA were ligated to a linearized vector pACYDuet using a seamless cloning kit to construct GFP reporter vectors pACYDuet-ParaB-gfp and pACYDuet-PlsrR-LsrR-PlsrA-gfp, respectively. The constructed plasmids pACYDuet-ParaB-gfp and pACYDuet-PlsrR-LsrR-PlsrA-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 PlsrA and the inducible promoter ParaB. 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.
Since the pACYDuet-PlsrR-LsrR-PlsrA-gfp had a weak fluorescence intensity when the transcription factor LsrR was expressed using a WT promoter PlsrR, a constitutive promoter PJ23119 was selected to express the transcriptional regulation factor LsrR. With the plasmid pACYDuet-PlsrR-LsrR-PlsrA-gfp as a template, the WT PlsrR promoter was replaced with a PJ23119 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-PJ23119-LsrR-PlsrA-gfp sequenced as positive and the plasmids pACYDuet-ParaB-gfp and pACYDuet-PlsrR-LsrR-PlsrA-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
The plasmid pACYDuet-ParaB-dCpf1-CRP constructed in Example 1 was linearized by PCR, and the template DNA was digested with a DpnI enzyme. A gene fragment PJ23119-LsrR-PlsrA was amplified with the plasmid pACYDuet-PJ23119-LsrR-PlsrA-gfp constructed in Example 3 as a template. Purified and recovered fragments of PJ23119-LsrR-PlsrA were ligated to a linearized vector pACYDuet-ParaB-dCpf1-CRP using a seamless cloning kit, and the arabinose-induced promoter ParaB in the CRISPR-dCpf1 system was replaced with the cell density-dependent starting element PJ23119-LsrR-PlsrA constructed in Example 3 to construct a plasmid pACYDuet-PJ23119-LsrR-PlsrA-dCpf1-CRP. The plasmid pACYDuet-PJ23119-LsrR-PlsrA-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-PlsrA-dCpf1-CRP could spontaneously activate GFP and inhibit RFP according to a change in cell density. As shown in
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110968581.8 | Aug 2021 | CN | national |
