WO2025063625A1 - Engineered retron comprising dna binding protein-specific binding site, and use thereof - Google Patents

Abstract

The present invention relates to: an engineered retron comprising a DNA binding protein-specific binding site; and a use thereof, wherein the engineered retron comprising a DNA binding protein-specific binding site has been used to achieve transcriptional control as well as a self-regulating feedback circuit and developed into a system for controlling the intracellular location and function of proteins and can thus be used as a biosensor, a memory device, an artificial adaptive circuit, a system for controlling the intracellular position of proteins, and a system for controlling the function and position of proteins at a post-translational level.

Description

Engineered retrones comprising DNA binding protein-specific binding sites and uses thereof

The present invention relates to engineered retrones comprising a DNA binding protein specific binding site and uses thereof.

Programmable biomolecular interactions among DNA, RNA, and proteins are key building blocks of synthetic biological systems. In particular, DNA-protein, RNA-protein, RNA-RNA, and protein-protein interactions have been extensively designed and used to create genetically encodeable synthetic biological devices in a myriad of applications, including gene regulation and editing biosensors, self-assembling nanostructures, complex logic computation, and metabolic engineering. These molecular interactions can be dynamically controlled, primarily by modulating gene expression or by changing the molecular structure of the RNA or protein components, thereby controlling the number or state of copies of the participating molecules.

However, a similar approach to DNA in living cells is difficult even in relatively simple bacteria. Unlike the proteome and transcriptome, where small subunits are physically separated, the subunits of the genome are covalently linked within larger DNA molecules such as chromosomes or plasmids. This limits the independent control of specific functional parts of the DNA, such as short (~20 bp) protein-binding sequences. Although DNA-protein interactions have played a central role since the beginning of synthetic biology due to their reliable modularity, tunability, orthogonality, and signaling responsiveness, their use in vivo remains limited to molecular events in the genome. In contrast, in vitro conditions, which do not require such limitations, have enabled a wider range of applications, including comprehensive characterization of DNA-protein interactions, real-time biosensors, and DNA-protein hybrid nanostructures. The pioneering work of J. Elbaz et al. (Nat Commun 7, 11179, 2016) demonstrated that the eukaryotic retroviral reverse transcription system can be designed to release the constraints of cellular DNA by producing DNA oligonucleotides with programmable sequences intracellularly. However, this system requires relatively heavy genetic components and requires the construction of complex DNA nanostructures to address low stability and functionality.

Retrons (Fig. 1a), a widespread prokaryotic simple reverse transcription system consisting of a non-coding template RNA and a reverse transcriptase (RT) pair, have recently attracted attention due to their small genome size, simple mechanism, programmability, and ability to produce up to 50,000 single-stranded DNA units per cell. Because of these engineerable properties, retrons have been reused to generate template DNA for homologous recombination and CRISPR-Cas adaptation, advancing dynamic genome engineering in bacteria, yeast, and even human cells. Retron-derived DNA (retron-DNA) has a little-known feature, a double-stranded hairpin structure, which is common to most retrons discovered so far. The lack of evolutionarily conserved sequences in this domain suggests that retron-DNA could be reprogrammed to mediate sequence-specific protein binding.

Accordingly, the inventors of the present invention completed the present invention by developing pretroDNA ( P protein-binding small DNA in situ generated by engineered RETRO n) by reprogramming the double-stranded domain of retron-DNA specific protein binding (Fig. 1b), thereby designing a new method to control, design, and utilize the interaction network of DNA and proteins in living cells (Figs. 1c and 1d).

The purpose of the present invention is to provide an engineered retron comprising an msr region; an msd region comprising a DNA-binding protein specific binding site; and a region encoding reverse transcriptase, an expression vector comprising the engineered retron, and a host cell transformed with the expression vector.

Another object of the present invention is to provide a gene expression system comprising a nucleic acid construct comprising an engineered retron comprising an msr region; an msd region comprising a DNA-binding protein-specific binding site; and a region encoding a reverse transcriptase; and a base sequence encoding a DNA-binding protein, an expression vector comprising the gene expression system, and a host cell transformed with the expression vector.

Another object of the present invention is to provide a gene expression system comprising a nucleic acid construct comprising an engineered retron comprising an msr region; an msd region comprising a DNA-binding protein-specific binding site; and a region encoding a fusion protein comprising a cargo protein and a reverse transcriptase; and a base sequence encoding a fusion protein comprising a DNA-binding protein and a protein that is localized within a cell, an expression vector comprising the gene expression system, and a host cell transformed with the expression vector.

Another object of the present invention is to provide a composition for regulating the intracellular localization or activity of a cargo protein, comprising a gene expression system comprising a nucleic acid construct comprising a base sequence encoding a fusion protein comprising a DNA binding protein and a protein having a designated intracellular localization; and a method for regulating the intracellular localization or activity of a cargo protein, comprising a step of expressing the gene expression system.

Another object of the present invention is to provide a gene expression system comprising an engineered retron comprising an msr region; an msd region comprising first and second DNA binding protein specific binding sites; and a region encoding a reverse transcriptase; a nucleic acid construct comprising a base sequence encoding a fusion protein comprising a first DNA binding protein and the first protein; and a nucleic acid construct comprising a base sequence encoding a fusion protein comprising a second DNA binding protein and the second protein; and a method for co-localization between proteins comprising a step of expressing the same.

To achieve the above object, the present invention provides an engineered retron comprising an msr region; an msd region including a DNA binding protein specific binding site; and a region encoding a reverse transcriptase, an expression vector comprising the engineered retron, and a host cell transformed with the expression vector.

In addition, the present invention provides a gene expression system comprising a nucleic acid construct comprising an engineered retron comprising an msr region; an msd region comprising a DNA-binding protein-specific binding site; and a region encoding a reverse transcriptase; and a base sequence encoding a DNA-binding protein, an expression vector comprising the gene expression system, and a host cell transformed with the expression vector.

In addition, the present invention provides a gene expression system comprising a nucleic acid construct comprising an engineered retron comprising an msr region; an msd region comprising a DNA-binding protein-specific binding site; and a region encoding a fusion protein comprising a cargo protein and a reverse transcriptase; and a base sequence encoding a fusion protein comprising a DNA-binding protein and a protein that is localized within a cell, an expression vector comprising the gene expression system, and a host cell transformed with the expression vector.

In addition, the present invention provides a composition for regulating the intracellular localization or activity of a cargo protein, comprising a gene expression system comprising an engineered retron comprising an msr region; an msd region comprising a DNA-binding protein-specific binding site; and a region encoding a fusion protein comprising a cargo protein and a reverse transcriptase; and a nucleic acid construct comprising a base sequence encoding a fusion protein comprising a DNA-binding protein and a protein having a cell-localized location; and a method for regulating the intracellular localization or activity of a cargo protein, comprising a step of expressing the gene expression system.

In addition, the present invention provides a gene expression system comprising an engineered retron comprising an msr region; an msd region comprising first and second DNA binding protein specific binding sites; and a region encoding a reverse transcriptase; a nucleic acid construct comprising a base sequence encoding a fusion protein comprising a first DNA binding protein and the first protein; and a nucleic acid construct comprising a base sequence encoding a fusion protein comprising a second DNA binding protein and the second protein; and a method for co-localization between proteins comprising a step of expressing the same.

The present invention relates to an engineered retron comprising a DNA-binding protein-specific binding site and uses thereof, and by using the engineered retron comprising a DNA-binding protein-specific binding site, not only transcription control but also a self-regulating feedback circuit is implemented, and a system for regulating the intracellular localization and function of a protein is developed, and therefore, it can be utilized as a biosensor, a memory device, an artificial adaptive circuit, a system for regulating the intracellular localization of a protein, and a system for controlling the function and localization at the post-translational level of a protein.

Figure 1a shows the transcriptional mechanism of bacterial retrons, which consists of msr-msd and reverse transcriptase (RT) genes. msr-msd is transcribed into a noncoding template RNA with a characteristic structure, and the long hairpin domain of the template RNA encoded by msd is converted into retron-DNA by RT and RNase H. The final product is a DNA/RNA/protein complex, in which the noncoding RNA is covalently linked to retron-DNA via a 2',5'-phosphodiester linkage, and RT remains bound to retron-DNA after reverse transcription is completed.

Figure 1b shows pretroDNA with reprogrammed msd region. The number of copies of pretroDNA in a cell can be controlled by controlling expression using pretroDNA encoding specific protein binding sequences in a fully double-stranded hairpin domain and a stimuli-inducible promoter.

Figure 1c shows an automatic feedback circuit using pretroDNA encoding a transcription factor binding “operator” sequence.

Figure 1d shows the mechanism of regulating the intracellular localization and function of proteins using allosteric transcription factors and pretroDNA.

Figure 2a shows the pRetro plasmid encoding the IPTG-inducible retron-DNA production genetic circuit. The red box indicates the hairpin region to be reprogrammed.

Figure 2b shows the results of quantitative PCR analysis showing that the number of copies of retron-DNA is increased by IPTG in E. coli harboring pRetro.

Figure 2c shows the results of capillary electrophoresis analysis of retron-DNA mutants produced in E. coli to analyze the programmability of the hairpin region. Peak areas indicate relative intracellular productivity compared to WT retron-DNA. 0.5 mM IPTG was used (nd, not detected).

Figure 2d shows pretroDNA_tetO and pretroDNA_luxO, which encode TetR and LuxR binding sequences, respectively, in the hairpin region.

Figure 2e shows the results of capillary electrophoresis analysis of pretroDNA produced in E. coli using 0.5 mM IPTG.

Figure 2f shows dynamic fine-tuning of the aTc-responsive TetR/P _LtetO-1 genetic circuit by pretroDNA_tetO-mediated TetR decoding.

Figure 2g shows the results of confirming GFP expression using the circuit of Figure 2f.

Figure 2h shows dynamic fine-tuning of the 3OC6HSL-responsive LuxR/P35LB10 genetic circuit by pretroDNA_luxO-mediated LuxR decoying. ASV is a degradation tag to rapidly synchronize the tuned transcriptional activity of P _35LB10 with measured GFP levels.

Figure 2i shows the results of confirming GFP expression using the circuit of Figure 2h.

Figure 2j shows the co-expression mechanism of pretroDNA_tetO and pretroDNA_luxO.

Figure 2k shows that co-expression of pretroDNA_tetO and pretroDNA_luxO demonstrates parallel TF-decoding functions without crosstalk.

Figure 3a shows the integration of pretroDNA_tetO-based positive feedback and TetR/P _LtetO-1- based aTc biosensor circuits.

Figure 3b shows that the A1/A2 region of the template RNA was expanded to increase pretroDNA production and enhance feedback.

Figure 3c shows that positive feedback maintains activation of P _LtetO-1 after aTc removal, thereby converting transient exposure to aTc into long-term GFP expression.

Figure 3d shows the pretroDNA_luxO based negative feedback and its integration with the LuxR/P _35LB10 circuit.

Figure 3e shows the results showing that negative feedback imparts signal recovery response dynamics to the LuxR/P _35LB10 circuit.

Figure 3f shows the results showing that negative feedback imparts signal recovery response dynamics to the LuxR/P _35LB10 circuit.

Figure 4a shows the results of evaluating PretroDNA_tetO generated by mCherry fusion RT in E. coli.

Figure 4b shows the binding of Tar-TetR-GFP localized to the membrane pole and mCherry-RT with the pretroDNA_tetO tag.

Figure 4c shows the results confirming co-localization of mCherry-RT with the PretroDNA_tetO tag and Tsr-TetR-GFP at the inner membrane pole.

Figure 4d shows the results confirming co-localization of mCherry-RT with the PretroDNA_tetO tag and Tsr-TetR-GFP at the inner membrane pole.

Figure 4e shows the co-localization of mCherry-RT with the PretroDNA_tetO tag and PopZ-TetR-GFP, which forms condensates at the poles.

Figure 5a is a schematic diagram to confirm that the subcellular localization of mCherry-RT with pretroDNA_tetO tag is reversed by aTc from the inner membrane pole with Tar-TetR-GFP to a uniform distribution throughout the cell.

Figure 5b shows the results of fluorescence microscopy observation according to the concentration of aTc.

Figure 5c shows the results of analyzing the Im/Ic ratio according to the concentration of aTc.

Figure 5d shows the results of time-dependent fluorescence microscopy observations upon exposure to aTc.

Figure 5e shows the results of analyzing the Im/Ic ratio over time when exposed to aTc.

Figure 5f shows that Cre monomers form a catalytically active tetrahedral complex to perform cleavage, reconstituting the reporter plasmid to constitutively express RFP.

Figure 5g shows the activity regulation mechanism of petroDNA_tetO-tagged Cre. PretroDNA_tetO-tagged Cre, which is locally concentrated in PopZ-TetR-GFP condensates, can promote cleavage, which can be counteracted by aTc. Cleavage activity can be confirmed by measuring RFP expression in subcultured cells.

Figure 5h shows RFP levels of subcultured cells measured by a plate reader. P _lux was activated by 100 nM 3OC ₆ HSL and Ptac was activated by 0.025 mM IPTG.

Figure 5i shows the results of flow cytometry analysis of subcultured cells to determine whether cells were exposed to aTc. P _lux was activated by 100 nM 3OC ₆ HSL, and P _tac was activated by 0.025 mM IPTG. NC represents the negative control without IPTG, and PC represents the positive control with 0.25 mM IPTG.

Figure 5j shows a representative image of Figure 5i.

Figure 6a is a schematic diagram showing the reassembly of ZF fused split YFPs (PBSII-nYFP and Zif268-cYFP) onto the petroDNA_ZF scaffold containing BSII and Zif268 binding sequences. PBSII-nYFP and Zif268-cYFP are expressed by the arabinose-inducible P _BAD promoter.

Figure 6b shows the results of fluorescence analysis of cells expressing each retron-DNA scaffold or control as described below. PBSII-nYFP and Zif268-cYFP expression were induced by 0.01% arabinose, and retron-DNA including pretroDNA_ZF expression was induced by 0.5 mM IPTG (**P<0.01, ***P<0.001, ****P<0.0001).

The present invention is described in detail below.

The present invention provides an engineered retron comprising an msr region; an msd region comprising a DNA binding protein specific binding site; and a region encoding a reverse transcriptase.

In the present invention, retron is a unique genetic element found in the bacterial genome, and retron DNA controls the transcription of a simple operon consisting of three loci: msr, msd, and ret . Retron encodes reverse transcriptase (RT) and ncRNA (non-coding RNA), and when retron ncRNA (msr-msd) is transcribed, it folds into a typical structure recognized by reverse transcriptase. Then, reverse transcriptase reversely transcribes a portion of ncRNA (msd) starting from the 2′ end of a conserved guanosine residue found immediately after the double-stranded RNA structure in ncRNA. During reverse transcription, cellular RNase H degrades the segment of ncRNA that serves as a template, but does not degrade the other portion of ncRNA (msr), thereby generating a mature RNA-DNA hybrid (multicopy single-stranded DNA, msDNA).

In the present invention, the msr gene, msd gene and ret gene used in the engineered retron can be derived from bacterial operons, preferably Escherichia coli retron (e.g., Ec48, E67, Ec73, Ec78, EC83, EC86, EC107, and Ec107), myxobacterial retron such as Myxococcus xanthus retron (e.g., Mx65, Mx162) and Stigmatella aurantiaca retron (e.g., Sa163); Salmonella enterica; Vibrio cholerae retron (e.g., Vc81, Vc95, Vc137); Vibrio parahaemolyticus (e.g., Vc96); and Nannocystis exedens letrone (e.g., Ne144).

In the present invention, the "DNA binding protein" is a protein having a DNA binding domain and a specific or non-specific affinity for single- or double-stranded DNA. DNA binding proteins are composed of many kinds of proteins that can physically interact with DNA. These proteins perform various functions, such as chromosomal DNA organization/compaction (e.g., histones), initiation and regulation of transcription, DNA replication, and/or DNA recombination (e.g., transcription factors, polymerases), and DNA modification (e.g., endonucleases, demethylases). Some DNA binding proteins exhibit DNA sequence-specific interactions, while others have no or minimal sequence recognition functions. In general, DNA binding proteins contain a DNA binding domain necessary for physical interaction between the protein and DNA, while enzymatic or regulatory functions are generally performed through separate domains within the protein. DNA binding proteins include domains such as zinc fingers, helix-turn-helix, and leucine zippers to further strengthen binding to nucleic acids. DNA binding proteins may include transcription factors that regulate the transcription process, various polymerases, nucleases that cleave DNA molecules, and histone proteins involved in chromosome condensation and transcription in the cell nucleus, and preferably, the DNA binding protein may be a transcription factor, but is not limited thereto.

In the present invention, a "transcription factor" is a DNA binding protein that regulates transcription by binding to a specific DNA sequence near or within the coding region of a gene. A transcription factor that promotes the initiation of transcription is called an activator, and a protein that prevents a transcription factor from binding to the transcription initiation site is called a repressor.

In the present invention, the "allosteric transcription factor" regulates transcription by undergoing a conformational change that changes its affinity for an operator DNA sequence in response to specific allosteric stimuli such as biomarkers, heavy metals, antibiotics, light, and heat. The conformational change of the transcription factor causes it to detach from or bind to a DNA sequence upstream of a target gene to activate or repress its expression. The transcription factor can be assembled with other DNA segments commonly used in synthetic biology, such as promoters, ribosome binding sites (RBSs), terminators, and reporter genes, to create a transcription factor-based biosensor circuit. Thus, such genetic devices can be used to sense and respond to various intracellular or environmental ligand concentrations. The above “allosteric transcription factor” may be, but is not limited to, TetR, LuxR, LacI, NahR, DmpR, XylR, AraC, PobR, TlpA, cI, EL222, ArsR or QacR.

In the present invention, the msr region, which is not reverse transcribed, forms 1 to 3 short stem loops or various sizes ranging from 3 to 10 bp, while the msd region folds into single/double long hairpins and exists in a wide variety of long stem forms ranging from 10 to 50 bp in length, which are also included in the final msDNA form. In one embodiment of the present invention, the stem region of the msd region can be completely hybridized. "Hybridization" refers to the formation of a complex between nucleotide sequences that are sufficiently complementary to form a complex through Watson-Crick base pairing. In one embodiment of the present invention, the length of the stem region may be 10-50 bp, and preferably 21 bp or more, but is not limited thereto.

In the present invention, the engineered retron can be operably linked to a promoter and can be implemented as a self-feedback circuit. In one embodiment of the present invention, the allosteric transcription factor TetR binds to tetO of P _LtetO-1 in the absence of anhydrotetracycline (aTc), which is an allosteric inducer, to repress a downstream gene. In another embodiment, the allosteric transcription factor LuxR is normally dissociated from LuxO in P _35LB10 , and 3OC ₆ HSL (P35LB1039, 3-oxohexanoyl-homoserine lactone) causes LuxR to bind to LuxO to repress a downstream gene.

Additionally, the present invention provides an expression vector comprising the engineered retron.

In the present invention, the "vector" is a composition of matter that can be used to deliver a nucleic acid of interest into the interior of a cell. The retron msr gene, the msd gene, and the ret gene can be introduced into a cell with a single vector or introduced in multiple separate vectors to produce msDNA in a host subject. The vector typically includes control elements operably linked to the retron sequences, which enable in vivo production of msDNA in a subject species. For example, the retron msr gene, the msd gene, and the ret gene can be operably linked to a promoter to allow expression of retron reverse transcriptase and the msDNA product.

In the present invention, the expression vector is a viral vector or a non-viral vector (e.g., a plasmid).

In addition, the present invention provides a host cell transformed with the expression vector.

In the present invention, an isolated host cell is provided, wherein the host cell comprises an engineered retron or vector system as described herein.

In the present invention, the host cell is a prokaryotic, archaeon, or eukaryotic host cell. For example, the host cell can be a bacterial, protist, fungal, animal, or plant host cell. In some embodiments, the host cell is a mammalian host cell. The host cell can be a human or non-human mammalian host cell. In other embodiments, the host cell is an artificial cell or a genetically modified cell.

Methods for introducing nucleic acids into host cells are well known in the art. Commonly used methods include chemically induced transfection, typically using divalent cations (e.g., CaCl ₂ ), dextran-mediated transfection, polybrene-mediated transfection, lipofectamine and LT-1-mediated transfection, electroporation, protoplast fusion, encapsulation of nucleic acids in liposomes, and direct microinjection of nucleic acids containing engineered retrons into the nucleus of cells.

The present invention also provides a gene expression system comprising a nucleic acid construct comprising an engineered retron comprising an msr region; an msd region comprising a DNA binding protein specific binding site; and a region encoding a reverse transcriptase; and a base sequence encoding a DNA binding protein.

In the present invention, the region encoding the reverse transcriptase may be linked to a base sequence encoding a cargo protein. The cargo protein may be linked to the N-terminus or the C-terminus of the reverse transcriptase. The cargo protein and the reverse transcriptase may be linked via a linker, and the linker may be, but is not limited to, GGGGS, (GGGGS) ₂ , (GGGGS) ₃ , EAAAK, (EAAAK) ₂ , or (EAAAK) ₃ .

In the present invention, the cargo protein is a functional regulatory substance that has biological activity that regulates all physiological phenomena in a living body by being delivered into cells, and refers to a chemical substance such as a compound that can regulate the function of a cell.

In the present invention, the base sequence encoding the DNA binding protein can be linked to a base sequence encoding a protein having a designated intracellular location. The protein having a designated intracellular location can be any one selected from the group consisting of Tar, Tsr, TatA, mreB, ftsZ, and PopZ, but is not limited thereto.

Additionally, the present invention provides an expression vector comprising the gene expression system.

In the present invention, the engineered retron and nucleic acid constructs can be introduced into cells with a single vector or introduced into multiple separate vectors.

In addition, the present invention provides a host cell transformed with the expression vector.

The present invention also provides a gene expression system comprising a nucleic acid construct comprising an engineered retron comprising an msr region; an msd region comprising a DNA binding protein specific binding site; and a region encoding a fusion protein comprising a cargo protein and a reverse transcriptase; and a base sequence encoding a fusion protein comprising a DNA binding protein and a protein that is localized within a cell.

Additionally, the present invention provides an expression vector comprising the gene expression system.

In addition, the present invention provides a host cell transformed with the expression vector.

The present invention also provides a composition for modulating the intracellular localization or activity of a cargo protein. For example, the composition can include a gene expression system comprising a nucleic acid construct comprising an engineered retron comprising an msr region; an msd region comprising a DNA binding protein specific binding site; and a region encoding a fusion protein comprising a cargo protein and a reverse transcriptase; and a base sequence encoding a fusion protein comprising a DNA binding protein and a protein having a designated intracellular localization.

The present invention also provides a method for regulating the intracellular localization or activity of a cargo protein. The method can include the step of expressing a gene expression system comprising a nucleic acid construct comprising an engineered retron comprising an msr region; an msd region comprising a DNA binding protein specific binding site; and a region encoding a fusion protein comprising a cargo protein and a reverse transcriptase; and a base sequence encoding a fusion protein comprising a DNA binding protein and a protein having a designated intracellular localization.

In addition, the present invention also provides a gene expression system comprising an engineered retron comprising an msr region; an msd region comprising first and second DNA binding protein specific binding sites; and a region encoding a reverse transcriptase; a nucleic acid construct comprising a base sequence encoding a fusion protein comprising a first DNA binding protein and the first protein; and a nucleic acid construct comprising a base sequence encoding a fusion protein comprising a second DNA binding protein and the second protein; and a method for co-localization between proteins comprising a step of expressing the same.

Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

Hereinafter, the present invention will be described in more detail through examples and experimental examples.

It will be apparent to those skilled in the art that these examples and experimental examples are only intended to illustrate the present invention and that the scope of the present invention is not to be construed as being limited by them.

<Example 1> Experimental method

Strains and growth conditions

All cloning, retron-DNA biosynthesis, genetic circuit characterization, and fluorescence microscopy experiments were performed using E. coli DH5a. The plasmids used in this experiment are detailed in Table 1, and the combinations of plasmids used in each experiment are listed in Table 2.

[Table 1]

[Table 2]

Cells were cultured in LB (Formedium) at 37°C with appropriate antibiotics and inducers. All antibiotics were purchased from GoldBio and used at concentrations of ampicillin 100 μg/ml, kanamycin 50 μg/ml, chloramphenicol 25 μg/ml, and spectinomycin 100 μg/ml.

For biosynthesis and extraction of retron-DNA (Figs. 2b, 2c, 2e, and 4a), a single colony from an LB plate was inoculated into 3 ml of LB and shaken overnight at 240 rpm. The culture was then diluted 1:100 into 6 ml of LB and cultured for 2 h. After 0.5 mM IPTG was added, the culture was further cultured for 5 h. The culture was then centrifuged to pellet, stored at -20°C, and used for retron-DNA extraction.

For genetic circuit characterization, qPCR analysis, and microscopic observation, transformants from single colonies transformed on LB plates were inoculated into 300 μl of LB in deep-well plates with breathable sealing films, covered with sealing films, and shaken at 1000 rpm overnight. Then, the culture was diluted 1:100 with 200 μl of LB in 96-well black plates (SPL) with transparent bottoms, cultured for 80 min, and then appropriate inducers were added and further cultured. Fluorescence and cell density measurements were performed 3 h after the addition of inducers (Figs. 2g, 2i, and 2k). Cell collection for qPCR analysis was performed at 5 h (Fig. 2b). Microscopic observations were performed at 8 h (Fig. 4b, c), 2 h (Fig. 4d), and 6 h (Fig. 4e) (Fig. 4b, c), and dynamic protein subcellular localization was performed at 8 h (Figs. 5b to 5e).

For positive feedback characterization to enhance aTc biosensor sensitivity (Fig. 3b), the overnight seed culture was diluted 1:100, aTc was added, and cultured in 200 μl of LB medium for 3 h. Then, the fluorescence and cell density of the culture were measured. For memory characteristic analysis (Fig. 3c), the overnight seed culture was diluted 1:100 in 200 μl of LB medium, 100 ng/mL aTc was added, and cultured for 3.5 h. After that, the cells were pelleted and washed with fresh LB medium. The cells were then diluted 1:100 again in fresh LB medium and cultured for 5–6 h. Then, they were diluted 1:100 again, and this process was repeated four times. The GFP level and OD were measured immediately before each dilution.

In the characterization of negative feedback (Fig. 3e and 3f), overnight seed cultures were diluted 1:100 in 200 μl of LB medium, cultured for 2.5 h, and treated with 3OC ₆ HSL. The cells were then further cultured for 16 h. GFP levels and OD were monitored every 10 min.

Plasmid production

Plasmids were constructed using standard molecular cloning techniques and Gibson assembly. The gene sequences used in the present invention are shown in Table 3. All primers (Table 4) were purchased from Bionics (Korea).

[Table 3]

[Table 4]

The Eco1 retron sequence of pFF753, the Ptac sequence (BBa_K864400), PJ23106 (BBa_J23106), and the terminator sequence of pXW117Ptet2-gfp (Addgene #160818) were cloned into pCDFDuet-1 to generate pRetro. The dRT sequence of pFF758 was cloned into pRetro to generate pRetro_dRT. pFF753 (Addgene #61453) and pFF758 (Addgene #61454) were generously provided by T. Lu at MIT. The msd variant sequences were cloned into pRetro to generate mutants of pRetro, including pPretro_tetO and pPretro_luxO. pRetro_str_1, pRetro_str_2, and pRetro_str_3 were generated by site-directed mutagenesis in pRetro.

pXW117Ptet2-gfp was used as the TetR/P tetO-1 circuit plasmid pTet_GFP. The ASV cleavage tag and P _35LB10 sequences were cloned into pXW117Ptet2-gfp (Addgene #160819) to generate the LuxR/P _35LB10 plasmid pLux_GFP. pXW117Ptet2-gfp and pXW101Plux2-gfp were generous gifts from B. Wang, University of Edinburgh. The Cherry sequence was cloned into the pSC101 plasmid to generate pTet_mCherry. The P _LtetO-1 sequence of pXW117Ptet2-gfp, the pretroDNA_tetO gene sequence of pPretroDNA_tetO, and the RT sequence of pFF753 were cloned into pCDFDuet-1 to generate pTet_pretroDNA_tetO. The dRT sequence of pFF758 was cloned into pTet_pretroDNA_tetO to generate pTet_pretroDNA_tetO_dRT.

The pretroDNA_tetO sequence PCR amplified with the extended a1/a2 region was cloned into pTet_pretroDNA_tetO and Tet_pretroDNA_tetO_dRT, generating pTet_pretroDNA_tetO_a23 and pTet_pretroDNA_tetO_a23_dRT, respectively. The P _J23115 sequence (BBa_J23115) was cloned into pRetro, pPretro_tetO, and pPretro_luxO, generating pRetro_115, pPretro_tetO_115, and pPretro_luxO_115, respectively. pretroDNA_tetO was PCR amplified with the substituted a1/a2 region and cloned into pPretro_luxO_115, generating pPretro_tetO_luxO_J115.

The tar sequence was PCR amplified from E. coli MG1655, and the tetR and gfp sequences from pXW117Ptet2-gfp and the luxR and P _lux sequences from pXW101Plux2-gfp were cloned into pCDFDuet-1, generating pTar-TetR-GFP. The tsr and tatA sequences of E. coli MG1655 and the popZ sequence of NSSCP (Addgene #183190) were cloned into pTar-TetR-GFP, generating pTsrTetR-GFP, pTatA-TetR-GFP, and pPopZ-TetR-GFP, respectively. NSSCP was provided by H. Huang, National Taiwan University. The retron cassette sequence of pRetro_115 and the mCherry sequence were cloned into the pACYCDuet-1 plasmid to generate pRetro_mCherry-RT and pRetro_RT-mCherry.

Retron-DNA extraction and analysis

To compare the relative productivity of retron-DNA mutants with that of WT retron-DNA per cell, equal amounts of cells were harvested for each experiment based on OD600 measurements. DNA was extracted from pelleted cells using a plasmid miniprep kit (Enzynomics) and then treated with RNase A/T1 mixture (Thermo Fischer) for 30 min at 37°C. Extracted retron-DNA was analyzed by capillary electrophoresis using a Qsep-1 (Bioptic) instrument with a standard quantitation cartridge (S2) and Q-Analyzer software (Figs. 2c, 2e; 4a). 25/100 bp Mixed DNA Ladder (Bioneer D-1020) was used as a marker. For sequencing analysis, retroDNA was separated by agarose electrophoresis and gel extraction, and then amplified using a PCR extender and Seq_1/2/3 primers (Table 3). Specifically, the extracted retroDNA and Seq_1 were annealed to each other, extended by a polymerase reaction, and then amplified by PCR using Seq_2/3 primers. The generated PCR products were analyzed by Sanger sequencing.

Quantitative PCR

The relative copy number of retron-DNA to the intracellular encoding plasmid was measured by qPCR analysis using two sets of primers. The region outside the msd site that was present only in the plasmid was analyzed using primer set 1 (qPCR_plasmid_F/R), and the region within the msd site that was present in both retron-DNA and the plasmid was analyzed using primer set 2 (qPCR_rtDNA_F/R) (Table 4). The difference in cycle threshold (Ct) between primer sets 1 and 2 was used as the ΔCt value. qPCR of a mixture of synoligo_rtDNA and purified pFF758 plasmid at a range of copy number ratios was performed to generate a standard curve of ΔCt values against copy number ratios. For analysis of copy number ratios in E. coli cells, 5 μl of cell culture was collected, mixed with 5 μl of dilution water, and incubated at 95 °C for 5 min. The heated culture was diluted 1:100 with water, and 1 μl of it was used in a 20 μl reaction with 2X qPCR premix (highQu ORA qPCR Green ROX L Mix) and 0.4 μM of each primer. qPCR was performed using a CFX Opus RealTime PCR system (Bio-rad). The copy number ratio was calculated using a standard curve from the measured ΔCt values.

Optical density and fluorescence measurements using a plate reader

GFP, mCherry, and cell density were measured using a plate reader (Agilent BioTek Synergy H1). The excitation and emission wavelengths for GFP are 479/520 nm, and for mCherry, 587/620 nm. The absorbance at 600 nm was measured as optical density to determine the cell density. Each measurement was performed with an equal volume of blank LB medium control for normalization. In Fig. 3b, a well of empty vector without aTc was used as a blank to distinguish subtle differences. For continuous time course analysis (Fig. 3c, 3e), fluorescence and optical density levels were measured every 10 min while shaking at 800 rpm and incubating at 37°C in a plate reader.

Fluorescence microscopy analysis

Agarose pads were prepared using TE buffer containing 1% agarose and placed on slide glasses. To observe live cells, 1 μl of cell culture was pipetted onto the agarose pad and covered with a cover glass. A Zeiss Axioscope A1 fluorescence microscope equipped with a 100x Plan-Neofluar oil immersion objective was used for microscopic observation. GFP signals were detected using Zeiss filter set 38 (470/40 nm excitation filter, 495 nm beam splitter, 525/50 nm emission filter), and mCherry signals were detected using Zeiss filter set 20 (546/12 nm excitation filter, 560 nm beam splitter, 575-640 emission filter). Digital images were captured using an AxioCam HRm camera. For dynamic control of protein subcellular localization (Fig. 5), cultured cells were pelleted and washed twice with M9 minimal medium (Difco M9 Minimal salts, 5x). The cells were then incubated with aTc and observed using a microscope.

Brightness adjustment, thresholding, and merging of images were processed using Adobe Photoshop. The same drawings were processed under identical conditions. The cell distribution in Fig. 4b represents the intracellular distribution of mCherry fluorescence along the medial axis and was generated using MicrobeJ. Normalized intensity profiles were used. At least 10 images (n = 3 biological replicates) from different fields of view were analyzed. To facilitate the analysis, clustered cells and falsely detected spots (area <0.01) were manually excluded. The detected cells were then sorted in ascending order of length. Im and Ic values were determined from half of the normalized mCherry intensity profile generated using MicrobeJ along the axis of each cell. Three images from n = 3 biological replicates were analyzed.

<Experimental Example 1> Reprogramming of retron-DNA

To perform experiments to program the stem region of retron-DNA to induce sequence-specific DNA-protein interactions, we used a plasmid encoding an engineered Eco1-retron derived from E. coli BL21 with a constitutively expressed reverse transcriptase and a template RNA driven by IPTG-inducible Ptac (Fig. 2a).

<1-1> Evaluation of copy number of retron-DNA

First, quantitative PCR analysis was performed using two primer sets to determine the relative copy-number ratio of retron-DNA to plasmid. The first primer anneals to both the retron-DNA and msd regions of the plasmid, and the second primer anneals only to the plasmid.

As a result, the ratio of retron to plasmid DNA in E. coli treated with IPTG reached 538 (±31.8 SD) (Fig. 2b), which was 84-fold higher than that in the case not treated with IPTG (6.4 ± 1.7 SD), indicating that the copy number of retron-DNA can be dynamically controlled. Considering that the copy number of plasmids with CloDF13 replication origin is known to be 20–40 per cell, the number of retron-DNA copies per cell amounts to 10,760–21,520.

<1-2> Evaluation of programmability of retron-DNA

Next, we generated retron-DNA mutants and evaluated their productivity to investigate the programmability of the stem region (Fig. 2c).

As a result, it was confirmed that when the base sequence was randomly mutated without structural change, the productivity was similar to that of the wild-type retron-DNA, whereas when the bases of the internal loop were mutated into hybridized base pairs, the productivity decreased. These results suggest that the copy number of retron-DNA is mainly determined by structural factors rather than the base sequence itself.

Since most protein-bound DNAs are completely double-stranded, we confirmed changes in productivity by varying the strand length (17–25 bp) while maintaining complete hybridization.

As a result, when it was 21 bp or longer, the productivity was reduced by 90% compared to the wild-type retron-DNA. However, the 90% reduced copy number was 1,076–2,125 per cell, which is still higher than the average protein copy number of E. coli (~1,000 per cell). In addition, considering that most transcription factor (TF) binding sequences are in the range of 5–20 bp, the available stem length range seems to be sufficient.

These results demonstrate that the double-stranded domain of retron-DNA has sufficient programmability to be re-encoded with specific protein-binding sequences.

<Experimental Example 2> Production of pretroDNA encoding a sequence that binds to a transcription factor

To investigate whether the copy number can be dynamically controlled using pretroDNA encoding a transcription factor (TF)-binding sequence, we used TetR and LuxR as target transcription factors. TetR binds to tetO of P _LtetO-1 in the absence of the allosteric inducer anhydrotetracycline (aTc) and represses downstream genes. In contrast, LuxR is normally dissociated from LuxO in P _35LB10 , and 3OC ₆ HSL (P35LB1039, 3-oxohexanoyl-homoserine lactone) causes LuxR to bind to LuxO and repress downstream genes.

Specifically, pretroDNA_tetO and pretroDNA_luxO, which encode tetO (19 bp) or luxO (20 bp) sequences that bind to TetR and LuxR, respectively, in the hairpin region, were produced. Sanger sequencing analysis of the pretroDNA produced in E. coli confirmed that the designed sequence (top) and the analyzed sequence (bottom) were identical (Fig. 2d).

Additionally, capillary electrophoresis analysis of pretroDNA produced in E. coli using 0.5 mM IPTG was performed to confirm intracellular production (Fig. 2e), confirming that sufficient copies were produced for protein binding.

<Experimental Example 3> Verification of decoy ability of pretroDNA

<3-1> Verification of decoy ability of pretroDNA_tetO

pretroDNA_tetO for TetR To verify the decoy ability, pretroDNA_tetO and P _LtetO-1 We implemented the TetR/P _LtetO-1 circuit, in which GFP is repressed by TetR under operation (Fig. 2f).

As a result, we confirmed that pretroDNA_tetO induced by IPTG resulted in GFP expression, and that various profiles, such as aTc-GFP dose response, were generated simply by changing the IPTG concentration (Fig. 2g).

This demonstrates that pretroDNA_tetO can be used to dynamically control the copy number of a target gene.

<3-2> Verification of decoy ability of pretroDNA_luxO

To verify the decoy ability of pretroDNA_luxO for LuxR, we implemented a LuxR/P3 _5LB10 circuit in which GFP is repressed by LuxR bound to 3OC ₆ HSL under the operation of pretroDNA_luxO and P _35LB10 (Fig. 2h). ASV degradation-tagged GFP was used to rapidly synchronize the coordinated transcriptional activity of P35LB10 with the measured GFP level.

As a result, we confirmed that pretroDNA_luxO induced by IPTG leads to GFP expression, and dynamic fine-tuning of the circuit is achieved by varying the IPTG concentration (Fig. 2i).

<3-3> Verification of compatibility and orthogonality of pretroDNA

Experiments were performed to investigate the compatibility and orthogonality of the two pretroDNAs using the TetR/P _LtetO-1 circuit expressing mCherry and the LuxR/P _35LB10 circuit expressing GFP (Fig. 2j).

As a result, we confirmed that both mCherry and GFP outputs were turned on only when both pretroDNAs were expressed together, whereas only mCherry or GFP output signals were generated when only pretroDNA_tetO or pretroDNA_luxO was expressed, respectively (Fig. 2k).

Overall, these results confirm that pretroDNA can specifically interact with target proteins and that pretroDNA binding to transcription factors may play a generalizable role as a post-translational transcription factor regulator for the dynamic fine-tuning of transcription factor-based gene networks.

<Experimental Example 4> Dynamic feedback circuit configuration using pretroDNA/aTF

Taking advantage of the dynamic copy number regulation capability of the pretroDNA decoy, we re-linked the pretroDNA decoy to the target transcription factor-responsive promoter to establish an automated feedback loop (Fig. 1c).

<4-1> Positive feedback loop design

First, we designed a positive feedback loop in which pretroDNA_tetO, which is operably linked to P _LtetO-1 , is induced by aTc and then decoys TetR to amplify its own expression (Fig. 3a). Since positive feedback is useful for controlling leakage in biosensor development to improve sensitivity, we integrated pretroDNA_tetO-based feedback into the optimized TetR-based aTc biosensor used in Figs. 2f and 2g. This combined biosensor showed a significant improvement in sensitivity up to 0.14 ng/ml compared to the dRT control (Fig. 3b).

<4-2> Adjusting feedback strength through a1/a2 area engineering

Additionally, we attempted to tune the feedback strength by engineering the a1/a2 region of template RNA, which is known to affect retron-DNA productivity. Strengthening the feedback with an extended a1/a2 (23 bp) resulted in increased background noise, while further improving the sensitivity to ~0.05 ng/ml.

These results suggest that pretroDNA-based positive feedback can enhance the sensitivity of TF-based biosensors, and further optimization can be achieved through fine-tuning of the feedback strength.

<4-3> Inducing long-term memory using positive feedback

We investigated whether pretroDNA_tetO-based positive feedback could convert transient aTc exposure into long-term memory.

To test this, cells were first cultured with 100 ng/ml aTc for 3.5 h, then washed and regularly diluted with fresh medium lacking aTc.

As a result, dRT and empty vector controls showed a rapid decrease in GFP to basal levels 5 h after aTc removal (Fig. 3c), but the positive feedback circuit (a12) showed a much slower decay, maintaining 82% and 29% at 5 and 22 h, respectively. Another circuit with stronger feedback (a23) showed an extended memory with no significant decrease in GFP up to 18 h.

<4-4> Negative feedback loop design

We constructed a negative feedback loop using pretroDNA_luxO and LuxR and integrated it with the LuxR/P _35LB10 circuit (Fig. 3d). In this coupled circuit, 3OC ₆ HSL represses GFP by P _35LB10 operation and simultaneously induces pretroDNA_luxO under the promoter P _lux , which is activated by LuxR bound to 3OC ₆ HSL. We expected that this circuit would exhibit a dynamic “decay-then-recovery” response in GFP expression due to the timing difference between immediate repression of GFP and delayed pretroDNA_luxO-mediated derepression (Fig. 3f). The expected response kinetics were observed in cells with negative feedback, whereas the dRT control only exhibited decay without recovery (Figs. 3e and 3f).

Taken together, these results demonstrate that the dynamic copy number controllability of pretroDNA can be utilized in combination with regulatory transcription systems to form automated feedback circuits.

<Experimental Example 5> Using pretroDNA/aTF Regulation of protein localization within cells

<5-1> Control of intracellular location

Experiments were performed to control protein subcellular localization using allosteric transcription factors (aTFs) and preroDNA.

It is known that reverse transcriptase remains bound to retron-DNA even after reverse transcription is completed (Fig. 1a), suggesting that retron-DNA can tag arbitrary protein “cargo” (Fig. 1d). To verify this concept, reverse transcriptase and cargo protein mCherry were fused with a (GGGGS) ₂ linker, and the reverse transcriptase performance of the fusion proteins was evaluated (Fig. 4a). We confirmed that pretroDNA_tetO was generated from both N- and C-terminal reverse transcriptase fusion mCherry, indicating that cargo proteins can be fused to both ends of reverse transcriptase.

We then verified whether mCherry (cargo) could be colocalized with the pre-localized TetR (bait) under the guidance of pretroDNA_tetO (prey). We used a Tar-TetR-GFP fusion protein as the pre-localized TetR, where Tar is a chemoreceptor protein localized at the poles. Colocalization of GFP and mCherry at the poles was observed in cells expressing pretroDNA_tetO and mCherry-RT (Fig. 4b), whereas in the controls using WT retorn-DNA or mCherry-dRT, the mCherry signal was uniformly distributed throughout the cytoplasm.

These results demonstrate that mCherry localization is induced by specific binding between the TetR domain and pretroDNA_tetO. Similar results were observed for RT-mCherry, suggesting that pretroDNA can be functionally tagged at both ends of the protein.

To investigate modularity, we then tested proteins present at other localizations, including Tsr, another inner membrane chemoreceptor similar to Tar, TatA, an inner membrane protein known to be evenly distributed throughout the membrane, and PopZ, a polar condensate-forming protein. Each protein was fused to TetR-GFP and co-expressed with pretroDNA_tetO and RT-mCherry. In contrast to WT retron-DNA, co-localization of GFP and mCherry at the expected locations was observed in all three cases ( Figures 4C–E ).

<5-2> Dynamic regulation of intracellular location

We performed experiments to dynamically switch the subcellular localization of specific stimulus-responsive proteins using allosteric transcription factors (aTFs) and preroDNA.

Specifically, we selected Tar-TetR-GFP, pretroDNA_tetO, and mCherry-RT to test whether the specific binding between Tar-TetR-GFP and pretroDNA_tetO/mCherry-RT complex could be post-translationally reversed by aTc (Fig. 5a).

To this end, cells were cultured in LB medium to express all components, then cultured for 1 h in M9 minimal medium without carbon source to attenuate further growth and gene expression before addition of aTc, and observed under a fluorescence microscope.

As a result, it was confirmed that the mCherry signal, which was concentrated at the poles, was relocated to a uniform distribution as the concentration of aTc increased (Fig. 5b).

To quantitatively analyze this, the ratio of the maximum intensity (I _m ) to the center intensity (I _c ) was calculated from the line-scan plot profiles of each cell (Fig. 5a). The I _m /I _c ratio is higher than 1 at the poles and equal to 1 for a uniform distribution. This ratio gradually decreased from 3.9 (±2.5, SD) when no aTc was used to 1.1 (±0.43, SD) when 10 ng/ml aTc was used (Fig. 5c).

Next, to investigate the time required for relocalization, cells were observed starting 5 minutes after the addition of aTc. As a result, as shown in Figures 5d and 5e, it was confirmed that relocalization was almost complete within 5 minutes and there was no noticeable change thereafter. This indicates that the transition occurs almost immediately at the post-translational level.

Overall, these results demonstrate that the specific stimulus-responsiveness of the aTF/pretroDNA pair can be used to dynamically regulate protein subcellular localization.

<Experimental Example 6> Using pretroDNA/aTF Regulation of protein function

<6-1> Regulation of protein function

To determine whether protein function can be regulated using an allosteric transcription factor (aTF) and pretroDNA, site-specific Cre recombinase was used as a cargo protein, an RFP-encoding reporter plasmid was used as a substrate for Cre, and PopZ-TetR-GFP condensate was used as a molecular hub. Cre excises the terminator sequence located between two loxP sites, modifying the reporter plasmid to constitutively express RFP.

Cre monomers sequentially bind to loxP sequences and then assemble into a catalytically active tetrahedral complex, where locally concentrated Cre promotes tetrahedralization and enhances recombination function. Recently, PopZ condensates have been repurposed as molecular scaffolds to locally concentrate the separase enzyme and enhance its catalytic activity in E. coli . Here, we investigated whether locally concentrating Cre in PopZ-TetR-GFP condensates via pretroDNA_tetO tagging would enhance excision activity, as confirmed by measuring RFP levels in cultured cells ( Fig. 5g ).

As a result, Cre tagged with pretroDNA_tetO showed higher RFP levels than the control without pretroDNA_tetO (Fig. 5h). Although the increase was offset by the addition of aTc, the RFP level was still slightly higher than the control without pretroDNA_tetO, possibly because post-reverse transcription reverse transcriptase dimerization partially promotes tetramerization.

These results demonstrate that the pretroDNA/aTF system can be used to post-transcriptionally control not only cargo localization but also its ability to respond to aTF inducers.

<6-2> Function and location control at the post-translational level of proteins

We performed experiments to determine whether the pretroDNA_tetO-mediated Cre control system can record brief aTc exposure events occurring in minimal medium without carbon sources. To do this, cells were cultured expressing all components in the absence of aTc, then transferred to M9 medium and exposed to aTc for various exposure times (Fig. 5i and 5j).

As a result, cultures of cells exposed to aTc showed significantly lower RFP levels compared to controls that were not exposed to aTc, indicating that aTc exposure almost irreversibly switches the post-translational functional state of pretroDNA_tetO-tagged Cre. In particular, a mere 1-min exposure was found to decrease RFP levels by nearly twofold. These results confirm that aTc can function as a recording device that converts immediate exposure to aTc for as short a time as 1 min into a genetic memory of readable fluorescence output changes in resource-poor environments.

Overall, these results suggest that application of pretroDNA/aTF can transform the inducible transcription system into a post-translational rapid effector that is readily activated by specific trigger stimuli and can perform predefined tasks even in resource-poor environments.

<Example 7> Co-localization of multiple proteins using pretroDNA

We selected PBSII and Zif268, representative zinc-fingers (ZFs) each having a 9-bp DNA-binding sequence, as targets, designed pretroDNA_PZ encoding two ZF-binding sequences in a single molecule, and performed a split YFP reassembly assay to verify whether pretroDNA_PZ can simultaneously interact with the two targets (Fig. 6a). PBSII and Zif268 were fused to the N-terminal and C-terminal parts of split YFP, respectively.

As a result, as shown in Fig. 6b, cells expressing pretroDNA_PZ showed a 6.4-fold increase in YFP fluorescence level compared to the dRT control, suggesting that co-localization with pretroDNA_PZ leads to proximity-induced reassembly of YFP. Interestingly, a slight increase in fluorescence was observed when pretroDNA encoding each ZF binding sequence was expressed, which may be due to enhanced stability or lifetime of the protein due to pretroDNA binding.

These results demonstrate that pretroDNA can not only interact with the ZF but also interact with two other proteins simultaneously, suggesting that pretroDNA may serve as a programmable molecular scaffold for co-localization of multiple proteins.

Claims (22)

An engineered retron comprising an msr region; an msd region comprising a DNA-binding protein specific binding site; and a region encoding reverse transcriptase. In the first paragraph, An engineered retron, characterized in that the DNA binding protein is an allosteric transcription factor. In the first paragraph, An engineered retron, characterized in that the engineered retron is utilized as a biosensor or a molecular scaffold. In the first paragraph, An engineered retron, characterized in that the stem region of the above msd domain is completely hybridized. In the second paragraph, An engineered letron, characterized in that the length of the stem portion is 21 bp or longer. An expression vector comprising the engineered retron of claim 1. A host cell transformed with the expression vector of claim 6. An engineered retron comprising an msr region; an msd region comprising a DNA binding protein specific binding site; and a region encoding a reverse transcriptase; and A nucleic acid construct comprising a base sequence encoding a DNA binding protein A gene expression system comprising: In Article 8, The above engineered retron is characterized in that it is used as a biosensor or molecular scaffold. In Article 8, A gene expression system, characterized in that the region encoding the above reverse transcriptase is linked to a base sequence encoding a cargo protein. In Article 10, A gene expression system, characterized in that the cargo protein is linked to the N-terminus or C-terminus of a reverse transcriptase. In Article 10. A gene expression system, characterized in that the cargo protein and reverse transcriptase are connected via a linker. In Article 12, A gene expression system, characterized in that the linker is GGGGS, (GGGGS) ₂ , (GGGGS) ₃ , EAAAK, (EAAAK) ₂ , or (EAAAK) ₃ . In Article 8, A gene expression system, characterized in that the base sequence encoding the DNA binding protein is linked to a base sequence encoding a protein whose intracellular location is specified. In Article 14, A gene expression system, wherein the intracellular localized protein is any one selected from the group consisting of Tar, Tsr, TatA, mreB, ftsZ, and PopZ. An engineered retron comprising an msr region; an msd region comprising a DNA binding protein specific binding site; and a region encoding a fusion protein comprising a cargo protein and a reverse transcriptase; and A nucleic acid construct comprising a base sequence encoding a fusion protein comprising a DNA binding protein and a cellular localization protein A gene expression system comprising: An expression vector comprising the gene expression system of claim 16. A host cell transformed with the expression vector of claim 17. A composition for regulating the intracellular location or activity of a cargo protein, comprising the gene expression system of claim 16. A method for regulating the intracellular location or activity of a cargo protein, comprising the step of expressing the gene expression system of claim 16. An engineered retron comprising an msr region; an msd region comprising first and second DNA binding protein specific binding sites; and a region encoding reverse transcriptase; A nucleic acid construct comprising a base sequence encoding a first DNA binding protein and a fusion protein comprising the first protein; and A nucleic acid construct comprising a base sequence encoding a fusion protein comprising a second DNA binding protein and a second protein; A gene expression system comprising: A method for co-localization between proteins, comprising the step of expressing the gene expression system of claim 21.

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