KR100658943B1 - Method for selecting microorganisms cloned with target gene using photochemical reaction - Google Patents

Abstract

A method for selecting microorganisms cloned with a target gene by using photochemical reaction is provided to remove undesired microorganism clones using simple photo illumination prior to the desired gene screening, so that the gene cloned with a desired gene fragment or its library is able to be simply screened. The method for selecting microorganisms cloned with a target gene by using photochemical reaction comprises the steps of: preparing a recombinant vector by inserting a target gene or its library and a promoter of the target gene into a recombinant plasmid containing origin, antibiotic makers, restriction enzyme sites and ferrochelatase gene(hemH); transforming a microorganism with the recombinant vector; culturing the transformed microorganism in a medium containing antibiotics with photo illumination; and validating the cloning of microorganism when the transformed microorganism is grown, wherein the transformed microorganism is obtained by inserting the ferrochelatase gene(hemH) into a photosensitive mutant microorganism obtained by deleting the ferrochelatase gene(hemH); the recombinant plasmid is p19hemH; and the microorganism is Escherichia coli.

Description

Screening method for cloned microorganisms using photochemical reactions {Method for Selecting Microorganisms Cloned with Target Gene Using Photochemical Reaction}

1 shows a genetic map of the recombinant plasmid p19hemH.

Figure 2 is a photograph when the mutant strain used in the present invention is plated on a solid medium and subjected to light irradiation, showing a black mask attached before smearing E. coli MEBLWLS.

Figure 3 is a photograph of the mutant strain used in the present invention plated on a solid medium and subjected to light irradiation, showing the result of culturing E. coli MEBLWLS on a solid medium and irradiated with a fluorescent lamp.

Figure 4 is a photograph confirmed by cutting the insertion sites of randomly selected colonies to prove the effectiveness of the present invention.

The present invention relates to a method for screening a microorganism cloned into a target gene or a library thereof using a photochemical reaction, and more particularly, to prepare a plasmid containing the hemH gene and a photosensitive mutant microorganism in which the hemH gene is deleted. The cloned plasmid was cloned to obtain a recombinant plasmid by cloning the target gene or a library thereof, and then inserting the recombinant plasmid into the prepared mutant microorganism, followed by culturing while irradiating light to a medium containing antibiotics. It relates to a method for screening microorganisms.

Escherichia coli is a microorganism generally inhabiting the human intestine, and the optimal temperature and acidity have been studied. In the early 1990s, Inoguchi et al. Discovered that a gene called visA was involved in E. coli's light sensitivity before the E. coli gene map was completed. Its mechanism of action has been shown to die because visA lacks a substance called protoporphyrin IX in Escherichia coli and fails to produce ham and thus does not remove free radicals in E. coli (Nakahigashi et al., Proc. Natl. Acad. Sci., 88: 10520, 1991). This gene was later found to be hemH involved in ham synthesis in E. coli (Frustaci et al., J. Bac., Apr. 2154, 1993).

In general, as a technique for discovering useful genes in microorganisms and the environment, a partial cutting of a gene library, introduced into an appropriate vector, inserted into a microorganism, and when properly expressed, a method of discovering a desired gene through a desired screening method has been used. However, in this method, the process of obtaining the right gene was very difficult because of the large number of self-combined vectors or the wrong genes. Recently, a technique for selecting a strain containing a gene correctly using a fluorescent protein and FACS, which is developed, is to express a fluorescent protein in E. coli when the desired gene is properly inserted, and according to the intensity of the fluorescence (Fu AY et al., Nat. Biotechnol., 17: 1109, 1999). However, this method has the disadvantage that it can be classified only in the liquid state.

Examples of searching for genes using photochemical reactions are those in which visA is deficient and fluorescent proteins are searched by light irradiation, but this method can be classified only in the solid state (Sruggs et al., Biotech. Bioeng., 84: 445, 2003).

Accordingly, the present inventors have made diligent efforts to improve the problems of the prior art, and confirmed that the effective clones can be easily and simply selected using only light source irradiation using photosensitive mutant microorganisms and recombinant plasmids to be inserted into the mutant microorganisms. Was completed.

It is an object of the present invention to provide photosensitive mutant microorganisms and recombinant plasmids useful for the selection of cloned microorganisms. Another object of the present invention is to provide a method for selecting a microorganism cloned into a gene of interest or a library thereof by using the above-described mutant microorganism and recombinant plasmid.

In order to achieve the above object, the present invention is the origin ( Ori ), antibiotic markers, restriction enzyme sites, and hemH Recombinant plasmids useful for the selection of cloned microorganisms containing genes and microorganisms transformed with the recombinant plasmids are provided.

In the present invention, the recombinant plasmid may be characterized in that p19hemH, the transformed microorganism may be characterized in that E. coli.

The invention also relates to the hemH in a microorganism having the hemH gene It provides a photosensitive mutant microorganism obtained by deleting a gene.

In the present invention, the photosensitive mutant microorganism is hemH using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 Amplifying a DNA fragment comprising a portion of a gene and inserting the amplified DNA fragment into Escherichia coli containing pKD46, thereby hemH by recombinase produced by pKD46. It may be characterized by being prepared through the step of inducing the deletion of the gene.

The present invention also provides a recombinant vector in which a target gene or a library thereof and a promoter of the target gene are inserted into the recombinant plasmid, and a microorganism transformed with the recombinant vector.

The present invention also comprises the steps of culturing the transgenic microorganisms while irradiating light in a medium containing antibiotics; And it provides a method for screening the microorganisms cloned into the target gene or its library comprising the step of identifying the cloned when the microorganisms grow.

In the present invention, the mutant microorganism is a light-sensitive mutant microorganism which is unable to produce perokylase , and the recombinant plasmid is a cloning vector having the hemH gene removed from the mutant microorganism from the shine- dalgano sequence. Therefore, when the genes partially cut to search for the effective genes are inserted into the vector, self-binding clones or error clones do not express hemH at the back, and these clones can be removed only by light irradiation.

In the present invention, there is no promoter in the plasmid used for recombination of the gene of interest or its library, and there is only a promoter derived from the inserted gene, so that it is possible to select a microorganism transformed with a recombinant vector containing the gene of interest.

In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. The vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no suitable restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods can facilitate ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. In the present invention, preferred host cells are prokaryotic cells. Suitable prokaryotic host cells are E. coli JM109, E. coli DH5α, E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL-1Blue (Stratagene), E. coli B, E. coli B21, and the like. However, E. coli such as FMB101, NM522, NM538 and NM539 Strains and other prokaryotic species and genera may also be used. In addition to the aforementioned E. coli , strains of the genus Agrobacterium, such as Agrobacterium tumefaciens, bacilli such as Bacillus subtilis , Salmonella typhimurium or Serratia marghesen another variety of enteric bacteria and Pseudomonas (Pseudomonas) in the strain, such as (Serratia marcescens) may be used as host cells. It is also possible to use eukaryotic cells such as yeast and fungi as hosts.

Prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra. Alternatively, electroporation (Neumann et al., EMBO J., 1: 841, 1982) can also be used for transformation of these cells.

Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to allow gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

In addition, "expression vector" generally refers to a fragment of DNA that is generally double stranded as a recombinant carrier into which fragments of heterologous DNA have been inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells. Once expression vectors are within a host cell, they can replicate independently of the host chromosomal DNA and several copies of the vector and their inserted (heterologous) DNA can be produced.

As is well known in the art, to raise the expression level of a transgene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the chosen expression host. Preferably, the expression control sequence and the gene of interest are included in one expression vector including the bacterial selection marker and the replication origin. If the host cell is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

Example  1: recombinant vector p19hemH Manufacture

P19hemH was prepared using pUC19 (New England Biolabs Inc. USA) as a backbone for use in light-sensitive E. coli strains. Chromosome was isolated from E. coli K-12, λ-, F-, prototrophic E. coli W3110, followed by primer 1 (SEQ ID NO: 1: 5'-) to secure the hemH gene including the shine- dalgano sequence. TCTAGAAATCGATAAGAGGCGGTAATGCG-3 ') and Primer 2 (SEQ ID NO: 2: 5'-GGAAGCTTTTAGCGATACGCGGCAACAA-3') were synthesized and PCR was performed using the same. PCR conditions are as follows. The first denaturation was done once at 94 ° C for 5 minutes, then the second denaturation was at 45 ° C for 94 seconds, the annealing for 45 seconds at 60 ° C, and the extension for 1 minute at 72 ° C. 30 seconds were performed and this was repeated 30 times. Thereafter, a final extension was made once for 7 minutes at 72 ° C. DNA obtained by PCR was subjected to agarose gel electrophoresis to separate DNA fragments of about 986 bp size, which were cleaved with two restriction enzymes Xba I and Hind III. At the same time, the recombinant plasmid pUC19 was cut with two restriction enzymes Xba I and Hind III, mixed with the DNA fragments obtained above, and then linked using T4 DNA ligase, which was then linked to E. coli XL1-Blue [ supE44]. hsdR17 recA1 endA1 gyrA96 thi by transfection in relA1 lacF '(proAB + lacIqlacZ Δ M15Tn (tetr))] to give a recombinant plasmid p19hemH. Figure 1 shows the recombinant plasmid p19hemH thus obtained.

Example  2: manufacture of light-sensitive E. coli mutant strains and Photosensitivity  Experiment

Mutations of E. coli sensitive to light were prepared using the method performed by Kirill et al. (Kirill, A. et al., Proc. Natl. Acad. Sci., 97: 6640, 2000).

To obtain a strain lacking the hemh gene in Escherichia coli W3110, primer 3 (SEQ ID NO: 3'-ATGCGTCAGACTAAAACCGGTATCTCGTCGGCAAACCTGGGTACGCCCGATGTGTAGGCTGGAGCTGCTTCGAAG-3 ') and Primer 4 (SEQ ID NO: 5'-TTAGAGATACGCCTCCTACTCTCTCTCACTCATCATTCAT) Using these primers, the pKD3 plasmid (Kirill A. et al., Proc. Natl. Acad. Sci., 97: 6640, 2000) was subjected to PCR using a template (first denaturation at 95 ° C for 5 minutes, second denaturation at 95 ° C for 30 seconds, hybridization). 60 ° C. 1 minute, extension 72 ° C. 1 minute 30 seconds, 30 repetitions). The result contains the middle chloramphenicol resistance gene, the gene that causes recombination outwards, and the end of the gene ( hemH ) to be removed. This amplified DNA fragment was electroporated into a strain containing pKD46 (Kirill A. et al., Proc. Natl. Acad. Sci., 97: 6640, 2000). In this case, recombinase produced by pKD46 induces a deletion of the hemH gene. The inserted strains were placed in Luria-Bertani medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L) in a solid phase medium containing 50 μg / L of ampicillin and 34 μg / L of chloramphenicol at 30 degrees. Incubated for 18 hours. Colonies PCR (first denaturation 95 ℃ 5 minutes, second denaturation 95 ℃ 30 seconds, hybridization 65 ℃ 1 minute, extension 72 ℃ 1 minute 30 seconds, 30 times) to find the strains missing genes from the colonies after culture Was performed. At this time, primer 5 and (SEQ ID NO: 5'-ATGCGTCAGACTAAAACCGGTATCCT-3 ') primer 6 (SEQ ID NO: 6: 5'-T CATCGCAGTACTGTTGTATTCATTAA-3') were synthesized and used. This transformed strain was named Escherichia coli MBELWLS, and cultured and preserved by adding 1 mg / mL of hemin (Sigma, USA) at the time of culture.

To experiment with photosensitivity, a black mask that does not transmit light is attached to a Petri dish, E. coli MBELWLS is applied to a Luria-Butani plate medium containing 34 µg / L of chloramphenicol, and then irradiated with a fluorescent lamp. Incubated at 37 ° C. for 2 days. FIG. 2 shows a black mask attached before smearing E. coli MBELWLS, and FIG. 3 shows a plate medium incubated at 37 ° C. for 2 days while irradiating light with a fluorescent lamp after smearing E. coli MBELWLS. As shown in FIG. 3, the light-sensitive E. coli MBELWLS was grown where the light was blocked by the black mask, and the E. coli MBELWLS did not grow at all when the light was irradiated.

Example  3: Photoreaction  Confirmation of insertion site of clones obtained using

In this example, metagenome was used as a gene population to select effective clones by photoreaction. Soil used to extract metagenome was collected from Yasan near Korea Advanced Institute of Science and Technology. The collected soil was removed from the grass roots and foreign substances with four layers of gauze and the gene was extracted by the following method. First, 1 g of the collected soil was well dispersed in 10 ml buffer (500 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mg / ml lysozyme). The dispersed suspension was incubated at 37 ° C. for 1 hour. Thereafter, 100 μl of 2 mg / ml proteinase K was injected, and then incubated at 30 ° C. for 1 hour. Next, 5 ml of 6% SDS solution was added and incubated at 65 ° C. for 3 hours. Next, only the supernatant was collected by centrifugation (6000 rpm) at 4 ° C., and then, the same volume of chloroform and isoamyl alcohol mixed solution (24: 1) was added thereto, and the supernatant was collected again by centrifugation. This process was repeated three times. Finally metagenome in aqueous solution was precipitated with isopropyl alcohol and washed with 70% ethanol. The finally obtained metagenome was used again by dissolving in distilled water.

Cloning of the gene was performed as follows. That is, the metagenome obtained above was partially cut for 10 minutes, 20 minutes, and 30 minutes, respectively, by restriction enzyme Sau3A I. The cleaved genes were cloned into the BamH I site of p19hemH prepared in Example 1, and then inserted into the E. coli MBELWLS prepared in Example 2 by electroporation. After incubation at 37 ° C. for 1 hour after insertion, the plate was plated on Luria-Butani solid plate medium containing 34 μg / L of ampicillin. After incubation at 37 ° C. for 10 hours, colonies shown were grown for 6 hours in a Luria-Butani liquid medium containing 50 μg / L ampicillin and then purified by inserting recombinant plasmids. The collected plasmids were cut with EcoR I, a restriction enzyme, and examined the trend of the inserted genes. As a result, as shown in Figure 4, the recombinant plasmid thus obtained was able to obtain metagenomic fragments of various sizes containing the promoter region of the inserted gene.

As described above in detail specific parts of the present invention, those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

The present invention has the effect that the plasmid and hemH gene containing hemH gene provides a deletion photosensitive mutant microorganism, to the thus prepared plasmid cloning a gene of interest or the library thus obtained recombinant plasmid, and then mutated Preparative After inserting the recombinant plasmid into the microorganism, there is an effect of providing a method for screening the cloned microorganism, characterized in that cultured while irradiating light to the medium containing antibiotics.

According to the present invention, the false clones that follow when searching for a useful gene can be removed by simple light irradiation without using a conventional fluorescent protein detection method. However, self-binding clones can be excluded, so that effective clones can be efficiently selected.

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Claims (12)

A recombinant plasmid containing an origin (Ori), an antibiotic marker, a restriction enzyme site, and a gene encoding a ferrochelatase ( hemH ) is transfected with a recombinant vector having a gene of interest or a library thereof and a promoter of the gene of interest. Culturing the converted microorganisms while irradiating light in a medium containing antibiotics; And When the transformed microorganism grows, the method for screening the microorganism cloned with the target gene or library comprising the step of identifying the cloned. The method of claim 1 wherein the transformed microorganism is obtained by deleting the genes (hemH) encoding the Faroe Killa hydratase (ferrochelatase) in a microorganism having the gene (hemH) encoding the Faroe Killa hydratase (ferrochelatase) photosensitive Obtained by introducing into a mutant microorganism. The method of claim 1, wherein said recombinant plasmid is p19hemH. The method of claim 2, wherein the photosensitive mutant microorganism comprises amplifying a DNA fragment comprising a portion of a gene ( hemH ) encoding a ferrochelatase using primers of SEQ ID NO: 3 and SEQ ID NO: 4, and The amplified DNA fragment is inserted into Escherichia coli containing pKD46, and is prepared by inducing the deletion of the gene ( hemH ) encoding a ferrochelatase by the recombinase produced by the pKD46. How to. The method for screening microorganisms cloned into the gene of interest or a library thereof according to claim 1, which is E. coli. delete delete delete delete delete delete delete

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