# Patent

## Title
Expression vector for cyanobacteria

## Abstract

The present disclosure discloses a vector that can be used for both cyanobacteria and E. coli, which contains, sequentially, a pUC replication origin as a replication origin; a spectinomycin-resistant gene as a selection marker; and a promoter selected from a group consisting of a trc promoter, a tetA promoter or a modified tetA promoter, a BAD promoter and a cbbL promoter. An industrially useful substance may be produced effectively using a host cell transformed with the vector. Also, the vector may be used to insert a variety of target genes through simple combination and, as a result, various vectors can be prepared effectively.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to Korean Patent Application No. 10-2014-0177540, filed on Dec. 10, 2014, and all the benefits accruing therefrom under 35 U.S.C. §119, the contents of which in its entirety are herein incorporated by reference.
BACKGROUND
1. Field
The present disclosure relates to an expression vector which is capable of producing desired substances by transforming cyanobacteria. The present disclosure also relates to a vector that can be used for both cyanobacteria and E. coli, a host cell transformed with the vector, a method for producing a substance using the transformed host cell, and a method for preparing a multivector using the vector.
[Description about National Research and Development Support]
This study was supported by Ministry of Science, ICT and Future Planning, Republic of Korea (Cooperation research project: KCRC CCS2020 Business, Project No. 2014M1A8A1049277) under the superintendence of the Korea Carbon Capture & Sequestration R&D Center.
2. Description of the Related Art
At present, cutting-edge metabolic engineering techniques such as next-generation genome sequencing and fast DNA synthesis are employed to produce useful bioproducts. The production of bioproducts using E. coli, yeast or Corynebacterium, which are the most widely used industrially at present, is being continuously developed. For the production of foreign metabolites, it is essential to reconstitute required heterologous genes in cells. Also, an optimization process for maximizing the production of the target substance is necessary. A generally adopted method for constructing a heterologous metabolic pathway for producing the target substance is to insert a specific gene into a plasmid or genome. More recently, advanced cloning techniques such as sequence and ligase-independent cloning (SLIC), Gibson DNA assembly, circular polymerase extension cloning (OPEC), etc. are being used.
SUMMARY
In an aspect, the present disclosure is directed to providing an expression vector which is operable in cyanobacteria.
In another aspect, the present disclosure is directed to providing a vector which can transform cyanobacteria after being inserted into the cyanobacterial genome.
In another aspect, the present disclosure is directed to providing a vector which is operable in both cyanobacteria and E. coli. 
In another aspect, the present disclosure is directed to providing a vector having two or more target genes being inserted and capable of expressing two or more kinds of proteins simultaneously.
In another aspect, the present disclosure is directed to providing a vector which operates effectively in the industrially useful Synechococcus elongatus PCC 7942.
In another aspect, the present disclosure is directed to providing an expression vector capable of effectively producing industrially useful substances and a host cell transformed with the vector.
In another aspect, the present disclosure is directed to providing a method for effectively producing industrially useful substances.
In another aspect, the present disclosure is directed to providing a method for preparing a multivector which expresses two or more target proteins that can invoke a mechanism for producing a useful substance or themselves can be industrially useful.
In another aspect, the present disclosure is directed to providing a vector which can maintain strong expression even when a host cell to which the vector is introduced is in a stationary phase.
In an aspect, the present disclosure provides an expression vector for cyanobacteria, the expression vector containing, sequentially, a pUC replication origin as a replication origin; a spectinomycin-resistant gene as a selection marker; and a promoter selected from a group consisting of a trc promoter, a tetA promoter or a modified tetA promoter, a BAD promoter and a cbbL promoter.
In another aspect, the present disclosure provides a host cell transformed with the vector.
In another aspect, the present disclosure provides a method for producing a substance, which includes culturing the transformed host cell.
In another aspect, the present disclosure provides a method for preparing a multivector containing two or more target genes, the method including inserting the target genes into the vector according to an exemplary embodiment of the present disclosure, wherein the vector already contains one or more preexisting target gene before the insertion of another target genes, and the insertion includes forming complementary binding between the BglII site located upstream of the preexisting target gene of the vector or the another target gene to be inserted into the vector and the BamHI site located downstream of the preexisting target gene of the vector or the another target gene to be inserted into the vector.
Cyanobacteria such as Synechococcus elongatus are photosynthetic microorganisms that can grow using light and carbon dioxide only like higher plants. Since the vector disclosed in the present disclosure is operable in cyanobacteria such as Synechococcus elongatus, it can produce a variety of biofuel substitutes or chemical products. Also, the vector may simultaneously express two or more target proteins that can invoke a mechanism for producing a useful substance or the target proteins themselves may be industrially useful. In another aspect, the present disclosure may provide a vector which can maintain strong expression even when its host cell is in stationary phase. Furthermore, time and cost can be saved remarkably and gene expression level can be controlled effectively because of simple gene assembly. In addition, mutation rate can be significantly reduced because transformation is conducted following restriction enzyme treatment and ligation without having to perform PCR. Furthermore, the vector can be usefully used as a gene expression tool since Synechococcus elongatus mainly transforms the target gene on its genome.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 schematically describes BglBrick cloning.
FIG. 2 schematically describes a procedure of obtaining a vector in which target genes A-B and target genes B-A are inserted using BglBrick cloning.
FIG. 3 schematically describes development of a pSe1Bb1s-GFP vector as one of SyneBrick vectors using the OPEC method.
FIG. 4 schematically describes assembly of pSe1Bb2s-GFP, pSe1Bb8s-GFP, pSe1Bb2Os-GFP and pSe1BbcbbLs-GFP vectors as SyneBrick vectors through PCR amplification, restriction enzyme treatment and ligation.
FIG. 5 schematically describes transformation of 5 SyneBrick vectors into Synechococcus elongatus strains.
FIG. 6 shows confocal microscopic images of green fluorescent protein (GFP) fluorescence by constructed strains.
FIG. 7 shows a result of measuring GFP fluorescence by SyneBrick vectors in the presence or absence of an inducer.
FIG. 8A ̃FIG. 8C show the full sequence (SEQ ID NO: 1) of a pSe1Bb1s-GFP vector.
FIG. 9A ̃FIG. 9C show the full sequence (SEQ ID NO: 2) of a pSe1Bb2s-GFP vector.
FIG. 10A ̃FIG. 10C show the full sequence (SEQ ID NO: 3) of a pSe1Bb8s-GFP vector.
FIG. 11A ̃FIG. 11C show the full sequence (SEQ ID NO: 4) of a pSe1BbcbbLs-GFP vector.
FIG. 12A ̃FIG. 12C show the full sequence (SEQ ID NO: 5) of a pSe1Bb2Os-GFP vector.



DETAILED DESCRIPTION
The vector disclosed in the present disclosure is also called a SyneBrick vector.
Synechococcus elongatus, one of cyanobacteria, is a photosynthetic microorganism that can grow using light and carbon dioxide only like higher plants. Due to this characteristic, the strain is gaining attentions worldwide as an environment-friendly microorganism. Unlike eukaryotic microalgae, the prokaryotic cyanobacteria are easy to be genetically modified to alter metabolic pathways or artificially regulate metabolites. A synthetic biological/metabolic engineering technique based on the genetic modification technology may be introduced to Synechococcus elongatus in order to produce a variety of biofuel substitutes or chemical products using metabolic pathways that have not existed.
In the present disclosure, a “multivector” refers to a vector which contains two or more target genes or two or more kinds of target genes desired to be overexpressed and is able to express two or more, or two or more kinds of target proteins simultaneously. The expressed two or more proteins may be industrially useful themselves, or the two or more proteins may invoke a mechanism for producing desired useful substances.
In the vector disclosed in the present disclosure, the genes are linked operably to each other. The term “operably” means that the target genes can be expressed normally.
In an aspect, the present disclosure provides an expression vector for cyanobacteria, which contains, sequentially: a pUC replication origin as a replication origin; a spectinomycin-resistant gene as a selection marker; and a promoter selected from a group consisting of a trc promoter, a tetA promoter or a modified tetA promoter, a BAD promoter and a cbbL promoter.
In an exemplary embodiment, the modified tetA promoter may include a sequence of SEQ ID NO 6.
Since the vector can be used for both cyanobacteria and E. coli, gene recombination can be easily achieved in E. coli and a desired protein may be obtained by transforming into cyanobacteria.
In an exemplary embodiment, the vector may further contain a repressor selected from a group consisting of a lacI repressor, a tetR repressor and an AraC repressor upstream of the promoter.
In an exemplary embodiment, the vector may further contain a neutral site (NSI) derived from Synechococcus elongatus PCC 7942 upstream or downstream of the replication origin. For example, the neutral site may include: NSIa including a sequence of SEQ ID NO 7; and NSIb including a sequence of SEQ ID NO 8. In an exemplary embodiment, the vector may be inserted onto the genome of Synechococcus elongatus PCC 7942 via the neutral site.
For example, a template vector of the expression vector for cyanobacteria may be a pBbE1c-RFP vector (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12). In addition, for example, the spectinomycin-resistant gene, the NSIa, the NSIb and the pUC may be derived from pSyn_1 (Invitrogen).
The vector may further contain a green fluorescent protein (GFP) gene downstream of the promoter. Since the green fluorescent protein (GFP) gene emits fluorescence when expressed in a host cell, it can tell whether the vector operates appropriately and the target gene is expressed normally. Another target gene may be inserted at the green fluorescent protein (GFP) gene site. A BglII site and a BamHI site, which are restriction enzyme sites, may be located on both sides of the green fluorescent protein (GFP) gene.
For example, the green fluorescent protein (GFP) gene may be derived from pBbB5k-GFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12).
In another exemplary embodiment, the vector may further contain a BglII site and a BamHI site as restriction enzyme sites. In another exemplary embodiment, the vector may further contain an EcoRI site, a BglII site, a BamHI and an XhoII site as restriction enzyme sites.
In another exemplary embodiment, the vector may further contain a target gene which encodes a target protein desired to be overexpressed.
In another exemplary embodiment, the vector may contain two or more target genes.
In an exemplary embodiment, the two or more target genes may be derived from different vectors. Each of the different vectors may have a BglII site and a BamHI site on both sides of the target gene, and the two target genes may be included in one vector by complementary binding between the BgIII site of one vector and the BamHI site of the other vector through restriction enzyme treatment.
BglBrick cloning is a cloning method not requiring a PCR amplification step. According to the method, target genes A and B included in different vectors may be easily cloned into one vector. For example, referring to FIG. 1, each vector has EcoRI, BgIII, BamHI and XhoI sites as restriction enzymes such that a target gene partB can be bound immediately downstream of a target gene partA. It is possible because, upon enzymatic treatment by BamHI and BglII, the complementary DNA strands can bind again to each other. In this way, various genes can be easily cloned into one vector.
In an exemplary embodiment, the BglII site and the BamHI site may be located on both sides of the target gene. In another exemplary embodiment, the order of the target gene and the restriction enzyme sites may be EcoRI→BgIII site→target gene→BamHI site→XhoII site. For example, let's suppose a vector A and a vector B as two vectors having such an order and having a target gene A and a target gene B, respectively. By treating the vector A with the restriction enzymes EcoRI and BamHI and treating the vector B with the restriction enzymes EcoRI and BglII, and then ligating them by complementarily binding the sites cleaved by BalII and BamHI, a vector in which the target genes A-B are inserted can be obtained (FIG. 2). Conversely, by treating the vector A with BgIII and XhoI and treating the vector B with BamHI and XhoI, and then performing ligation, the target genes may be inserted in the order of B-A (FIG. 2).
In an exemplary embodiment, the vector may be a pSe1Bb1s-GFP vector containing a lacI repressor and a trc promoter. In an exemplary embodiment, the vector may have a sequence of SEQ ID NO 1.
The locations of major parts of the pSe1Bb1s-GFP vector (SEQ ID NO 1) are as follows:

    
    
        pUC origin: 58-673
        NSIa: 745-1543
        spectinomycin-resistant gene: 1684-2694
        lacI repressor: 2790-3881
        trc promoter: 3938-4172
        green fluorescent protein: 4187-4923
        NSIb: 5132-5907.
    
    


In an exemplary embodiment, the vector may be a pSe1Bb2s-GFP vector containing a tetR repressor and a tetA promoter. In an exemplary embodiment, the vector may have a sequence of SEQ ID NO 2.
The locations of major parts of the pSe1Bb2s-GFP vector (SEQ ID NO 2) are as follows:

    
    
        pUC origin: 58-673
        NSIa: 745-1543
        spectinomycin-resistant gene: 1684-2694
        tetR repressor: 2721-3347
        tetA promoter: 3348-3420
        green fluorescent protein: 3436-4172
        NSIb: 4381-5156.
    
    


In an exemplary embodiment, the vector may be a pSe1Bb8s-GFP vector containing an AraC repressor and a BAD promoter. In another exemplary embodiment, the vector may have a sequence of SEQ ID NO 3.
The locations of major parts of the pSe1Bb8s-GFP vector (SEQ ID NO 3) are as follows:

    
    
        pUC origin: 58-673
        NSIa: 745-1543
        spectinomycin-resistant gene: 1684-2694
        AraC repressor: 2721-3599
        BAD promoter: 3875-3902
        green fluorescent protein: 3940-4676
        NSIb: 4885-5660.
    
    


In an exemplary embodiment, the vector may be a pSe1BbcbbLs-GFP vector containing a cbbL promoter. In another exemplary embodiment, the vector may have a sequence of SEQ ID NO 4.
The locations of major parts of the pSe1BbcbbLs-GFP vector (SEQ ID NO 4) are as follows:

    
    
        pUC origin: 58-673
        NSIa: 745-1543
        spectinomycin-resistant gene: 1684-2694
        cbbL promoter: 2724-2934
        green fluorescent protein: 2950-3686
        NSIb: 3895-4670.
    
    


In an exemplary embodiment, the vector may be a pSe1Bb2Os-GFP vector containing a TetR repressor and a modified tetA promoter including a sequence of SEQ ID NO 6. In another exemplary embodiment, the vector may have a sequence of SEQ ID NO 5.
The locations of major parts of the pSe1Bb2Os-GFP vector (SEQ ID NO 5) are as follows:

    
    
        pUC origin: 58-673
        NSIa: 745-1543
        spectinomycin-resistant gene: 1684-2694
        tetR repressor: 2721-3347
        modified tet A promoter: 3348-3459
        green fluorescent protein: 3475-4211
        NSIb: 4420-5195.
    
    


For example, the pSe1Bb1s-GFP SyneBrick vector may be constructed by constructing a pSe1Bb1s-RFP vector using the BglBrick vector pBbE1c-RFP published in the literature and pSyn_1 purchased from Invitrogen through OPEC cloning and then ligating pBbB5k-GFP whose GFP gene has been cleaved using a specific restriction enzyme to the pSe1Bb1s-RFP except for the RFP part. Subsequently, in order to construct various SyneBrick vectors by changing the gene expression regulating part, pSe1Bb2s-GFP and pSe1Bb8s-GFP may be obtained by cleaving the promoter and repressor parts of pBbE2c-RFP and pBbE8c-RFP respectively using specific restriction enzymes (AatII/EcoRI) and then inserting them after removing the promoter and repressor parts of pSe1Bb1s-GFP.
In addition, for example, after amplifying the promoter part possessed by a Synechococcus strain using a primer [5′-AAA GAC GTC ATC TCG CTT CTG G-3′ (SEQ ID NO 9)/5′-TTT GAA TTC GTC GTC TCT CCC T-3′ (SEQ ID NO 10)] and then treating with the restriction enzymes AatII and EcoRI, it may be inserted into the pSe1Bb1s-GFP vector having the promoter and repressor parts removed.
In order to improve the induction rate of the SyneBrick vector pSe1Bb2s-GFP containing the tetA promoter, an operator sequence may be further added so as to enhance the function of the repressor. The tetR part and the promoter part including the modified operator sequence may be prepared through DNA synthesis and then inserted into a pSe1Bb1s-GFP vector having the promoter and repressor parts removed using the AatII/EcoRI restriction enzyme to construct pSe1Bb2Os-GFP.
In another aspect, the present disclosure provides a transformed host cell transformed with one of the above-described vectors. The host cell may be cyanobacterium. Cyanobacteria such as Synechococcus elongatus are photosynthetic microorganisms that can grow using light and carbon dioxide only like higher plants. Since the vector disclosed in the present disclosure is operable in cyanobacteria such as Synechococcus elongatus, it can produce a variety of biofuel substitutes or chemical products. In an exemplary embodiment, the cyanobacterium may be Synechococcus elongatus PCC 7942.
In another aspect, the present disclosure provides a method for producing a substance, which includes culturing the transformed host cell. Two or more kinds of proteins may be produced simultaneously by the method. The produced two or more proteins may invoke a mechanism for producing industrially useful substances. In addition, the proteins themselves may be industrially useful.
In another aspect, the present disclosure provides a method for preparing a multivector containing two or more target genes. For example, the method may include inserting the target genes into the vector according to an exemplary embodiment of the present disclosure. The vector may already contain one or more preexisting target gene before the insertion of the new target genes. And, the insertion may include forming complementary binding between the BglII site located upstream of the preexisting target gene of the vector or another target gene to be inserted into the vector and the BamHI site located downstream of the preexisting target gene of the vector or another target gene to be inserted into the vector.
In an exemplary embodiment, the complementary binding may be formed between the BglII site located upstream of the preexisting target gene of the vector and the BamHI site located downstream of another target gene to be inserted into the vector, and, as a result of the complementary binding, the inserted target gene may be inserted upstream of the preexisting target gene in the vector. For example, referring to the left side of FIG. 2, a procedure whereby a target gene A is inserted into a vector already having a target gene B in the order of A-B is described. The target gene A to be inserted is designed to have EcoRI and BglII restriction enzyme sites upstream thereof and a BamHI restriction enzyme site downstream thereof. For this purpose, a vector having the target gene A and having EcoRI and BglII restriction enzyme sites upstream of A, and BamHI and XhoI restriction enzyme sites downstream thereof may be used. By treating the vector with restriction enzymes EcoRI and BamHI, the target gene A having EcoRI and BglII restriction enzyme sites upstream thereof and a BamHI restriction enzyme site downstream thereof may be obtained. Meanwhile, a vector having the target gene B and having EcoRI and BglII restriction enzyme sites upstream of B, and BamHI and XhoI restriction enzyme sites downstream thereof may be treated with restriction enzymes EcoRI and BglII. When the vectors containing the target gene A and the target gene B are mixed, complementary binding may occur between the BglII site located upstream of the target gene B of the vector and the BamHI site located downstream of the target gene A to be inserted into the vector and, as a result, a vector wherein the target genes are inserted in the order of A-B may be obtained.
In another exemplary embodiment, the complementary binding may be formed between the BglII site located upstream of another target gene to be inserted into the vector and the BamHI site located downstream of the preexisting target gene of the vector, and, as a result of the complementary binding, the inserted target gene may be inserted downstream of the preexisting target gene in the vector. For example, referring to the right side of FIG. 2, a procedure whereby a target gene A is inserted into a vector already having a target gene B in the order of B-A is described. The target gene A to be inserted is designed to have a BamHI restriction enzyme site upstream thereof and BamHI and XhoI restriction enzyme sites downstream thereof. For this purpose, a vector having the target gene A and having EcoRI and BglII restriction enzyme sites upstream of A, and BamHI and XhoI restriction enzyme sites downstream thereof may be used. By treating the vector with restriction enzymes BglII and XhoI, the target gene A having a BglII restriction enzyme site upstream thereof and BamHI and XhoI restriction enzyme sites downstream thereof may be obtained. Meanwhile, a vector having the target gene B and having EcoRI and BglII restriction enzyme sites upstream of B and BamHI and XhoI restriction enzyme sites downstream thereof may be treated with restriction enzymes BamHI and XhoI. When the vectors containing the target gene A and the target gene B are mixed, complementary binding may occur between the BamHI site located downstream of the target gene B of the vector and the BglII site located upstream of the target gene A to be inserted into the vector and, as a result, a vector wherein the target genes are inserted in the order of B-A may be obtained.
In another exemplary embodiment, the method may further include, before the insertion, preparing a restriction enzyme site-containing target gene having a BglII site upstream of the target gene to be inserted and a BamHI site downstream of the target gene to be inserted. The restriction enzyme site-containing target gene may be prepared according to any method known in the art. For example, as described above, a vector having the target gene to be inserted and having EcoRI and BglII restriction enzyme sites upstream of the target gene and BamHI and XhoI restriction enzyme sites downstream of the target gene may be used.
[Example 1] Construction of Novel SyneBrick Vector pSe1Bb1s-GFP
pBbE1c-RFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12) and pSyn_1 from Invitrogen were used to construct a SyneBrick vector pSe1Bb1s-GFP. Specifically, the lacI, ptrc and RFP parts of pBbE1c-RFP were amplified by PCR and the spectinomycin-resistant gene, NSIa, NS1b and pUC replication origin parts of pSyn_1 were amplified by PCR. Then, the novel SyneBrick vector was completed by combining the two PCR products through OPEC cloning (Quan J, Tian J (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4(7): e6441. doi:10.1371/journal.pone.0006441). In order to replace the RFP part of the vector with GFP, the RFP part was removed using EcoRI/XhoI restriction enzymes and the GFP part of another BglBrick vector pBbB5k-GFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12) was inserted using EcoRI/XhoI restriction enzymes and a ligase. The vector was transformed into E. coli HIT-DH5α (Cat# RH617-J80, RBC Bioscience) and then extracted by mini-prep. Since the assembled vector might have been mutated during the PCR procedure, its full sequence was identified through plasmid sequencing. The final SyneBrick vector with the DNA sequence confirmed was named as pSe1Bb1s-GFP (FIG. 3).
[Example 2] Construction of pSe1Bb2s-GFP, pSe1Bb8s-GFP, pSe1BbcbbLs-GFP and pSe1Bb2Os-GFP Using SyneBrick Vector pSe1Bb1s-GFP
The pSe1Bb1s-GFP prepared in Example 1 and previously known pBbE2c-RFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12) and pBbE8c-RFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12) were used to construct SyneBrick vectors pSe1Bb2s-GFP, pSe1Bb8s-GFP, pSe1BbcbbLs-GFP and pSe1Bb2Os-GFP. The meaning of the alphabets and numbers in the plasmid name can be understood from FIG. 4, B.
Since the repressor and promoter parts of the BglBrick vectors pBbE2c-RFP and pBbE8c-RFP and the SyneBrick vector pSe1Bb1s-GFP share AatII/EcoRI restriction enzymes, the promoter and repressor parts of pSe1Bb1s-GFP were removed with AatII/EcoRI restriction enzymes and the promoter and repressor parts of pBbE2c-RFP and pBbE8c-RFP were inserted (FIG. 4, C).
In order to construct a SyneBrick vector using a promoter possessed by a Synechococcus strain, the part presumed to be the promoter of the cbbL gene was amplified using primers [5′-AAA GAC GTC ATC TCG CTT CTG G-3′ (SEQ ID NO 9)/5′-TTT GAA TTC GTC GTC TCT CCC T-3′ (SEQ ID NO 10)] and inserted into the pSe1Bb1s-GFP vector whose promoter and repressor parts had been removed by treating with AatII and EcoRI restriction enzymes. The resulting SyneBrick vector was named as pSe1BbcbbLs-GFP (FIG. 4, C).
When fluorescence emission was measured, pSe1Bb2s-GFP showed little difference in gene expression levels between in the presence and in the absence of an inducer. Therefore, in order to construct a modified model, a DNA was synthesized by attaching one more operator sequence of the promoter part (see FIG. 4, D showing a nucleotide of positions 52-109 of SEQ ID NO: 6) and then inserted using AatII/EcoRI restriction enzymes. The constructed SyneBrick vector was named as pSe1Bb2Os-GFP.
Each of the prepared 5 SyneBrick vectors was transformed into a wild-type Synechococcus elongatus PCC 7942 strain (ATCC® 33912TM). FIG. 5 schematically describes the transformation of a wild-type Synechococcus elongatus PCC 7942 strain using the SyneBrick vectors having different promoters. Since the wild-type strain has a neutral site (NSI) which usually does not function, the spectinomycin-resistant gene, the repressor, the promoter and the GFP gene were inserted directly onto the Synechococcus elongatus PCC 7942 genome based on the complementary matching of NSIa (SEQ ID NO 7) and NSIb (SEQ ID NO 8) sequences of SyneBrick with the NSI sequence (FIG. 5).
[Example 3] Testing of SyneBrick Vector for Gene Expression in Synechococcus elongatus PCC 7942
It was tested whether the constructed 5 SyneBrick vectors are appropriate for gene expression in Synechococcus elongatus PCC 7942. First, the expression of the GFP gene of the newly constructed SyneBrick vectors was observed by confocal microscopy.
Referring to FIG. 6, the left lane shows original red autofluorescence characteristic of the strain and the middle lane shows green fluorescence originating from the added GFP gene. The topmost row shows images of the wild-type Synechococcus elongatus PCC 7942 strain and green fluorescence is not observed in the middle image. Olympus Fluoview FV1000 was used for the confocal microscopy. The green fluorescence images were obtained using 488 nm/500-530 nm (excitation/emission) lasers and the red fluorescence images were obtained using 543 nm/570-640 nm (excitation/emission) lasers. The strain was induced using an inducer and the images were obtained after reaching an exponential phase.
Also, the intensity of fluorescence by the GFP located downstream of the promoter was measured using an automatic microplate reader (Tecan Infinite M200 pro, Tecan Group Ltd., Switzerland) at wavelengths of 485/535 nm. The measured fluorescence intensity was divided by the optical density at 730 nm. The GFP fluorescence intensity per unit cells was determined on days 4 and 8 after culturing.
The culturing condition was as follows. 100 mL of BG-11 medium (UTEX The Culture Collection of Algae at The University of Texas at Austin) with a composition described in Table 1 was added to a 250-mL Erlenmeyer flask and then the mutant strain transformed with each of the 5 SyneBrick vectors was added after diluting to O.D. 0.6.












TABLE 1







Ingredients
Contents
Stock solution conc.
Final conc.




















1
NaNO3 (Fisher BP360-500)
10 mL/L
 30 g/200 mL dH2O
17.6 mM


2
K2HPO4 (Sigma P 3786)
10 mL/L
 0.8 g/200 mL dH2O
0.23 mM


3
MgSO4•7H2O (Sigma 230391)
10 mL/L
 1.5 g/200 mL dH2O
 0.3 mM


4
CaCl2•2H2O (Sigma C-3881)
10 mL/L
0.72 g/200 mL dH2O
0.24 mM


5
Citric acid•H2O (Fisher A 104)
10 mL/L
0.12 g/200 mL dH2O
0.031 mM 


6
Ferric ammonium citrate
10 mL/L
0.12 g/200 mL dH2O
0.021 mM 


7
Na2EDTA•2H2O (Sigma ED255)
10 mL/L
0.02 g/200 mL dH2O
0.0027 mM 


8
Na2CO3 (Baker 3604)
10 mL/L
 0.4 g/200 mL dH2O
0.19 mM


9
BG-11 trace metals solution
 1 mL/L


10
Sodium thiosulfate
 1 mL/L
49.8 g/200 mL dH2O
  1 mM



pentahydrate (agar media only,



sterile; Baker 3946)









Also, the strain was cultured at 30 and 130 rpm while supplying 5% CO2 continuously after adding 10 μg/mL of spectinomycin and an inducer required for GFP expression (0.1 mM IPTG, 200 nM aTC, 1 mM arabinose).
On days 4 and 8 after the culturing was initiated, the growing cells were added to a 96-well plate (flat bottom 96-well solid black plate, Corning, USA) with 200 μL per each, and GFP fluorescence was measured using an automatic microplate reader.
From FIG. 7, A, it can be seen that the Se1Bb1s-GFP strain induced with 0.1 mM IPTG showed 2.2-fold increased GFP fluorescence on day 4 and 1.7-fold increased GFP fluorescence on day 8, as compared to when it was not induced.
Referring to FIG. 7, D, the Se1BbcbbLs-GFP strain showed consistent GFP fluorescence without recurring induction. The fluorescence intensity was similar to that of Se1Bb1s-GFP before induction.
From FIGS. 7, B and C, it can be seen that the fluorescence by the Se1Bb2s-GFP and Se1Bb8s-GFP strains is not regulated by the inducer. Therefore, the same experiment was conducted after preparing a new strain using pSe1Bb2Os-GFP. In the presence of the inducer, the fluorescence intensity increased 1.4-fold on day 4 and 1.5-fold on day 8 (FIG. 7, E).

## Claims

1. An expression vector which is operable in cyanobacteria comprising, sequentially:
a pUC replication origin as a replication origin;
a spectinomycin-resistant gene as a selection marker; and
a promoter selected from a group consisting of a trc promoter, a tetA promoter or a modified tetA promoter, a BAD promoter and a cbbL promoter,
wherein the modified tetA promoter comprises a sequence of SEQ ID NO 6.

2. The vector according to claim 1, wherein the vector further comprises a repressor selected from a group consisting of a lad repressor, a tetR repressor and an AraC repressor upstream of the promoter.

3. The vector according to claim 1, wherein the vector further comprises a green fluorescent protein (GFP) gene downstream of the promoter.

4. The vector according to claim 1, wherein the vector further comprises a neutral site (NSI) comprising the neutral site comprising:
NSIa comprising a sequence of SEQ ID NO 7; and
NSIb comprising a sequence of SEQ ID NO 8.

5. The vector according to claim 1, wherein the vector further comprises a BglII site and a BamHI site as restriction enzyme sites.

6. The vector according to claim 5, wherein the vector further comprises a target DNA sequence which encodes a target protein desired to be overexpressed.

7. The vector according to claim 6, wherein the BglII site and the BamHI site are located on both sides of the target DNA sequence.

8. The vector according to claim 7, wherein the target DNA sequence encodes two or more proteins.

9. The vector according to claim 8, wherein the target DNA sequence is included in the vector by complementary binding between the BglII site of the upstream side of a first portion of the target DNA sequence and the BamHI site of the downstream side of a second portion of the target DNA sequence.

10. The vector according to claim 1, wherein the vector is
a pSe1Bb1s-GFP vector comprising a lad repressor and a trc promoter;
a pSe1Bb2s-GFP vector comprising a tetR repressor and a tetA promoter;
a pSe1Bb8s-GFP vector comprising an AraC repressor and a BAD promoter;
a pSe1BbcbbLs-GFP vector comprising a cbbL promoter; or
a pSe1Bb2Os-GFP vector comprising a tetR repressor and a modified tetA promoter comprising a sequence of SEQ ID NO 6.

11. The vector according to claim 10, wherein the vector comprises a sequence of SEQ ID NO 1; SEQ ID NO 2; SEQ ID NO 3; SEQ ID NO 4; or SEQ ID NO 5.

12. A host cell transformed with the vector according to claim 1.

13. The host cell according to claim 12, wherein the host cell is cyanobacterium.

14. The host cell according to claim 13, wherein the cyanobacterium is Synechococcus elongatus.

15. A host cell transformed with the vector according to claim 10.

16. A host cell transformed with the vector according to claim 11.

17. Transformed Synechococcus elongates PCC 7942 in which the vector according to claim 4 is inserted onto the genome of Synechococcus elongates PCC 7942 via the neutral site between NSIa and NSIb.