KR101659732B1 - Protein Expression System Induced by Heavy Metals And Biosensor for Detecting Heavy Metals - Google Patents
Protein Expression System Induced by Heavy Metals And Biosensor for Detecting Heavy Metals Download PDFInfo
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- KR101659732B1 KR101659732B1 KR1020150146913A KR20150146913A KR101659732B1 KR 101659732 B1 KR101659732 B1 KR 101659732B1 KR 1020150146913 A KR1020150146913 A KR 1020150146913A KR 20150146913 A KR20150146913 A KR 20150146913A KR 101659732 B1 KR101659732 B1 KR 101659732B1
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Abstract
The present invention relates to a protein expression system and a biosensor for detecting heavy metals, which are induced by heavy metals. By using a cadC gene which reacts with heavy metals, a gene expression inducing system having a high economic efficiency can be realized by using a heavy metal, It can be utilized as a biosensor capable of detecting heavy metals with high sensitivity.
Description
The present invention relates to a heavy metal-induced protein expression system and a transformant for heavy metal detection using a cadC gene which reacts with heavy metals.
In the case of a conventional biosensor for detecting heavy metals, a promoter and a gene region that are responsive to heavy metals are extracted and inserted into a vector, and the transformed host cell is developed as a biosensor using a transformed host cell as a biosensor Research has been conducted.
Whole-cell biosensors that detect cadmium, a typical harmful heavy metal, mainly use prokaryotic microorganisms. Examples include (1) a recombinant cadmium-resistant gene cadA promoter of Staphylococcus aureus and a firefly luciferase Plasmid transformants (Tauriainen, S., et al. , Biosens (Wu, CH, et al., Bioelectron , 931-938, 1998), (2) a recombinant plasmid transformant ( pseudomonas putida ) in which a cadR gene promoter regulating cadmium resistance was combined with a green fluorescent protein, et al., Biotechnol Prog, 898-903 , 2009), (3) a combination of a promoter and green fluorescent protein gene cadAC of E. coli recombinant plasmid transformants (Shetty, RS, et al. , Anal Bioanal Chem, 11-17 , 2003) have been reported. It has also been reported that the sensitivity of the biosensor is increased by eliminating the C-terminal amino acid of CadR protein, which is specific for cadmium and mercury, thereby improving the specificity for cadmium (Tao, HC, et al., Biotechnol Lett . 1253-1258, 2013).
A patent for the development of gene recombinant strains capable of detecting toxic substances by manipulating genetic engineering techniques for genes that exhibit selective responses to toxic substances and cell-based biosensors using the same, Based biosensor for cadmium (Cd2 (II)), which is a kind of cadmium (Cd2 (II)), utilizes a DNA microarray and utilizes a cadmium inducible promoter of Hansenula polymorpha , There is a patent using Saccharomyces cerevisiae as a host of a biosensor (Korean Patent Laid-open Publication No. 10-2006-0104939), and DR_0070, a gene of radiation-resistant microorganism Deinococcus radiodurans , And the expression of this gene and the promoter region < RTI ID = 0.0 > There is a patent application bar of a biosensor for detecting the cadmium body genetically modified transgenic (Republic of Korea Patent Publication 10-2012-0047635) including.
The above-described techniques have developed a biosensor in which a transformed host cell is used as a biosensor in which a promoter and a gene region which are responsive to heavy metals are extracted and inserted into a vector and the transformed host cell is used as a biosensor. It was necessary to develop a gene promoter having high reactivity to heavy metals and to apply a signal amplification module to improve the sensitivity of the biosensor.
Therefore, the present inventors discovered six cadC genes expected to be involved in heavy metal resistance from the genome information of Bacillus oceanisediminis 2691, and by using the transcription regulation system of these genes, The present invention has been accomplished by confirming that a biosensor for detecting heavy metals can be realized which can realize a system in which protein expression is induced and furthermore, the sensitivity is remarkably improved.
It is an object of the present invention to provide a protein expression inducing system which can utilize heavy metals as an inducing agent.
Another object of the present invention is to provide a transformant for detecting heavy metals capable of detecting heavy metals with high sensitivity.
It is another object of the present invention to provide a method for detecting and quantifying cadmium using the recombinant microorganism for detecting heavy metals.
In order to accomplish the above object, one aspect of the present invention is a first gene construct comprising a first promoter, a cadC gene whose expression is regulated by the first promoter and which encodes a CadC protein; And a second promoter, a multiple cloning site (MCS) in which a gene of a target protein whose expression is regulated by the second promoter is cloned, and a second cloning site interposed between the second promoter and the multiple cloning site, And an operator cadO in which the CadC protein binds and the expression of the target protein by the second promoter is inhibited.
According to another aspect of the present invention, there is also provided a first gene construct comprising a first promoter, a cadC gene whose expression is regulated by the first promoter, and which encodes a CadC protein; And a reporter gene encoding a reporter protein whose expression is regulated by the second promoter, and a reporter gene interposed between the first promoter and the reporter gene, wherein the CadC protein binds to the second promoter A second gene construct comprising an operator cadO in which the expression of the reporter protein is inhibited; and a transformant for detecting heavy metals in which the host cell is transformed.
Another aspect of the present invention provides a method for detecting or quantifying cadmium, wherein the recombinant microorganism for detecting heavy metals is cultured in a medium containing a suspected sample containing cadmium.
Using the cadC gene according to the present invention, it is possible to develop a biosensor capable of detecting a heavy metal with a high sensitivity as well as a gene expression inducing system which can be used as an expression inducing agent by using a low-cost heavy metal as an induction agent .
1 is a recombinant vector pCadC538 (cadC 538- egfp), pCadC551 (cadC 551- egfp), pCadC595 (cadC 595- egfp), pCadC640 (cadC640-egfp), pCadC1945 (cadC 1945- egfp), and pCadC4657 (cadC 4657- (egfp ).
2 is a recombinant vector pCadC538 (cadC 538- egfp), pCadC551 (cadC 551- egfp), pCadC595 (cadC 595- egfp), pCadC640 (cadC 640- egfp) pCadC1945 (cadC 1945- egfp), and pCadC4657 (cadC 4657- (eg, arsenic, cadmium, lead, zinc) in the transformant transformed with E. coli .
FIG. 3 shows the results of the amplification of the HK760 strain having the gene construct of? LacI and galM-KmR- cadC640-T7RNAP, the strain HK744 having the gene construct of? LacI and galM-KmR- cadC1945- T7RNAP , and the? LacI and galM-KmR- cadC4657- And shows the structure of the first gene construct homologously recombined on the chromosome of HK739 strain having the gene construct of T7RNAP .
Figure 4 is a second gene construct the truck bit is cloned into a recombinant vector pET21a (P T7 _cadO1945_egfp), pET21a (cadO640-egfp & P hce - cadC640) and 2-plasmid system, the recombinant vector pET21a (P T7 _cadO4657_egfp) and pACYC184 (P hce - < / RTI > cadC4657 ).
FIG. 5 shows the results of transformation of the recombinant vectors cloned in the second gene construct shown in FIG. 4 into the HK744 strain, HK760 strain and HK739 strain homologously recombined on the chromosome of the first gene construct of FIG. 3 And shows the structure in the biosensor.
6 is a T7 RNA polymerase to introduce / T7 promoter system as before, two-plasmid system in the recombinant vector pET21a (P T7 _cadO4657_egfp) with the pACYC184 (P hce - cadC4657) by transforming the HK739 the T7 RNA gene expression process And the expression efficiency of EGFP protein after introduction of the polymerase / T7 promoter system.
7 is HK744 (P T7 _cadO1945_egfp) transformants and HK760 - a graph showing the fluorescence amount according to the concentration of lead and cadmium in transgenic (cadO640-egfp & P hce cadC640 ).
Figure 8 is a graph comparing the expression of EGFP under the IPTG concentration to each other in the transformant of EGFP expression and egfp-pET21a / BL21 (DE3) transformant of the lead concentration of the HK744 (P T7 _cadO1945_egfp).
Hereinafter, the present invention will be described in detail.
One. An expression vector in which the expression of the target protein is regulated by heavy metals
A first aspect of the present invention is a first gene construct comprising a first promoter, a cadC gene whose expression is regulated by the first promoter and which encodes a CadC protein; And a second promoter, a multiple cloning site (MCS) in which a gene of a target protein whose expression is regulated by the second promoter is cloned, and an operator cado between the second promoter and the multiple cloning site ; And a second gene construct comprising the expression vector.
The expression vector of the present invention comprises a first promoter and a first gene construct whose expression is regulated by the first promoter and which comprises a cadC gene encoding a CadC protein.
The first promoter regulates the expression of the CadC gene encoding the CadC protein, and any known promoter may be used as long as it can regulate the expression of the downstream cadC gene. In particular, The promoter of the cadC gene derived from Oseani CDINISM 2691 may be used, but is not limited thereto.
The cadC gene is a gene encoding a transcription factor of CadC protein, the CadC protein is responsible for the binding to the operator cadO thereof in an environment that does not exist in the heavy metals inhibit the transcription of a gene of interest located downstream of the cadO on the other hand, in environments where heavy metals are present in the CadC protein it is combined with the heavy metals removed from cadO to thereby serve to activate transcription of a desired gene located downstream from the cadO.
CadC538 having the amino acid sequence of SEQ ID NO: 1, CadC551 having the amino acid sequence of SEQ ID NO: 3, CadC595 having the amino acid sequence of SEQ ID NO: 3, CadC640 having the amino acid sequence of SEQ ID NO: 4, CadC540 having the amino acid sequence of SEQ ID NO: CadC1945 having the amino acid sequence of SEQ ID NO: 6 and CadC4657 having the amino acid sequence of SEQ ID NO: 6. However, deletion, insertion, substitution or deletion of amino acid residues within the range of not affecting the functions of the above- Mutations or fragments of amino acids having different sequences by the combination of the amino acid sequences of SEQ ID NOs. Amino acid exchange at the level of proteins and peptides that do not globally alter the activity of the CadC protein is known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like. Accordingly, the CadC protein of the present invention comprises a protein having substantially the same amino acid sequence as the protein comprising any one of amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, a mutant thereof, or an active fragment thereof. The substantially same protein means those having an amino acid sequence homology of not less than 80%, preferably not less than 90%, and most preferably not less than 95%, but is not limited thereto, and has homology of not less than 80% Are included within the scope of the present invention if they have transcription factor activity.
The expression vector of the present invention comprises a second promoter, a multi cloning site where a gene of a target protein whose expression is regulated by the second promoter is cloned, and an operator cadO interposed between the second promoter and the multiple cloning site And a second genetic construct comprising
The second promoter regulates expression of a gene encoding a target protein present downstream of the second promoter, Any known type of promoter can be used as long as it can regulate the expression of the gene.
The multiple cloning site (MCS) is a site in which a target protein to be overexpressed through the expression vector of the present invention is inserted, and is a portion containing many restriction enzyme sites. During the process including cloning or subcloning Can be generally used. The restriction enzyme sites in the cloning site can be variously configured depending on the purpose, and the flexibility of the restriction enzyme sites makes it possible to clone the target gene for various applications.
In the second gene construct, an operator cadO is interposed between the second promoter and the multiple cloning site. Wherein the cadO is a site to which an CadC protein as an electron regulator is bound, and when the CadC protein is bound to the cadO interposed between the second promoter and the multiple cloning site, the RNA polymerase bound to the second promoter binds CadC The protein is clogged and the gene of the target protein present in the downstream can not be transcribed. On the other hand, as described above, the CadC protein binds with heavy metals in the presence of heavy metals and is separated from the cadO . At this time, the gene of the downstream target protein blocked by the CadC protein can be transcribed. CadC538 having the amino acid sequence of SEQ ID NO: 1 has a nucleotide sequence of SEQ ID NO: 69 or cadO having a nucleotide sequence substantially the same as the nucleotide sequence of SEQ ID NO: CadC551 having the amino acid sequence of SEQ ID NO: 2 has the base sequence of SEQ ID NO: 70 or cadO having the base sequence substantially identical thereto , CadC595 having the amino acid sequence of SEQ ID NO: 3 has the nucleotide sequence of SEQ ID NO: CadO consisting of substantially the same base sequence, CadC640 having the amino acid sequence of SEQ ID NO: 4 has the base sequence of SEQ ID NO: 72 or substantially the same base sequence cadO and CadC1945 having the amino acid sequence of SEQ ID NO: cadO and made of the same base sequence as the base sequence and substantially or the number 73, standing CadC4657 has the amino acid sequence of No. 6 is coupled to cadO made of the same base sequence as the base sequence and substantially or of SEQ ID NO: 74 and each specific. The term "gene comprising substantially the same base sequence" as used herein refers to those having 80% or more, preferably 90% or more, and most preferably 95% or more of sequence homology, but is not limited thereto, and 80% or more of the sequence homology And the encoded protein has an activity capable of binding to the same CadC protein. As described above, the cadO of the present invention can be mutated by substitution, deletion, insertion or a combination of one or more nucleotide bases, and these are also included in the scope of the present invention.
In the case of the expression vector comprising the first gene construct and the second gene construct, the CadC protein is expressed by the first promoter of the first gene construct, and the CadC protein thus expressed is expressed by the second gene cone And binds to the cadO of the tortoise to inhibit the expression of the target protein by the second promoter. In this situation, when the heavy metal is treated, the CadC protein bound to the cadO is bound to the heavy metal and separated from the cadO . As a result, the transcription and expression of the target protein are initiated by the second promoter. And increasing the concentration of heavy metals that are processed as described above, it may be increased with the transfer and expression of a target protein by separation and a second promoter of CadC protein from cadO.
In order to further improve the efficiency and sensitivity of the transfer and expression of a target protein by separation and a second promoter of CadC protein from cadO as described above, the expression vectors of the invention, the T7 RNA polymerase / T7 promoter system (hereinafter referred to as Quot; T7 system ") may be introduced.
First, the first gene construct may further comprise a T7RNAP gene encoding T7 RNA polymerase, wherein the second promoter of the second gene construct may be a T7 promoter, In addition, other RNA polymerase / promoter systems with high specificity can be applied to increase the expression of the target protein.
When the first gene construct includes the T7 RNA polymerase as described above, the first gene construct has the cadC Gene and the T7RNAP And further include a ribosome binding site (RBS) between the genes, and any known kind of RBS can be used.
In addition, the first gene construct may further include cadO between the first promoter and the cadC gene or the T7RNAP gene. When cadO is present between the first promoter and the cadC gene or the T7RNAP gene, the expression of CadC protein or T7 RNA polymerase by the first gene construct is also affected by the presence of heavy metals. Therefore, in the case where cadO exists in the first gene construct as described above, the target protein can not be expressed due to clogging of cadO by CadC protein in the second genetic construct in an environment where heavy metals are not present In addition, since the cadO in the first gene construct is also blocked by the CadC protein, the T7 RNA polymerase that transcribes the target protein in the second gene construct is also not expressed, and the expression of the target protein Can be more completely suppressed. On the other hand, in an environment in which heavy metals are present, the T7 RNA polymerase can be expressed by the first gene construct and the expressed T7 RNA polymerase can express the target protein in the second gene construct, With the introduction of the T7 system as described above, the expression vector of the present invention can control the expression of the target protein with higher efficiency or sensitivity.
In the first gene construct, the first promoter, cadO , cadC gene and T7RNAP gene may be sequentially provided, wherein the first promoter, the cadO operator, and the cadC gene are represented by SEQ ID NOS: 7 to 12 , Or a base sequence substantially identical thereto, but is not limited thereto.
The heavy metal may be, but is not limited to, cadmium or lead. Particularly, CadC538 having the amino acid sequence of SEQ ID NO: 1, CadC551 having the amino acid sequence of SEQ ID NO: 2, and CadC1945 having the amino acid sequence of SEQ ID NO: 5 exhibited specific reactivity to lead and CadC640 having the amino acid sequence of SEQ ID NO: CadC4657 having the amino acid sequence of SEQ ID NO: 6 exhibits specific reactivity to cadmium, and CadC595 having the amino acid sequence of SEQ ID NO: 3 shows specific reactivity to both lead and cadmium.
In addition, the second gene construct may further include a gene encoding a tag for separation and purification in order to facilitate the purification of the expressed target protein. As the tag for separation and purification, GST, poly-Arg, FLAG, His-tag or c-myc can be used. The expression vectors of the invention can be transformed into suitable host cells, which can replicate and function independently of the genome of the host cell, or, in some cases, integrate into the genome itself. Particularly, in the present invention, a plasmid vector can be used, which comprises (a) a cloning start point for efficiently making replication so as to include several hundred plasmid vectors per host cell, (b) a plasmid vector transformed with a plasmid vector And (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.
2. Transformants for heavy metal detection
Another aspect of the present invention is a method for regulating expression of a CadC gene, comprising: a first promoter; a first gene construct whose expression is regulated by the first promoter and which comprises a cadC gene encoding a CadC protein; And a second gene construct comprising a second promoter, a reporter gene encoding a reporter protein whose expression is regulated by the second promoter, and an operative cadO interposed between the first promoter and the reporter gene Wherein the transformant is transformed into a host cell.
The first gene construct teuneun T7 RNA and the T7RNAP gene encoding the polymerase may further comprise, the first can 1 further comprising a gene construct teueun first cadO between the promoter and the cadC gene or T7RNAP gene, the The second promoter of the second gene construct may be, but is not limited to, the T7 promoter.
All the constituents of the 'first gene construct' and the 'second gene construct' are the same as those described in ' 1. Expression vector in which the expression of the target protein is regulated by heavy metals ' Description is given here. In this item, only the constitution specific to the transformant for detecting heavy metals will be described.
The reporter gene may be a fluorescent gene or an antibiotic resistance gene. However, the reporter gene is not limited thereto, and those known in the art may be used. For example, the reporter gene may be (1) a gene encoding a fluorescent protein (e.g., GFP); (2) a gene encoding a colored protein; (3) a gene encoding a color-promoting or inducible protein; And (4) an antibiotic resistance gene. The reporter gene corresponding to the above (3) may include, for example, a luminizing agent, an alkaline phosphatase, and a beta-galactosidase. Alkaline phosphatase hydrolyzes BCIP to produce blue, and hydrolyzes PNPP to produce yellow. Beta-galactosidase hydrolyzes X-gal to produce blue derivatives, and hydrolyzes ONPG to produce yellow. Fluorescent substrates can also be used for alkaline phosphatase and beta -galactosidase. The antibiotic resistance gene corresponding to (4) above may be selected from tetracycline and kanamycin resistance genes.
The host cell may be any one selected from the group consisting of microorganisms such as Gram-negative bacteria, Gram-positive bacteria, actinomycetes, yeast and fungi. In addition, it may be any one selected from the group consisting of Staphylococcus aureus , Pseudomonas putida , Saccharomyces cerevisiae , and Deinococcus radiodurans , Preferably E. coli, and more preferably E. coli BL21 (DE3) or E. coli DH5.alpha., But is not limited thereto.
The first gene construct may be inserted into the chromosome of the host cell, and the second gene construct may be present in the recombinant expression vector in the transformed host cell. However, the present invention is not limited thereto. No.
A third gene construct comprising a cadC gene encoding a CadC protein whose expression is regulated by the third promoter and the third promoter is additionally transformed into the host cell while being contained in a recombinant expression vector, The third genetic construct may be divided into a recombinant expression vector distinct from the second genetic construct and may be contained in the same recombinant expression vector as that of the second genetic construct, May be contained within a vector.
The third promoter may be an expression promoter, but is not limited thereto.
Another aspect of the present invention provides a method for detecting or quantifying heavy metals, which comprises bringing the recombinant microorganism for detecting heavy metals into contact with a suspect sample containing heavy metals.
Another aspect of the present invention provides a method for expressing a protein by contacting a protein-expressing transformant containing the heavy metal as an expression-inducing agent with a solution containing heavy metals to obtain a desired protein.
According to a specific embodiment of the present invention, using the protein expression method of the present invention, expression efficiency similar to or better than that of commercially available IPTG can be achieved despite the use of inexpensive heavy metals (FIG. 8).
Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.
However, the following examples and experimental examples are provided only for illustrating the present invention, and the content of the present invention is not limited by the following examples and experimental examples.
[ Example And Experimental Example ]
Identification of CadC Protein Reactivity for Various Heavy Metals
[1-1] P cadC -cadO-cadC Production of genetic constructs
From the genome information of B. oceanisediminis 2691 (http://www.ncbi.nlm.nih.gov/nuccore/ALEG00000000) opened in NCBI, six CadCs having the amino acid sequences of SEQ ID NOS: 1 to 6 listed in Table 1 below protein (cadC1945, cadC4657, cadC538, cadC551, cadC595 and cadC640) of the gene coding for the six CadC protein in the genome of the B. oceanisediminis 2691 (cadC1945, cadC4657, cadC538, cadC551, cadC595 and cadC640) was identified as the A gene construct ( P cadC- cadO-cadC gene constructs) as shown in Table 2 was derived , including the nucleotide sequences of promoters ( P cadC ) and operator ( cadO ) that regulate the expression of these genes in the upstream.
Based on the nucleotide sequence of the thus-derived gene construct, primer pairs for each gene were prepared as shown in Table 3 and the genome constructs of Table 2 were obtained from the entire genome of B. oceanisediminis 2691 P cadC- cadO-cadC gene construct) was amplified and constructed.
(SEQ ID NO)
(aa)
Sequence homology (%)
(SEQ ID NO: 1)
(SEQ ID NO: 2)
(SEQ ID NO: 3)
(SEQ ID NO: 4)
(SEQ ID NO: 5)
(SEQ ID NO: 6)
(SEQ ID NO)
(Underline: start codon and termination codon)
(SEQ ID NO: 7)
(SEQ ID NO: 8)
(SEQ ID NO: 9)
(SEQ ID NO: 10)
(SEQ ID NO: 11)
(SEQ ID NO: 12)
[1-2] P cadC -cadO-cadC-egfp Production of transformants of genetic constructs
T7 ribosome binding site of primer pairs, including (T7 ribosome binding site) (for cadC538 SEQ ID NO primer pair 14 and 31; for cadC551 SEQ ID NO primer pair 17 and 31; for cadC595 SEQ ID NO: 20, and primer pair 31 ; for cadC640 SEQ ID NO: 23, and primer pair 31; for cadC1945 SEQ ID NO: 26, and primer pair 31; using for cadC4657 SEQ ID NO: 29, and primer pair 31) amplifies the egfp gene is a fluorescent gene by PCR two DNA to obtain a fragment, with a to and as a template to perform the PCR overlap the P-cadC -cadO cadC gene construct and egfp gene prepared in the above examples 1-1 fused, six P-cadC -cadO cadC -egfp gene constructs, respectively.
One of the six P cadC -cadO-cadC-egfp gene construct produced as described above, P cadC -cadO-cadC538-egfp gene construct, P cadC -cadO-cadC511-egfp gene construct, P cadC a -cadO-cadC595-egfp gene construct, P cadC -cadO-cadC1945-egfp gene construct and P cadC -cadO-cadC4657-egfp gene construct teuneun junction (ligation) to Tblunt ™ vector (Solgent, Korea) was inserted to create a recombinant vector pCadC538 (cadC 538- egfp), pCadC551 (cadC 551- egfp), pCadC595 (cadC 595- egfp), pCadC1945 (cadC 1945- egfp), pCadC4657 (cadC 4657- egfp), P cadC - cadO-cadC640-egfp gene construct teuneun by inserting a joint (ligation) to JET1.2 vector (Thermo Fischer Scientific, USA ) to prepare a recombinant vector pCadC640 (cadC 640- egfp) (Fig. 1).
By the six recombinant vector produced as described above and transformed into E. coli DH5α strain was produced transformants of P cadC -cadO-cadC-egfp gene construct.
[1-3] Determination of the reactivity of CadC protein to various heavy metals
The colonies of the six transformants prepared in Example 1-2 were inoculated into an LB liquid medium containing 50 μg / ml of ampicillin and cultured at 37 ° C. and 180 rpm for 12 hours. Increase, while the culture solution by 250㎖ in LB liquid medium in the Erlenmeyer flask containing the
200 μl of the culture was transferred to a 96 well black clear bottom plate (Greiner, Austria), and the absorbance at 595 nm was firstly measured using an absorbance filter in a fluorescent plate (Infinite 2000, Tecan, Austria) After 4 hours, 200 μl of the culture was again taken, and the absorbance was measured with the same filter to analyze the change in absorbance. Similarly, 200 μl of the culture was transferred to a 96 well black, clear bottom plate (Greiner, Austria), and the fluorescence intensity was measured using a fluorescent filter (Infinite 2000, Tecan, Austria) The amount of fluorescence was measured. After 4 hours, the amount of fluorescence was analyzed by measuring the amount of fluorescence with 200 쨉 l of the culture solution using the same filter. And obtaining a variation amount of the measured fluorescence (fluorescence increase) divided by the change in absorbance (increase in absorbance) specific fluorescence change (ΔF / ΔA) value as described above by calculating the concentration of specific reactivity for each heavy metal, heavy metal arsenic (As 3+) , Cadmium (Cd 2+ ), Cobalt (Co 2+ ), Cr (Cr 6+ ), Lead (Pb 2+ ) and Zinc (Zn 2+ ).
The specific fluorescence change value according to each heavy metal concentration of each transformant is shown in Fig. 2, and it was confirmed that each CadC protein exhibited different reactivity to each heavy metal (Fig. 2). In particular, CadC538, CadC551 and CadC1945 showed specific reactivity to lead, CadC640 and pCadC4657 showed specific reactivity to cadmium, and CadC595 showed similar reactivity to lead and cadmium.
Cadc640 protein, which is specific for cadmium, has a minimum detection concentration of about 10-20 μM for cadmium, and specific fluorescence change value increases with concentration. In addition, the CadC1945 protein exhibiting a specific reactivity to lead was found to have a minimum detection concentration of about 10-20 μM in lead, and a specific fluorescence change value was increased depending on the concentration (FIG. 2).
Implementation of Transformant for Sensitive Heavy Metal Detection
[2-1] Production of a first gene construct containing T7 RNA polymerase
B. from the dielectric of oceanesediminis 2691, SEQ ID NO: 36 and 37 SEQ ID NO: 10 using the primer pairs of P-cadC -cadO cadC640 of the gene construct, SEQ ID NO: SEQ ID NO: 11 using the
[2-2] Production of a transformant into which a first gene construct is introduced on a chromosome
Approximately 1000 bp of the portion of the galM gene upstream of the T7 RNA polymerase of E. coli BL21 (DE3) was amplified by PCR using the primer pairs of SEQ ID NOS: 32 and 34, and the PCR product was amplified in the same manner as in Example 2-1 the manufactured respective gene cassette (KmR- cadC640, KmR- cadC1945 and KmR- cadC4657) and by overlap PCR to prepare a galM-KmR- cadC640, galM-KmR- cadC1945 and galM-KmR- cadC4657 gene cassette.
Then, about 800 bp of the gene portion of the T7 RNA polymerase of E. coli BL21 (DE3) was amplified by PCR using the primer pair shown in Table 3 (the pair of primers of SEQ ID NOs: 38 and 47 for cadC640 and the pair of primers of SEQ ID NOs: 42 and 47 for cadC1945 primer pair; for cadC4657 SEQ ID NO: 46, and primer pair 47) by PCR PCR products obtained by amplification (T7RNAP fragment) and the galM-KmR- cadC640, galM-KmR- cadC1945 and galM-KmR- cadC4657 gene cassette SEQ ID NO: primer pair 32 and 47 to overlap PCR by the dielectric of the BL21 (DE3) homologous recombination (homologous recombination) a form of a linear galM-KmR- cadC640-T7RNAP fragment, galM-KmR- cadC1945-T7RNAP fragment and galM-KmR- cadC4657 fragment gene cassette was constructed.
On the other hand, the Δ lacI :: KmR strain of the KEIO collection was cultured to inoculate the P1 phage, and the BL21 (DE3) strain was used as a recipient cell to transform the cell lysate of P1 phage into P1- transduction) by Escherichia coli BL21 (DE3) :: Δ lacI was produced KmR strains, E. coli BL21 (DE3) Δ lacI :: KmR strains replaced with the kanamycin resistance gene Δ lacI gene is kanamycin is added to the 25㎍ / ㎖ concentration , And it was confirmed by PCR that the? LacI gene site was replaced with the kanamycin resistance gene. A pCP20 plasmid, which is a plasmid encoding the FRT flippase (FRT flippase), was transformed into the Escherichia coli BL21 (DE3) Δ lacI :: KmR strain and homologous recombination between FRT sequences located at both ends of the kanamycin resistance gene yielded kanamycin The resistance gene was deleted and BL21 (DE3) Δ lacI strain with kanamycin sensitivity was obtained. The BL21 (DE3)? LacI strain prepared as described above was inoculated into 100 ml LB liquid medium supplemented with ampicillin at a concentration of 50 占 퐂 / ml and cultured at 30 占 폚 and 180 rpm to prepare an algebra grower (OD 600 nm = 0.4 ~ 0.6), L-Arabinose was added to a final concentration of 0.1 M, followed by further incubation for 3 hours. The culture was transferred to a 50 ml conical tube and centrifuged at 4 ° C and 3000 rpm for 10 minutes to recover the cells. The cells were resuspended twice with cold distilled water and finally resuspended in 50 μl of cold distilled water. A public competent cell was prepared.
10 ng of the prepared linear galM-KmR- cadC640-T7RNAP fragment , galM-KmR- cadC1945-T7RNAP fragment and galM-KmR- cadC4657 fragment gene cassette were added to the prepared competent cells, The transformants were plated on LB solid medium supplemented with kanamycin at a concentration of 25 / / ml, and the resulting colonies were selected. From the resulting colonies, specificities were determined for the chromosome of BL21 (DE3) Δ lacI primers of SEQ ID NO: 48 and galM-KmR- cadC640-T7RNAP fragment, galM-KmR- cadC1945-T7RNAP fragment and galM-KmR- cadC4657-T7RNAP gene fragment specific primers (primers of SEQ ID NO: 37 for the cassette cadC640; cadC1945 primer of SEQ ID NO: 41 for; by using the primer of SEQ ID NO: 45) for cadC4657 confirmed by PCR, and the gene of ΔlacI galM-KmR- cadC4657-T7RNAP construct Having a strain HK739, and HK744 strain ΔlacI having galM-KmR- HK760 strain with the gene constructs of cadC640-T7RNAP, and ΔlacI and galM-KmR- gene construct of cadC1945-T7RNAP was prepared, respectively (Fig. 3) .
[2-3] operator cado To The second gene Constructed making
[2-3-1] CadC1945 Operators for proteins cado To The second gene Constructed Produce
Nde I from the plasmid pGESS (E135K) (Choi SL et al ., ACS Synthetic Biology 3: 163-171, 2014) and the pair of primers of SEQ ID NOS: 49 and 50 containing the Xho I restriction enzyme recognition sequence were used to amplify the egfp gene the amplified by PCR, and inserted it was purified by reacting with Nde I, Xho I restriction enzyme in the following, this vector pET21a (+) was cleaved by reacting with the same restriction enzyme to prepare a pET21a (egfp).
Were remove the lacO in the pET21a (egfp) prepared as described above and to insert the in place with the operator cadO be inserted into a restriction enzyme seat (T7 BamHI oligomer base sequence), the lacO is Bgl the pET21a (egfp) II, Xba I restriction enzyme, and the T7 BamHI oligomer base sequence was designed so as to be complementary in forward and reverse directions, and the 47 bp long T7 BamHI oligomer sequence of SEQ ID NOS: 52 and 53 was reacted at 95 ° C for about 5 minutes Status was then made double-stranded is allowed to stand for 20 minutes at room temperature, was then inserted into the seat lacO the removal of the pET21a (egfp) preparing a pET21a (T7BamHIoligo-egfp), insertion of the T7 BamHI oligomer base sequence as described above The recombinant vector obtained from the transformant transformed with pET21a ( T7BamHIoligo-egfp ) was confirmed as a template by performing PCR reaction.
In order to remove the lacI gene from the pET21a (T7BamHIoligo-egfp) prepared as described above, so that it does not contain a lacI gene regions in the pET21a (T7BamHIoligo-egfp) as a template, the 5 'and the terminal is phosphorylated SEQ ID NO: 53 and 54 primers ( T7BamHIoligo-egfp-ΔlacI ) recombinant vector in which the lacI gene was removed from the pET21a (+) vector by ligation of the ends of the PCR product, and the removal of the lacI gene Was confirmed by carrying out a PCR reaction.
Then, were inserted into the operator cadO (cadO1945) for CadC1945 protein in T7 BamHI oligomers edge of the pET21a (T7BamHIoligo-egfp), cutting the pET21a (T7BamHIoligo-egfp-ΔlacI) as prepared above into BamHI, XbaI restriction enzyme The oligonucleotide sequences of SEQ ID NOS: 57 and 58 having a length of 32 bp synthesized by being designed to be complementary in forward and reverse directions including the nucleotide sequence of cadO1945 were prepared in the same manner as the T7 BamHI oligomer, PET21a ( cadO1945-egfp ) was inserted into the cleaved site of pET21a ( T7BamHIoligo-egfp ), and the insertion of the cadO1945 oligomer base sequence as described above was carried out from the transformant transformed with pET21a ( cadO1945-egfp ) confirmed by performing a PCR reaction as a template to obtain a recombinant vector to be finally lacI gene was removed from the pET21a (+) vector lacO instead be included in the operator cadO1945 for CadC1945 protein was produced in a recombinant vector pET21a (P T7 _cadO1945_egfp) of the egfp gene insertion (Figure 5).
[2-3-2] Preparation of a second gene construct of the 2-plasmid system, including the operator cadO for the CadC4657 protein
Were inserted into the operator cadO (cadO4657) for the protein in the T7 CadC4657 BamHI oligomers place of the pET21a (T7BamHIoligo-egfp) prepared in Example [2-3-1], the pET21a (T7BamHIoligo-egfp) as prepared above It was digested with BamHI, XbaI restriction enzyme, to prepare a nucleotide sequence and comprises a sequence number of 32bp in length synthesized designed to have the forward and reverse complementary 59 and 60 the oligomer base sequence of the cadO4657 in the same manner as the T7 BamHI oligomers by the inserts in the cutting position were prepared pET21a (cadO4657-egfp), whether cadO4657 insertion of the oligomer nucleotide sequence as described above is the pET21a (cadO4657-egfp) of the next, the pET21a (T7BamHIoligo-egfp) made of double-stranded The recombinant vector obtained from the transformant was confirmed as a template by carrying out a PCR reaction.
Example lacI gene was removed in the same manner as in [2-3-1], and finally, pET21a (+) The lacI gene removed from the vector and lacO instead CadC4657 protein in pET21a (cad4657-egfp) as prepared above containing the operator of cadO4657 for and was produced with a recombinant egfp gene transfer vector pET21a (P T7 _cadO4657_egfp) (Fig. 5).
Further, from the pHCE vector (Poo, HR et al., 2002. Novel high-level constitutive expression system, pHCE vector, for a convenient and cost-effective soluble production of human tumor necrosis factor-alpha. Biotechology Letters 24: 1185-1189) SEQ ID NO: 61 and using a primer pair 62 were amplified with the HCE promoter (P hce) permanent expression promoter as PCR, B. oceanisediminis 2691 cadC4657 the gene using the primer pair SEQ ID NO: 63 and 64 from genomic DNA PCR in then, these two DNA fragments (P hce and cadC4657 gene) by performing the PCR overlap with SEQ ID NO: 61 and 64 primers and as the template to amplify hce P - was produced cadC4657 gene construct. The P hce - cadC4657 gene construct was reacted with Bam H1 and Hin dIII restriction enzymes and purified and then inserted into a pACYC184 vector (Catalog Number E4152S, New England Biolabs, Inc.) cut by reaction with the same restriction enzyme, The vector pACYC184 ( P hce - cadC4657 ) was prepared (Fig. 4).
[2-3-3] Operator for CadC640 protein cado Lt; RTI ID = 0.0 > 1-plasmid < / RTI > system,
An operator cadO ( cadO640 ) for the CadC640 protein was inserted into the T7 BamHI oligomer site of pET21a ( T7BamHIoligo-egfp-ΔlacI ) prepared in the above Example [2-3-1]. The pET21a ( T7BamHIoligo-egfp ) Was cleaved with BamHI and XbaI restriction enzymes. Oligomeric nucleotide sequences of SEQ ID NOS: 55 and 56 having a length of 32 bp synthesized so as to be complementary in forward and reverse directions including the nucleotide sequence of cadO640 were subjected to the same method as the T7 BamHI oligomer PET21a ( cadO640- egfp) was prepared by inserting it into the cleaved site of the pET21a ( T7BamHIoligo-egfp ). The insertion of the cadO640 oligomer base sequence as described above was performed using the pET21a ( cadO640-egfp ) Was transformed with a recombinant vector obtained from a transformant as a template.
Then, the HCE promoter ( P hce ), which is a constant expression promoter, was amplified by PCR using the primer pairs of SEQ ID NOs: 65 and 66 from the pHCE vector used in the above Example [2-3-2], and B. oceanisediminis 2691 The cadC640 gene was amplified by PCR using the primer pairs of SEQ ID NOs: 67 and 68 from the genomic DNA, and then overlap PCR was carried out using the two DNA fragments ( P hce and cadC640 genes) as primers and SEQ ID NO: 65 and 68 primer pairs To construct the P hce - cadC640 gene construct. The P hce - cadC640 gene constructs a reaction and purification of the truck bit to PspOM I restriction enzyme, and then, reacted with the same restriction enzymes and then inserted into a pET21a (P T7 _cadO640_egfp-ΔlacI) recombinant vector digested recombinant vector pET21a (cadO640-egfp & P hce - cadC640 ).
Implementation of biosensor and confirmation of reactivity to heavy metals
[3-1] Implementation of biosensor
To implement the detection of heavy metals for the biosensor of the present invention, the embodiment [2-2] in Example HK744 strain with the gene constructs of the ΔlacI galM-KmR- cadC1945-T7RNAP produced in [2-3 - 1] was produced by transforming a pET21a (P T7 _cadO1945_egfp) transformants HK744 (P T7 _cadO1945_egfp) produced from. The recombinant vector pET21a (SEQ ID NO: 2) prepared in the above Example [2-3-2] was added to HK739 strain having the gene construct of? LacI and galM-KmR- cadC4657-T7RNAP prepared in the above Example [ P T7 _cadO4657_egfp) and pACYC184 (P hce - transformed by transforming the cadC4657) each body HK739 (P T7 _cadO4657_egfp / P hce - the cadC4657) was produced. Finally, the embodiment [2-2] with a ΔlacI galM-KmR- Example [2-3-3] A pET21a (cadO640- produced in the HK760 strain with the gene construct produced in the cadC640-T7RNAP egfp & amp ; Pce - cadC640 ) was transformed to construct a transformant HK760 ( cadO640- egfp & pcece - cadC640 ).
Specifically, the above-mentioned transformation was carried out by inoculating each of the colonies of strain HK744, HK739 and HK760 prepared in Example 2-2 in 5 ml of LB liquid medium, culturing them at 37 DEG C and 180 rpm for 12 hours, , And this was inoculated in a 100 ml LB of 500 ml Flask at 1% (v / v) and cultured at 37 ° C. until the logarithmic increasing period (OD 600 nm = 0.4-0.6 ). Then, The cells were transferred to a conical tube and centrifuged at 4 ° C and 3000 rpm for 10 minutes to collect the cells. The recovered cells were resuspended in 50 ml of 80 mM MgCl 2 , 20 mM CaCl 2 solution, centrifuged at 4 ° C and 3000 rpm for 10 minutes, and finally resuspended in 5 ml of 100 mM CaCl 2 solution. Competent cells were prepared. In - (cadC640 cadO640-egfp & P hce) for 42 ℃ addition by each 50ng the HK744 and competent cells prepared as described above 100㎕ of HK760 the embodiment [2-3] The pET21a (P T7 _cadO1945_egfp) and pET21a produced in 1
[3-2] Check the reactivity to heavy metals
First, the embodiment [3-1] Conversion of the two transformants prepared in body HK744 (P T7 _cadO1945_egfp) and HK739 - the same as in Example 1 to confirm the reactivity with the heavy metals in the (P T7 _cadO4657_egfp / P hce cadC4657 ) The transformant was cultured and the lead or cadmium was added at a concentration of 0 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 20 μM, 30 μM, 50 μM and 100 μM, Were used to measure the absorbance and fluorescence of the transformant. T7 RNA polymerase / T7 promoter system (the "T7 system, it will), the embodiment [1-3] pCadC1945 (cadC 1945- egfp ) in order to confirm the degree of sensitivity enhancement through the introduction of and pCadC4657 (cadC 4657- (egfp ) were introduced.
As a result, in the existing embodiment [1-3] pCadC1945 (cadC 1945- egfp ) and the pCadC4657 (cadC 4657- egfp) introduced transformants, but starts to detect cadmium at levels of 10μM (Fig. 2, Fig. 6 ), the T7 embodiment the system is introduced for example, [3-1] of HK744 (P T7 in the transformant of _cadO1945_egfp) (Fig. 7), in the embodiment of the lead concentration of 10-100 nM trace level [3-1] In the transformant of HK739 ( P T7 _cadO4657_egfp / P hce - cadC4657 ), it was confirmed that the GFP fluorescence signal was amplified even at a trace amount of cadmium concentration of 10-100 nM level (FIG. 6) -1000 times more sensitive or sensitive.
Next, in order to confirm the reactivity of the transformant HK760 ( cadO640- egfp & pcece - cadC640 ) prepared in Example [3-1] to heavy metals, the transformant HK760 ( cadO640- egfp & pcece - cadC640 ) The colonies were inoculated into an LB liquid medium containing 50 μg / ml of ampicillin, cultured at 37 ° C. and 180 rpm for 12 hours, and used as a preculture. The culture solution was inoculated into a 250 ml Erlenmeyer flask at a concentration of 1% (v / v) in 50 ml of M9 liquid medium containing 50 μg / ml of ampicillin and 0.3% of glucose and cultured at 37 ° C. and 180 rpm, When the logarithmic increment (OD 600nm = 0.4-0.6) is reached, 4 ml of each solution is dispensed into a 14 ml round bottom tube, and Cadmium chloride (CaCl 2 ), Lead (II) chloride and PbCl 2 The final concentration was adjusted to be 0 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 20 μM, 30 μM, 50 μM and 100 μM and the absorbance and fluorescence intensity Respectively. Is compared with the introduction of transgenic, embodiment [1-3] pCadC640 (cadC 640- egfp ) in order to confirm the degree of sensitivity enhancement through the introduction of the T7 system.
As a result, this conventional embodiment [1-3] of pCadC640 (cadC 640- egfp) is introduced in the transformant but begins to detect cadmium at levels of 10μM (Fig. 2), T7 system introduced in Example 3 ( CadO640- egfp & pcece - cadC640 ) of HK760 ( cadO640- egfp & pcece - cadC640 ) at the concentration of 10-100 nM (FIG. 7) -1000 times more sensitive or sensitive.
Development of high protein expression system using heavy metals as expression inducer
Example [3-1] was the HK744 induce the expression of egfp gene in the downstream of the T7 promoter using the derived zero-lead expressed in transformant of (P T7 _cadO1945_egfp), an IPTG inducible expression as a control for this with zero to induce the egfp-pET21a / BL21 (DE3) expression of egfp gene in the transformant, and the two transformants compared to the non-emitting hyeonryang (Specific fluorescence, Fluoresence / OD 595nm ) of eGFP.
Colonies of the two transformants were inoculated into an LB liquid medium containing 50 μg / ml of ampicillin and cultured for 12 hours at 37 ° C. and 180 rpm for preculture. The culture medium to the 250㎖ while in LB liquid
As a result, when the concentration of IPTG and lead was 10 μM, it was confirmed that the amount of EGFP was almost the same, and the amount of protein expressed in both transformants was found to be similar. However, as the concentration of IPTG increased in the pET21a-egfp / BL21 (DE3) transformants, the amount of EGFP light decreased, while in HK744 ( P T7 _cadO1945_egfp ) transformants, the amount of EGFP increased (Fig. 8). From the results as described above, HK744 than using a lead-in (P T7 _cadO1945_egfp) transformants by using induction zero expression, zero pET21a-egfp / BL21 (DE3) transformed expression induction of IPTG in the transformants, the same or a number of proteins Can be produced.
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
<110> Korea Research Institute Bioscience and Biotechnology <120> Microbial Biosensor for Detecting Heavy Metals <130> 2015-DPA-1178 <150> KR 1020140148483 <151> 2014-10-29 <160> 74 <170> Kopatentin 2.0 <210> 1 <211> 121 <212> PRT <213> Bacillus oceanisediminis 2691 <400> 1 Met Gln Asn Asp Ala Cys Glu Val Thr Cys Ile Asp Glu Glu Lys Val 1 5 10 15 Asn Arg Gly Lys Asn Glu Leu Leu Gln Gln Asn Pro Leu Glu Val Ala 20 25 30 Lys Val Phe Lys Ala Leu Ser Asp Asp Thr Arg Ile Lys Ile Ala Tyr 35 40 45 Ala Leu Ser Leu Glu Asp Glu Leu Cys Val Cys Asp Val Ala Asn Ile 50 55 60 Val Gly Ala Thr Thr Ala Thr Thr Ser His His Leu Arg Leu Leu Lys 65 70 75 80 Asn Leu Gly Leu Ala Lys Tyr Arg Lys Glu Gly Lys Leu Val Tyr Tyr 85 90 95 Ser Leu Asp Asp Asp His Val Lys Gln Leu Ile Gln Val Ala Phe Ala 100 105 110 His Gln Lys Glu Val Val Lys Ile Val 115 120 <210> 2 <211> 126 <212> PRT <213> Bacillus oceanisediminis 2691 <400> 2 Met Ser Leu Val Asp Lys Asn Met Lys Asp Thr Leu Lys Asp Glu Cys 1 5 10 15 Glu Ile Tyr Cys Tyr Asp Pro Val Lys Val Ser Lys Leu Gln Asp Leu 20 25 30 Leu Ser Ser Lys Ser Thr Leu His Leu Ser Lys Lys Phe Lys Leu Leu 35 40 45 Ala Asp Glu Thr Arg Leu Lys Ile Ile Leu Ser Leu Ala Ile Glu Gly 50 55 60 Glu Leu Cys Val Cys Asp Val Ala Asn Ile Ile His Ser Ser Ile Ala 65 70 75 80 Thr Ala Ser Tyr His Leu Arg Phe Leu Lys Lys Ser Gly Val Ala Asn 85 90 95 Tyr Arg Lys Glu Gly Lys Leu Ala Phe Tyr Tyr Ile Asp Asp Glu Ile 100 105 110 Phe Lys Ser Met Val Leu Leu Ser Phe His His Lys Glu Pro 115 120 125 <210> 3 <211> 132 <212> PRT <213> Bacillus oceanisediminis 2691 <400> 3 Met Ser Asp Lys Val Leu Asn Lys Ser Ser Gln Asp Thr Cys Glu Thr 1 5 10 15 Phe Cys Tyr Asp Glu Glu Lys Val Ser Arg Val Ser Gly Arg Ile Asp 20 25 30 Glu Ile Met Gly Val Glu Gln Leu Phe Lys Ala Leu Ser Asp Ser Thr 35 40 45 Arg Ile Lys Ile Ala Tyr Ala Leu Thr Leu Glu Glu Glu Leu Cys Val 50 55 60 Cys Asp Val Ala Asn Ile Ile Gly Ser Ser Thr Ala Thr Ala Ser His 65 70 75 80 His Leu Arg Leu Leu Arg Lys Met Gly Leu Ala Lys Tyr Arg Lys Glu 85 90 95 Gly Lys Met Val Phe Tyr Ser Leu Ser Asp Glu His Val His Gln Leu 100 105 110 Val Ser Ile Ala Leu Ile His Ser Lys Glu Gly Glu Gly Asp Gly Glu 115 120 125 Ser Thr Gly Lys 130 <210> 4 <211> 122 <212> PRT <213> Bacillus oceanisediminis 2691 <400> 4 Met Lys Gln Asp Asp Val Cys Glu Val Thr Cys Val Asp Glu Glu Lys 1 5 10 15 Val Arg Arg Val Lys Glu Ser Val Lys Gln Gln Asn Thr Leu Ala Val 20 25 30 Ser Gln Ile Phe Lys Ala Leu Ser Asp Asp Thr Arg Val Lys Ile Thr 35 40 45 Phe Ser Leu Tyr Glu Glu Glu Gly Leu Cys Val Cys Asp Val Ala Asn 50 55 60 Ile Val Gly Cys Thr Thr Ala Thr Ala Ser His His Leu Arg Leu Leu 65 70 75 80 Arg Asn Met Gly Leu Ala Lys Tyr Arg Lys Glu Gly Lys Leu Val Phe 85 90 95 Tyr Ser Leu Asp Asp Asp His Val Arg Gln Leu Ile Gln Ile Ala Phe 100 105 110 Ala His Gln Lys Glu Val Glu Asn Tyr Glu 115 120 <210> 5 <211> 122 <212> PRT <213> Bacillus oceanisediminis 2691 <400> 5 Met Ser Lys Lys Asp Thr Cys Asp Ile Tyr Cys Tyr Asp Glu Ala Lys 1 5 10 15 Val Lys Arg Ile Gln Gly Glu Met Gln Lys Glu Asp Ile Ser Ser Val 20 25 30 Ser Gln Leu Phe Lys Ala Leu Ala Asp Glu Asn Arg Ala Lys Ile Ser 35 40 45 Tyr Ala Leu Cys Gln Asp Asp Glu Leu Cys Val Cys Asp Val Ala Asn 50 55 60 Ile Ile Gly Ala Thr Val Ala Thr Thr Ser His His Leu Arg Thr Leu 65 70 75 80 His Lys Gln Gly Ile Val Lys Tyr Arg Lys Glu Gly Lys Leu Ala Phe 85 90 95 Tyr Ser Leu Asp Asp Glu His Ile Arg Gln Leu Met Val Ile Ala Leu 100 105 110 Thr His Lys Lys Glu Met Lys Val Asn Val 115 120 <210> 6 <211> 122 <212> PRT <213> Bacillus oceanisediminis 2691 <400> 6 Met Lys His Glu Asp Val Cys Glu Val Thr Cys Val Asp Glu Glu Lys 1 5 10 15 Val Arg Arg Val Lys Glu Ser Ala Ser His His Asn Thr Ser Ala Val 20 25 30 Ala Gln Ile Phe Lys Ala Leu Ser Asp Glu Thr Arg Val Lys Ile Ala 35 40 45 Phe Ser Leu Cys Glu Glu Glu Glu Leu Cys Val Cys Asp Val Ala Asn 50 55 60 Ile Val Gly Cys Thr Thr Ala Thr Ala Ser His His Leu Arg Leu Leu 65 70 75 80 Arg Asn Met Gly Leu Ala Lys Tyr Arg Lys Glu Gly Lys Leu Val Phe 85 90 95 Tyr Ser Leu Asp Asp Asp His Val Lys Gln Leu Ile Gln Ile Ala Phe 100 105 110 Thr His Gln Lys Glu Val Ala His Tyr Glu 115 120 <210> 7 <211> 566 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 7 aaaactggca cccttacact attcctagtg tatccaacat agaaaggaaa ttaaatttag 60 ttataagaat aattaattac tgcaaactaa agaaaacctg ctttttatgg gaattatatt 120 caaacgtata tttgactatt tgtttttgtt cacatataat aatattcaaa tgaatatttg 180 attgatctga ggtgaaaaat ttgcaaaatg atgcatgtga agttacgtgt atagatgaag 240 aaaaagtcaa tagaggtaaa aatgaactac tccaacaaaa tcctttggaa gtagccaaag 300 tttttaaagc tttatcggat gatactagga ttaagattgc gtatgctctt tctttggaag 360 atgaattgtg tgtatgtgat gtagcaaata ttgttggtgc tacaactgcc acaacatctc 420 atcatttgag gttacttaaa aatcttggat tagctaaata tcgaaaagaa ggtaagttag 480 tttactattc attagatgat gatcatgtaa agcaactaat acaggttgca tttgcacatc 540 aaaaggaggt tgtaaaaatt gtctga 566 <210> 8 <211> 581 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 8 taagttatga ttttttggtt attctcacgc gtttttgtta agagccgata taaagtaaca 60 cgtgaatcag atatacttac gtcttcagac ataagagaat atgaaacact tcgcttcttt 120 attcaagtaa atatttgaat gttgacatga aagcttactc attataagct aatcacatat 180 tttttaatga ggtgggagaa ttgtctttag ttgataaaaa tatgaaagat actctaaaag 240 atgaatgtga aatctattgt tatgaccctg taaaagtatc aaagttacaa gatttgttaa 300 gttctaaaag tacacttcat ctatcaaaaa aatttaaact tcttgctgat gagacaagat 360 taaaaattat tctttcatta gctatagaag gggaactttg cgtttgtgat gtcgctaata 420 ttattcattc ctcaatagct acagcttctt atcatttacg gtttttaaaa aaatcaggtg 480 tagctaatta tagaaaggaa ggtaaattag cattttacta tatcgatgat gaaattttca 540 agtcgatggt attactctct tttcatcata aggagccttg a 581 <210> 9 <211> 599 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 9 acgataaaaa gtataaaggt aattaagaat agctttttaa tcataagaaa cctcctaata 60 gtattaacga actttatttt aacagagtaa atataattat tcaaaagtac atttgaataa 120 atttttgaca aatttgttaa atcagcatag actaacgata taacatattc aaacaaatat 180 ttgaataaag ggtgacaaat atgagtgata aagtattgaa caaatcttcc caagatacat 240 gtgagacgtt ttgctacgat gaagaaaagg ttagccgagt aagcgggcgg atagatgaga 300 taatgggagt agagcaactg tttaaggcgc tatccgattc aacaagaata aagattgcat 360 acgcacttac acttgaagaa gaattgtgtg tatgtgatgt ggctaatatt atcggatcca 420 gtactgccac cgcttcccac catttgcgtt tactgcgtaa aatgggcttg gctaaatatc 480 ggaaagaggg aaagatggta ttttactctt tatcagatga gcacgtccat caactagttt 540 ctattgcttt aatacactca aaggaaggtg aaggagatgg ggaaagcact ggcaagtag 599 <210> 10 <211> 565 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 10 cttattttct gacattgttt tacctcctct taaaaagttt ccttaatcca aaaatattag 60 tacttgccaa taatttatga caaggacaac ctttttgtca acaaacattt tacgaaaaca 120 ttcaaataat tatttgacca aataaaagga attacatata ctaataatca aatgattgtt 180 tgagtatgga ggggtttaat ttgaaacaag atgatgtttg tgaagttacc tgtgtggatg 240 aagagaaagt aaggcgtgtt aaggaatcgg taaaacagca gaataccttg gctgtttctc 300 aaatatttaa agcattatct gatgacacta gagtaaagat tactttttcc ctatatgaag 360 aagaggggtt atgtgtctgt gatgttgcga atatcgtcgg atgtacgaca gcaaccgcat 420 cacaccattt gcgtttgtta cgcaatatgg gactagcgaa atatcgaaag gaaggaaaac 480 tagtttttta ttcgctggat gatgatcatg tcagacagct tatccaaatc gcatttgcac 540 atcaaaagga ggtggagaac tatga 565 <210> 11 <211> 569 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 11 acttctttga taaatctatt caaataatgt aacagttttg atcgttatga tcatttgcta 60 atataagtag gtttggatat ggaatttatt gagttattta attttttatc cattattcaa 120 ataatcattt gaataatgtt agggtaataa tatataatat attcaaacat acacttgaat 180 aaaaggaggg ataatgagta atgagtaaga aagatacttg tgatatttat tgttatgacg 240 aagcaaaagt caaacgaata caaggtgaga tgcaaaaaga agatatatct agtgtttccc 300 aattatttaa agcacttgca gatgaaaata gggcaaaaat ttcctatgca ttatgtcaag 360 atgatgaact ttgtgtgtgt gatgtagcta atatcattgg ggctactgtt gcaacgacat 420 ctcaccattt aaggaccctt cataaacaag ggattgtaaa ataccgaaaa gaaggcaaac 480 tagcctttta ttcgcttgat gatgaacata ttagacagtt aatggttatt gcattaactc 540 ataaaaagga gatgaaagtc aatgtctga 569 <210> 12 <211> 569 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 12 aatttaagat agaataaagc aaataataat tttcagttaa tggttcatcc ttcaatttgg 60 atgaaccatt tgtgctgttg gatactcatt ttcttatgac ataaaaatat cttcctaata 120 atcaaatgaa tgtttgactg tttggtaata gatgcatata ctaataatca aataatcgtt 180 tgattaagga ggtggcttcc ttgaagcatg aagatgtttg tgaagtaacc tgcgtagatg 240 aagaaaaagt aagacgtgtt aaagaatctg cctcacacca caatacctct gctgttgccc 300 aaatatttaa agcattatct gatgaaacta gggttaagat tgccttttcc ctatgtgagg 360 aagaagaact gtgcgtctgt gatgtagcca atattgtggg gtgcacaacg gctacggctt 420 ctcaccattt acggcttctt cggaatatgg gattggcgaa gtatcgtaag gagggcaaat 480 tggtctttta ttcactggat gatgatcatg tcaaacagct gattcaaatt gcctttacac 540 atcaaaagga ggtggcgcac tatgagtga 569 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Cad538F <400> 13 gaaaactggc acccttacac tattcctagt 30 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 538_OF <400> 14 ttgtaaaaat tgtctgagga gatatacata tggtg 35 <210> 15 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 538_OR <400> 15 tatgtatatc tcctcagaca atttttacaa cctcc 35 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Cad551F <400> 16 gtaagttatg attttttggt tattctcacg 30 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 551_OF <400> 17 catcataagg agccttgagg agatatacat atggt 35 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 551_OR <400> 18 atgtatatct cctcaaggct ccttatgatg aaaag 35 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Cad595F <400> 19 gacgataaaa agtataaagg taattaagaa 30 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 595_OF <400> 20 gaaagcactg gcaagtagag gagatataca tatgg 35 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 595_OR <400> 21 tgtatatctc ctctacttgc cagtgctttc cccat 35 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Cad640F <400> 22 gcttattttc tgacattgtt ttacctcctc 30 <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 640_OF <400> 23 tggagaacta tgaatgagga gatatacata tggtg 35 <210> 24 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 640_OR <400> 24 tatgtatatc tcctcattca tagttctcca cctcc 35 <210> 25 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Cad1945F <400> 25 gacttctttg ataaatctat tcaaataatg 30 <210> 26 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 1945_OF <400> 26 tgaaagtcaa tgtctgagga gatatacata tggtg 35 <210> 27 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 1945_OR <400> 27 tatgtatatc tcctcagaca ttgactttca tctcc 35 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Cad4657F <400> 28 gaatttaaga tagaataaag caaataataa 30 <210> 29 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 4657_OF <400> 29 tggcgcacta tgagtgagga gatatacata tggtg 35 <210> 30 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> 4657_OR <400> 30 tatgtatatc tcctcactca tagtgcgcca cctcc 35 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> rbs_eGFPR <400> 31 gttacttgta cagcttgtcc atgccg 26 <210> 32 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> galM_F <400> 32 gagccagccc ttgccagtcg gcgtaagg 28 <210> 33 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> galMKmR_OF <400> 33 gcaccgtcgc gccgaggaat tctgtaggct gg 32 <210> 34 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> galMKmR_OR <400> 34 gcagctccag cctacagaat tcctcggcgc gacg 34 <210> 35 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Km640OR <400> 35 ggaggtaaaa caattccggg gatccgtcga c 31 <210> 36 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Km640OF <400> 36 gatccccgga attgttttac ctcctcttaa aaag 34 <210> 37 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> T7_640OR <400> 37 tagtaaatcc ggatcattca tagttctcca cctc 34 <210> 38 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> T7_640OF <400> 38 ctatgaatga tccggattta ctaactggaa g 31 <210> 39 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Km1945OR <400> 39 tatcaaagaa gtattccggg gatccgtcga c 31 <210> 40 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Km1945OF <400> 40 gatccccgga atacttcttt gataaatcta ttc 33 <210> 41 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> T7_1945OR <400> 41 gttagtaaat ccggatcaga cattgacttt c 31 <210> 42 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> T7_1945OF <400> 42 caatgtctga tccggattta ctaactggaa g 31 <210> 43 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> K4657OF <400> 43 gatccccgga atataaaaat atcttcctaa taatc 35 <210> 44 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> K4657OR <400> 44 ggaagatatt tttatattcc ggggatccgt cgac 34 <210> 45 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> T74657tga_OR <400> 45 atccggatca ctcatagtgc gccacctcct tttg 34 <210> 46 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> T74657tga_OF <400> 46 tggcgcacta tgagtgatcc ggatttacta actg 34 <210> 47 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> T7R2 <400> 47 agaccacgcc tcgccaccga gtagaccc 28 <210> 48 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> gpmA_F <400> 48 cgccatgacg aaccagaacc agcttag 27 <210> 49 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> egfp_XhoI_R <400> 49 tggtgctcga gttacttgta cagcttgtcc atgc 34 <210> 50 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> egfp_NdeI_F <400> 50 agaaggagat atacatatgg tgagcaaggg cgag 34 <210> 51 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> T7-For-BamHI <400> 51 gatctcgatc ccgcgaaatt aatacgactc actataggat cccgggt 47 <210> 52 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> T7-Rev-BamHI <400> 52 ctagacccgg gatcctatag tgagtcgtat taatttcgcg ggatcga 47 <210> 53 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> del_lacI_F <400> 53 cccttacacg gaggcatcag tgacc 25 <210> 54 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> del_lacI_R <400> 54 ccctgcatta ggaagcagcc cagtag 26 <210> 55 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CadO640-F <400> 55 gatccaataa tcaaatgatt gtttgagtat gt 32 <210> 56 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CadO640-R <400> 56 ctagacatac tcaaacaatc atttgattat tg 32 <210> 57 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CadO1945-F <400> 57 gatcctatat tcaaacatac acttgaataa at 32 <210> 58 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CadO1945-R <400> 58 ctagatttat tcaagtgtat gtttgaatat ag 32 <210> 59 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CadO4657-F <400> 59 gatccaataa tcaaataatc gtttgattaa gt 32 <210> 60 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CadO4657-R <400> 60 ctagacttaa tcaaacgatt atttgattat tg 32 <210> 61 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> hceF_HindIII <400> 61 ggaagcttca tgaccccatc tggaatcgcc 30 <210> 62 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> hCEOR <400> 62 atcttcatgc ttcaatgata tctccttttt ccag 34 <210> 63 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> 4657OF <400> 63 aaaaggagat atcattgaag catgaagatg tttg 34 <210> 64 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 4657R_BamHI <400> 64 ccggatccac ttgcatcaga aacaccatcc 30 <210> 65 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> hCE_F <400> 65 gggcccgatc tctccttcac agattcccaa tc 32 <210> 66 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> hce640_OR <400> 66 cccctccatt gatatctcct ttttccagaa g 31 <210> 67 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hce640_OF <400> 67 ggagatatca atggaggggt ttaatttg 28 <210> 68 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 640R <400> 68 gggcccttca tagttctcca cctccttttg 30 <210> 69 <211> 27 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 69 taatattcaa atgaatattt gattgat 27 <210> 70 <211> 26 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 70 tttattcaag taaatatttg aatgtt 26 <210> 71 <211> 26 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 71 catattcaaa caaatatttg aataaa 26 <210> 72 <211> 26 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 72 aataatcaaa tgattgtttg agtatg 26 <210> 73 <211> 26 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 73 tatattcaaa catacacttg aataaa 26 <210> 74 <211> 26 <212> DNA <213> Bacillus oceanisediminis 2691 <400> 74 aataatcaaa taatcgtttg attaag 26
Claims (19)
A second promoter, a multiple cloning site (MCS) in which a gene of a target protein whose expression is regulated by the second promoter is cloned, and a second cloning site (MCS) interposed between the second promoter and the multiple cloning site, A second gene construct comprising an operator cadO in which a protein binds and the expression of a target protein by the second promoter is inhibited,
The cadO and cadC genes are derived from Bacillus oceanisediminis ,
The CadC protein is any amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 6,
In the presence of heavy metals, the CadC protein expression vector, characterized in that by being separated from the cadO, the expression of a target protein by the second promoter activation.
A second promoter, a multiple cloning site (MCS) in which a gene of a target protein whose expression is regulated by the second promoter is cloned, and a second cloning site (MCS) interposed between the second promoter and the multiple cloning site, A second gene construct comprising an operator cadO in which a protein binds and the expression of a target protein by the second promoter is inhibited,
Wherein the first gene construct further comprises a T7RNAP gene encoding a T7 RNA polymerase,
The first gene construct teuneun further comprising a cadO between the first promoter and the gene or cadC T7RNAP gene,
The second promoter of the second gene construct is a T7 promoter to which the T7 RNA polymerase binds,
In the presence of heavy metals, the CadC protein expression vector, characterized in that by being separated from the cadO, the expression of a target protein by the second promoter activation.
Wherein the first gene construct further comprises a ribosome binding site (RBS) between the cadC gene and the T7RNAP gene.
Wherein the first gene construct is comprised of a first promoter, a cadO , a cadC gene, and a T7RNAP gene in this order.
The CadC proteins with the CadC cadO the protein bonds are shown in SEQ ID No. 1 and SEQ ID NO: 69, SEQ ID NO: 2 and SEQ ID NO: 70, SEQ ID NO: 3 and SEQ ID NO: 71, SEQ ID NO: 4 and SEQ ID NO: 72, SEQ ID NO: 5 and SEQ ID NO: 73, or SEQ ID NO: 6 and SEQ ID NO: 74, respectively.
Wherein said heavy metal is cadmium or lead.
A second promoter, a reporter gene encoding a reporter protein whose expression is regulated by the second promoter, and a reporter gene which is interposed between the second promoter and the reporter gene, A second gene construct comprising an operator cadO in which the expression of the protein is inhibited;
Is transformed into a host cell,
The cadO and cadC genes are derived from Bacillus oceanisediminis ,
The CadC protein is any amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 6,
A transformant for detecting heavy metals, characterized in that in the presence of a heavy metal, the CadC protein is separated from the cadO , whereby the reporter protein is expressed by the second promoter.
A second promoter, a reporter gene encoding a reporter protein whose expression is regulated by the second promoter, and a reporter gene which is interposed between the second promoter and the reporter gene, A second gene construct comprising an operator cadO in which the expression of the protein is inhibited;
Is transformed into a host cell,
Wherein the first gene construct further comprises a T7RNAP gene encoding a T7 RNA polymerase,
The first gene construct teuneun further comprising a cadO between the first promoter and the gene or cadC T7RNAP gene,
The second promoter of the second gene construct is a T7 promoter to which the T7 RNA polymerase binds,
A transformant for detecting heavy metals, characterized in that in the presence of a heavy metal, the CadC protein is separated from the cadO , whereby the reporter protein is expressed by the second promoter.
Wherein the first gene construct further comprises an internal ribosome entry point (IRBS) between the cadC gene and the T7RNAP gene.
Wherein the first gene construct comprises a first promoter, a cadO gene, a cadC gene, and a T7RNAP gene in this order.
The CadC proteins with the CadC c adO the protein bonds are shown in SEQ ID No. 1 and SEQ ID NO: 69, SEQ ID NO: 2 and SEQ ID NO: 70, SEQ ID NO: 3 and SEQ ID NO: 71, SEQ ID NO: 4 and SEQ ID NO: 72, SEQ ID NO: 5, and SEQ ID NO: 73, or SEQ ID NO: 6 and SEQ ID NO: 74, respectively.
Wherein the reporter gene is a fluorescent gene or an antibiotic resistance gene.
Wherein the heavy metal is cadmium or lead.
Wherein the first gene construct is present in a state inserted into a chromosome of the host cell and the second gene construct is present in a state transformed into the host cell while being contained in a recombinant expression vector. Transformant for detection.
A third gene construct comprising a cadC gene encoding a CadC protein whose expression is regulated by the third promoter and the third promoter is additionally transformed into the host cell while being contained in a recombinant expression vector, Wherein said heavy metal is present in a state of being in a state of being in a state of being in a state of being in a state of being present.
And the third promoter is always an expression promoter.
Wherein the second gene construct and the third gene construct are contained in the same recombinant expression vector.
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