Patent Application
20080044829
The present invention relates to a biosensor using a specific microbe and, more particularly, it relates to a biosensor for a visual detection of harmful substances in environment by a simple means utilizing pigment-synthesizing gene of a purple non-sulfur bacterium and to a method for preparing such a biosensor. The present invention particularly relates to a biosensor for detection of arsenic in environment in low cost and by highly sensitive and simple means utilizing pigment-synthesizing gene of a purple non-sulfur bacterium and also to a method for preparing such a biosensor.
At present, in an aqueous environment such as the ocean, rivers and lakes/marshes, pollution by various harmful substances such as agricultural chemicals, heavy metals and endocrine disrupting chemicals is one of the serious problems and there has been a continued demand for removal thereof, improvement of quality of water, etc. in view of environmental cleanup and improvement in public hygiene. In the removal of various harmful substances as such those existing in environment and in the monitoring of the affection of specific chemical substances on environment, means for detecting them and means for measuring the concentration thereof are important at first. With regard to the means for detection and for grasping the concentration as such, a process where samples are collected in the field and subjected to measurement by instruments in laboratories is common but, in that case, it is not possible to trace the changes in concentration within short time. In addition, it is usual that objects to be measured are present as a mixture and, therefore, means such as chromatography is sometimes necessary for their separation and, in addition, since concentration of the object to be measured is usually much diluted, large amount of sample is sometimes necessary. Furthermore, depending upon the type of the aimed chemical substance, there are problems that its analytical operation is troublesome or that powerful chemicals or hazardous reagent may be necessary and, in measuring plural samples, there are problems that instruments become large and cost is high. In this way, when applying an instrumental analysis to environmental monitoring, there are many problems [“Biosensing” by Ikuo Karube (1988) published by Keigaku Shuppan]. For such problems, biosensors are receiving public attention in recent years and studies have been conducted therefor.
A biosensor stands for a device by which physiological or biochemical changes are detected, identified and recorded. The function is based on biochemical specificity of a substance having a biological activity and cells and tissues of higher animals and plants, enzymes, antibodies, DNA, receptors, cells of microbes, etc. are biological measuring elements. As in the examples such as enzyme vs. substrate, antibody vs. antigen and receptor vs. ligand, specificity of the reaction is a main advantage of a biosensor. Additional advantages of a biosensor are that it has a sensitivity keenly responding even to a substance in small amount, that it achieves the function even in a non-transparent solution unlike the optical instrumental analysis means, that a direct measurement is possible whereby separation analysis and purification of sample are not necessary, that the system is able to be made small due to the above whereby conveyance is good, that powerful chemicals and hazardous reagents are unnecessary since it is derived from biological element and monitoring is possible under a mild environment of ambient temperature and pressure, that a real-time analysis is possible due to quick response and that operation is simple [“Biosensing” by Ikuo Karube (1988) published by Keigaku Shuppan; S. F. D'Souza, (2001) Biosens. Bioelectron., 16:337-53].
Incidentally, in elements of a biosensor, enzymes and antibodies have very high specificity and are useful but, on the other hand, there are disadvantages that purification thereof needs much time and high cost and that inactivation due to instability of the molecule itself and to toxic substance in environment such as heavy metal is apt to happen. In view of environmental monitoring, a microbial biosensor is believed to be advantageous as compared with other types of biosensor due to the characteristics that genetic operation such as mutation and recombination is easy, that cost is able to be reduced because incubation is easy, that there are microbes suitable even for severe environmental condition due to acquirement of resistance, etc.
Microbial biosensors fundamentally comprise the following elements, i.e. a recombinant microbe as a host, a gene promoter sequence specifically responding to a specific chemical substance and a reporter gene located at downstream region of the promoter sequence and, in some cases, a physico-chemical device necessary for detecting the signal from the sensor is combined therewith (FIG. 1, [S. Belkin, (2003) Cur. Opin. Microbiol., 6:206-12]). When environmental stress or substance to be analyzed is present, its signal is transmitted into the microbe cells in such a system whereby the function of the promoter sequence becomes specifically “on” and gene transfer at the downstream region of the promoter sequence is induced. By detecting and measuring the expression signal of the reporter gene, an environmental monitoring is possible. When a microbial biosensor which has been actually conducted up to present is taken as an example, representative ones are that, with regard to a promoter, binary control system or repressor which is originally owned by a host such as heavy metal-responding promoter or DNA damage-responding promoter is mediated for response regulation and that, with regard to reporter gene, the gene which is able to detect and measure the signal with high sensitivity such as fluorescent protein or gene participating in luminescence is used [S. F. D'Souza, (2001) Biosens. Bioelectron., 16:337-53].
Particularly with regard to reporter gene, various comparative investigations have been carried out already and problems for each of them have been found as well [K. Hakkila, et al., (2002) Anal. Biochem., 301:235-42]. In green fluorescent protein (GFP) and red fluorescent protein (DsRed), protein itself expressed from each gene emits fluorescence without addition of co-factor, substrate, etc. whereby simple measurement and high sensitivity can be expected but they have a disadvantage that long time is needed until the translated protein is transformed into an active type where its conformation is able to emit fluorescence. Although lucFF which is firefly luciferase gene and luxCDABE which is microbioluminescence gene operon are also reporter genes which are frequently used for microbial biosensors, the former is required that a reaction substrate is added thereto while, in the latter, it codes for enzyme which produces a substrate of luminous reaction and enzyme which catalyzes the luminous reaction whereby there is no need of adding the substrate, etc. although, the fact that bacteria cells are always exposed to optical stress and that, since production of five polypeptides is inevitable, load is applied to cell growth and metabolism depending upon copy numbers of plasmid having reporter gene has been pointed out. In addition, in both luminous and fluorescent reporter genes, measuring requirements such as light-resistant chamber, optical fiber and high-sensitivity photometer are necessary for the measurement of a sample.
Among the above-mentioned hazardous substance polluting the aqueous environment, a particularly serious one is arsenic. Although its concentration is as very low as 0.0001%, arsenic is widely distributed on the surface of the earth and is present by bonding to minerals of metals such as copper, lead and gold. Most of arsenic is eluted into a hydrosphere by action of rivers and rain and accumulated in underground water or marine water [R. S. Oremland, et al., (2003) Science 300:939-44]. As such, arsenic has a characteristic that it is accumulated in a specific hydrosphere from the geological point of view. However, accumulation of arsenic affects a serious influence on the health of persons living the area around there and causes health troubles such as arteriosclerosis, myocardial infarction and peripheral circulatory troubles of the limbs. At present, the areas where severe affection by arsenic is noted are found throughout the world. Places where many patients suffering from arsenic intoxication are noted are whole area of Bangladesh, West Bengal areas of India and Inner Mongolia Autonomous Region of China and, besides them, presence of patients suffering from arsenic intoxication has been also confirmed in the State of Códoba of Argentina and Torreón of Mexico. Most of the causes thereof are use of underground water polluted with arsenic. A fundamental solving means therefor is to secure water source which is not polluted with arsenic but, due to the problem of economic circumstance, etc. of such areas, the current status is that it has not been solved yet.
On the other hand, detection of arsenic has been usually conducted by means of an inductively coupled plasma emission spectrometry (ICP-AES) where a measurement with high sensitivity is possible. However, the apparatus used for this analytical method is very expensive (several ten million yens) whereby an enormous cost is needed. Therefore, relatively less expensive (but less sensitive) kits for the detection of arsenic which utilize chemical reaction have been developed and actually used. In such a detection kit however, an operation process is troublesome and powerful chemicals such as hydrochloric acid or tin chloride are needed for the reaction and, therefore, there is a problem that arsenic is unable to be measured easily [M. M. Rahman, et al., (2002) Environ. Sci. Technol., 36:5385-94].
The present invention has been created in view of the above-mentioned current status of the prior art and its object is to provide a biosensor where measurement is able to be conducted by simpler operations, cost is less expensive and specific chemical substance is able to be visually detected. Another object of the present invention is to provide an apparatus by which arsenic is able to be detected in low cost, with high sensitivity and by easy operations.
In order to achieve such objects, the present inventors have carried out intensive studies paying their attention to pigment synthesis system of purple non-sulfur bacteria belonging to the family of photosynthetic bacteria and, as a result, they have found that enzyme gene carrying a leading role in the pigment synthesis system of this bacterium is very useful as a reporter gene of a biosensor whereupon the present invention has been achieved. The present inventors have also thought that arsenic is able to be detected in low cost, with high sensitivity and by simple operations when an inductive promoter showing a specific response to arsenic is adopted as an inductive promoter for the biosensor and have carried out intensive studies for an appropriate arsenic-inductive promoter sequence and, as a result, they have achieved the present invention.
Thus, in accordance with the present invention, there is provided a biosensor for the detection of a specific chemical substance, characterized in that it comprises a recombinant purple non-sulfur bacterium wherein sensor vector is introduced into a pigment variant of a purple non-sulfur bacterium being deficient in gene coding for a spheroidene monooxygenase enzyme, and that the sensor vector is a sensor vector where inductive promoter showing a specific response to a specific chemical substance is connected in an operable manner to the upstream region of gene coding for the spheroidene monooxygenase enzyme.
In accordance with the present invention, there is also provided a method for the preparation of a biosensor for detection of a specific chemical substance, characterized in that it comprises the steps of:
preparing a pigment variant of a purple non-sulfur bacterium being deficient in gene coding for spheroidene monooxygenase enzyme;
cloning an open reading frame of gene coding for spheroidene monooxygenase enzyme being deficient in promoter on vector;
connecting an inductive promoter showing a specific response to a specific chemical substance in an operable manner to the upstream region of gene coding for spheroidene monooxygenase enzyme in the vector to construct a sensor vector; and
introducing the sensor vector into the pigment variant of a purple non-sulfur bacterium to prepare a recombinant purple non-sulfur bacterium.
According to preferred embodiment of the present invention, the above-mentioned purple non-sulfur bacterium is Rhodovulum sulfidophilum.
According to preferred embodiment of the present invention, the above-mentioned specific chemical substance is dimethyl sulfide and the inductive promoter showing a response to the above-mentioned specific chemical substance is dimethyl sulfide-inductive promoter.
According to more preferred embodiment of the present invention, the above-mentioned dimethyl sulfide-inductive promoter is a promoter which is present in an upstream region of operon of gene coding for dimethyl sulfide dehydrogenase enzyme.
According to the particularly preferred embodiment of the present invention, the above-mentioned promoter is DNA consisting of a base sequence of SEQ ID No. 1 or DNA consisting of a base sequence which is at least 60% homologous to the base sequence of SEQ ID No. 1 and having an activity of dimethyl sulfide-inductive promoter.
According to another preferred embodiment of the present invention, the above-mentioned specific chemical substance is arsenic and the inductive promoter showing a response to the above-mentioned specific chemical substance is DNA consisting of a base sequence of SEQ ID No. 29 or DNA consisting of a base sequence which is at least 60% homologous to the base sequence of SEQ ID No. 29 and having an activity of arsenic-inductive promoter.
In the biosensor of the present invention, gene coding for spheroidene monooxygenase enzyme is used as a novel reporter gene as mentioned above whereby the measured result is able to be expressed as a clear color tone change of the culture liquid of the biosensor. Accordingly, when the biosensor of the present invention is used, no measuring equipment such as a highly sensitive photometer in the case of biosensors in the prior art using luminous or fluorescent reporter gene is necessary and the hazardous substances in environment are able to be visually detected by a simple means. Particularly, the biosensor in the preferred embodiment of the present invention adopts an inductive promoter showing a specific response to arsenic as an inductive promoter of the biosensor whereby it is now possible to detect arsenic in low cost, with high sensitivity and by a simple operation.
a shows LC-MS spectrum of a carotenoid pigment. Peak numbers of each sample correspond to
b shows LC-MS spectrum of a carotenoid pigment. Peak numbers of each sample correspond to
In
A purple non-sulfur bacterium which is the main constituting element of the biosensor of the present invention synthesizes carotenoid pigment from geranylgeranyl diphosphate which is a common product of isoprenoid biosynthesis route. In the freshwater genus Rhodobacter which is a type of purple non-sulfur bacterium, it has been already known that eight kinds of enzymes concerning carotenoid synthesis route are present and that genes coding therefor form cluster [H. P. Lang, et al., (1995) J. Bacteriol., 177(8):2064-73]. A purple non-sulfur bacterium accumulates spheroidenone which is the final product of carotenoid or a derivative thereof and the culture liquid shows reddish brown color derived from the pigment.
Production of spheroidenone is carried out by oxidation of spheroidene which is a precursor yellow pigment (or a derivative thereof) and this reaction is catalyzed by CrtA enzyme (spheroidene monooxygenase). Accordingly, in a purple non-sulfur bacterium strain where CrtA enzyme is deficient, the culture liquid turns yellowish brown as a result of accumulation of spheroidene. The idea which exists in the base of the present invention is that, as a result of regulation of the expression of this CrtA enzyme, changes in color tone of the culture liquid from yellow to red are achieved and, as a result of its utilization, construction of “visual biosensor” is made possible. Particularly, the idea which exists in the base of the present invention is that, as a result of regulation of expression of this CrtA enzyme using an arsenic-inductive promoter, changes in color tone of culture liquid from yellow to red are achieved only in the presence of arsenic whereby presence of arsenic is visually detected.
Flow of construction of the “visual biosensor” of the present invention is in accordance with the process as shown in
Firstly, in a purple non-sulfur bacterium, there is constructed a pigment variant which is a variant where gene (crtA) coding for CrtA is deficient and yellow color is resulted as spheroidene is accumulated. After that, there is constructed a crtA vector where crtA gene ORF being deficient in promoter sequence is cloned on plasmid. The so-called inductive promoter showing a specific response to a specific chemical substance is inserted into the crtA vector whereupon a sensor plasmid is constructed. Finally, the sensor plasmid is introduced into the pigment variant to construct the aimed biosensor strain. In the biosensor strain constructed as such, the inductive promoter in the upstream region of the crtA gene does not work in the absence of the target compound and spheroidene is accumulated due to deficiency of CrtA to give yellowish brown color. On the contrary, in the presence of the target compound, the inductive promoter is activated and transcription of the crtA gene arranged in the downstream region is promoted. The accumulated spheroidene is converted to spherdoidenone by the expressed CrtA and the cells turn reddish brown color. As a result of the changes in color tone as such, detection of the target compound is now possible.
With regard to the purple non-sulfur bacterium used for the biosensor of the present invention, any species may be used so far as it is a purple non-sulfur bacterium which has gene coding for spheroidene monooxygenase enzyme acting as a reporter gene in the biosensor in genome and has an ability of producing reddish brown pigment spheroidenone by oxidation of yellow pigment spheroidene and of accumulating it in the bacterium cell and, for example, Rhodovulum sulfidophilum which is a marine purple non-sulfur bacterium is able to be used. As to the R. sulfidophilum, cloning of gene crtA coding for spheroidene monooxygenase enzyme and decoding of its base sequence have been completed already in the previous study by the present inventors. Accordingly, R. sulfidophilum is convenient for using as a purple non-sulfur bacterium containing reporter gene.
In the biosensor of the present invention, the above-mentioned purple non-sulfur bacterium is not used as it is but a pigment variant where gene coding for a spheroidene monooxygenase is deficient is prepared and, into this pigment variant, a sensor vector in which an inductive promoter specifically responding to a specific chemical substance which is an object to be detected by the biosensor is connected, in an operable manner, to the upstream region of reporter gene by means of a genetic engineering technique is introduced and the recombinant prepared thereby is used as a biosensor. With regard to an inductive promoter which specifically responds to a specific chemical substance, that which is appropriate corresponding to the object to be detected by the biosensor of the present invention may be appropriately selected and, when the object to be detected by the sensor is dimethyl sulfide for example, it is possible to use a promoter which is present in the upstream region of operon of gene coding for a dimethyl sulfide dehydrogenase enzyme as a dimethyl sulfide-inductive promoter showing a specific response to dimethyl sulfide. To be more specific, this promoter is DNA consisting of a base sequence of SEQ ID No. 1. It is also possible to use DNA consisting of the base sequence having at least 60% of homology to the base sequence of SEQ ID No. 1 and having a dimethyl sulfide-inductive promoter activity or, in other words, DNA within an equivalent range to the base sequence of SEQ ID No. 1 as a promoter in the biosensor of the present invention.
Preferred range of homology of the DNA within an equivalent range to the base sequence of SEQ ID No. 1 is at least 70%, more preferably at least 80%, still more preferably at least 90%, further preferably at least 95% and, most preferably, at least 98%. The homologous DNA as such is able to be easily prepared by, for example, a well-known site-specific mutagenesis (Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982). Further, the fact whether the resulting DNA has a dimethyl sulfide-inductive promoter activity is able to be confirmed, for example, by such a manner that a transformed Escherichia coli into which the resulting DNA is introduced into an upstream region of an appropriate reporter gene is prepared and incubated in the presence of dimethyl sulfide to check whether the reporter gene is expressed.
In the present invention, the term reading “connected in an operable manner” means that a promoter is arranged on the upstream region of the gene in such a manner that expression control of gene is made possible.
The object to be detected by the biosensor of the present invention is determined by the specificity of the inductive promoter used and any chemical substance may be used as an object to be detected so far as an inductive promoter showing a specific response to the target chemical substance is present. The biosensor of the present invention is able to be used particularly advantageously for the detection of agricultural chemicals, heavy metals and endocrine disrupting chemicals in aqueous environments such as the ocean, rivers and lakes utilizing its simplicity for measuring operations and visibility of the measured results. When, for example, a dimethyl sulfide-inductive promoter is selected as an inductive promoter, the biosensor of the present invention is able to be used in the detection of dimethyl sulfoxide in the ocean which has a risk of contributing in climate change of the earth and to investigate the changes of its concentration.
In the biosensor of the present invention, a constitutive promoter which is inherently present in an upstream region of gene coding for a spheroidene monooxygenase enzyme of a purple non-sulfur bacterium is substituted with an inductive promoter which specifically responds to a specific chemical substance by some way and, to be more specific, it is able to be prepared by the following steps. Thus:
gene coding for a spheroidene monooxygenase enzyme is substituted with other gene such as, for example, kanamycin-resisting gene to prepare a pigment variant of non-sulfur bacterium where gene coding for spheroidene monooxygenase enzyme is deficient;
an open reading frame of gene coding for a spheroidene monooxygenase enzyme being deficient in promoter is cloned onto a vector such as plasmid;
an inductive promoter specifically responding to a specific chemical substance is connected in an operable manner to the upstream region of gene coding for a spheroidene monooxygenase enzyme in the vector to construct a sensor vector; and
the sensor vector is introduced into a pigment variant of a purple non-sulfur bacterium to prepare a recombinant purple non-sulfur bacterium.
All of those steps may be appropriately carried out by common methods which have been known well by persons skilled in the art and detailed illustration therefor will be omitted.
In order to detect the chemical substance to be detected using the biosensor of the present invention, the recombinant purple non-sulfur bacterium constituting the biosensor of the present invention is incubated under a photoaerobic condition, a sample having a possibility of containing the chemical substance to be detected is added thereto and incubation is further continued. If no chemical substance to be detected is contained in the sample, the inductive promoter still remains inactive whereby the purple non-sulfur bacterium cell is unable to transcribe/express the spheroidene monooxygenase enzyme gene and, due to the deficiency of the enzyme, a yellow pigment spheroidene is accumulated in the cell and the color still remains yellow brown. On the contrary, when a chemical substance to be detected is contained in the sample, the inductive promoter is activated, transcription of the spheroidene monooxygenase enzyme gene existing in the downstream region is promoted, the spheroidene monooxygenase enzyme is expressed in the purple non-sulfur bacterium cell, the yellow pigment spheroidene accumulated in the cell is oxidized due to the catalytic action of the enzyme so that it is converted to a reddish brown pigment spheroidenone and the color tone of the cell turns from yellowish brown to reddish brown. As a result of the color tone change as such, a visual detection of the chemical substance to be detected is now possible.
Now the biosensor for detection of arsenic which is the particularly preferred embodiment of the present invention will be illustrated as hereunder.
Flow of the construction of the biosensor for detection of arsenic by the present invention is in accordance with the process as shown in
Firstly, there is constructed a pigment variant which is a variant being deficient in gene (crtA) coding for CrtA in the purple non-sulfur bacterium and showing yellow color as spheroidene is accumulated. After that, there is constructed a crtA vector in which crtA gene ORF being deficient in promoter sequence is cloned onto a plasmid. When the so-called inductive promoter showing a specific response to arsenic is inserted into the crtA vector, a sensor plasmid is constructed. Finally, the sensor plasmid is introduced into the pigment variant whereupon the aimed biosensor strain is constructed. In the biosensor strain constructed as such, the inductive promoter in the upstream region of the crtA gene in the absence of arsenic does not work and spheroidene is accumulated due to deficiency of CrtA to give yellowish brown color. On the other hand, in the presence of arsenic, the inductive promoter is activated and transcription of crtA gene arranged in the downstream region is promoted. Due to the expressed CrtA, the accumulated spheroidene is converted to spheroidenone and the cells turn reddish brown. As a result of the changes in color tone as such, detection of arsenic is possible.
The arsenic-inductive promoter used in the biosensor for the detection of arsenic according to the present invention consists of a promoter region of upstream region of arsenic-resisting gene operon of Escherichia coli and gene region of the most upstream region of arsenic-resisting gene operon. The arsenic-resisting gene operon of E. coli is constituted from three genes—arsR, arsB and arsC—and Pars which is a promoter region is ligated to the upstream region of arsR gene which is the most upstream region. This operon is characterized in being controlled that, only when arsenic is present, it is strongly expressed and this control is carried by an interaction of ArsR which is an expression control protein for which arsR gene is coded with Pars which is a promoter region of arsRBC. ArsR protein has a sequence showing a bonding ability to arsenic and a sequence having a bonding ability to Pars which is a promoter region of arsRBC and, when no arsenic is present, it is bonded to Pars to suppress the transcription of arsRBC. On the other hand, when arsenic is present, ArsR protein is bonded to arsenic, causes a conformation change and is dissociated from Pars whereupon transcription from Pars takes place. As such, in the arsenic-resisting gene operon of E. coli, response to arsenic and control of transcription are carried out due to an interaction of ArsR protein with Pars which is a promoter region of arsRBC. In the present invention, the Pars and arsR regions of E. coli as such are used as a control system of the biosensor whereby construction of biosensor which is able to detect arsenic is made possible.
Now the principle of detection of arsenic using the arsenic-detecting biosensor of the present invention will be illustrated in more details by referring to
To be more specific, the arsenic-inductive promoter which is able to be used in the arsenic-detecting biosensor of the present invention is DNA consisting of the base sequence of SEQ ID No. 29. In addition, DNA consisting of the base sequence which is at least 60% homologous to the base sequence of SEQ ID No. 29 and having an arsenic-inductive promoter activity or, in other words, DNA which is within an equivalent range to the base sequence of SEQ ID No. 29 is also able to be used as an arsenic-inductive promoter in the arsenic-detecting biosensor of the present invention.
Preferred range of homology of DNA within an equivalent range to the base sequence of SEQ ID No. 29 is at least 70%, more preferred range is at least 80%, still more preferred range is at least 90%, further preferred range is at least 95% and the most preferred range is at least 98%. Incidentally, such a homologous DNA may be easily prepared by, for example, a well-known site-specific mutagenesis [Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982]. The fact that whether the resulting DNA has an arsenic-inductive promoter activity is able to be confirmed by such a manner, for example, that a transformant E. coli where the resulting DNA is introduced into an upstream region of an appropriate reporter gene is prepared and incubated in the presence of arsenic to check whether the reporter gene is expressed.
In the present invention, the term “arsenic-inductive promoter” or “an inductive promoter showing a specific response to arsenic” means DNA which makes, only in the presence of arsenic, transcription of gene ligated to downstream region thereof possible and that is a conception including, if necessary, a gene sequence of additional control protein which interacts to a promoter region such as arsR gene of E. coli in addition to the common promoter region.
The present invention will now be specifically illustrated by way of the following Examples. Incidentally, descriptions in the Examples are purely for understanding of the invention only and the present invention is not limited thereby at all.
1: Strains and Media Used in this Example
Strains used in this Example are shown in Table 1 while media used therein are shown in Table 2. Rhodovulum sulfidophilum W-1S train has been deposited as FERM P-15320 at the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology [Center No. 6, 1-1, Higashi-1-chome, Tsukuba-shi, Ibaraki Prefecture, Japan (post office code: 305-8566)] (old name: the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI [1-3, Higashi-1-chome, Tsukuba-shi, Ibaraki Prefecture, Japan (post office code: 305)] (date of deposition: Dec. 11, 1995).
A 4CyMOM medium was used for incubation of Rv. sulfidophilum W-1S strain and variants and transformant strains thereof. For the incubation of the biosensor strain, 4CMOM medium was prepared as a 4CyMOM medium containing no yeast extract and then used therefor. For the incubation of E. coli JM109 strain, E. coli S17-1 strain, E. coli S17-1 λ-pir strain and transformant strain for each of them, an LB medium was used. If necessary, ampicillin (Amp), kanamycin (Km) or tetracycline (Tc) was added to each medium so as to make the final concentration thereof 50 μg/ml, 30 μg/ml or 20 μg/ml, respectively. With regard to a photosynthetic bacterium however, the amount of Tc to be added was adjusted so as to make its final concentration 5 μg/ml.
An SOC medium was used during a temporary incubation after electroporation and during the transformation process.
2: Plasmids Used in this Example
The plasmids used in this Example will be summarized later. Hereinafter, basic ones for construction of each plasmid and vector will be mentioned.
A fragment amplified by a PCR was incorporated into pGEM-T Easy Vector (Promega). For the preparation of Rv. sulfidophilum pigment variant, pSM3065 [S. Masuda, et al., (1999) J. Bacteriol., 181(14):4205-15] which is a suicide vector was utilized. For the expression of crtA in Rv. sulfidophilum pigment variant, pRK415 [N. T. Keen, et al., (1988) Gene, 70:191-7] which is a broad host range vector was used. With regard to a kanamycin cassette (KmR), a sequence contained in pUC4K (Amersham Pharmacia Biotech) was utilized.
3: Extraction of Total DNA
Extraction of total DNA from Rv. sulfidophilum W-1S strain was carried out according to the protocol of the reference document [“Illustrated Bio-Experiments, (ii) Fundamental Gene Analysis” (1995) by Hiroki Nakayama and Takahito Nishikata, published by Shujunsha].
4: PCR
With regard to PCR, an operation was conducted using TaKaRa LA Taq™ with GC Buffer (TaKaRa) according to the protocol attached thereto. With regard to a template, each total DNA or plasmid DNA was used or a direct PCR from a culture liquid or a liquid in which colonies were suspended was carried out. In a direct PCR, 50 μl of a culture liquid or a liquid where colonies were suspended in 50 μl of H2O was heated at 95° C. for 5 minutes and the supernatant liquid after a centrifugal separation was used as a template.
5: Recovery of DNA Fragment from Gel, Ligation and Transformation
When the fragment amplified by a PCR is cloned, it was incorporated into pGEM-T Easy Vector and then introduced into E. coli JM109. The method was carried out in accordance with the protocol for pGEM-T Easy Vector System. In case a plasmid DNA was introduced into E. coli S17-1, the method therefor also followed said protocol.
The DNA fragment treated with a restriction enzyme was subjected to electrophoresis on agarose gel and then recovered by an excision from the gel. In extracting the DNA from the gel fragment, that was conducted using a dialysis tube [“A Laboratory Manual—Genetic Engineering” edited by Masahiro Muramatsu, (1988), published by Maruzen]. The liquid in the dialysis tube was recovered by paying careful attention so that gel did not come therein and the DNA was recovered by extracting with PCI (phenol-chloroform-isoamyl alcohol) [“Illustrated Bio-Experiments, (i) Fundamental Molecular Biochemical Experiments” (1995) by Takahito Nishikawa, published by Shujunsha] and by precipitating with ethanol. In the operation of precipitating with ethanol, glycogen (Nakarai Tesk) was used as a co-precipitating agent for the purpose of enhancing the recovering efficiency of DNA.
When the excised DNA fragment is subjected to ligation, it was conducted using DNA Ligation Kit Ver. 2 (TaKaRa).
6: Extraction of Plasmid DNA
Extraction of plasmid DNA was carried out using CONCERT Rapid Plasmid Miniprep System (GIBCO BRL), QIAprep Spin Miniprep Kit (QIAGEN) or QIAGEN Plasmid Midi Kit (QIAGEN).
7: Conjugation: Conjugation was carried out upon introduction of the plasmid DNA into Rv. sulfidophilum from Escherichia coli into which a recombinant plasmid was introduced. The method therefor is as shown below. Incidentally, all of the things used in the following operation were subjected to sterilization using an autoclave.
E. coli was subjected to a shake culture at 37° C. in 10 ml of an LB medium in a test tube while Rv. sulfidophilum was incubated under a bright condition by stirring with a stirrer in 30 ml of 4CyMOM medium in a Roux bottle. E. coli was incubated until OD660 became nearly 0.2 and Rv. sulfidophilum was incubated until OD660 became nearly 1.6 and then 170 μl and 60 μl, respectively, of each microbe liquid was mixed in an Eppendorf tube. After collecting the cells, they were suspended again in 230 μl of an LB medium and the resulting liquid was spread on an MF-Millipore Type HA Membrane Filter (Millipore) on 2% NaCl LB agar plate. After the plate was allowed to stand overnight at 32° C., the membrane was recovered into a vial. An LB medium (250 μl) was added into the vial and subjected to vortex whereupon the cells on the membrane were recovered and transferred into an Eppendorf tube. To the LB suspension was added 50 μl of T4 phage liquid [Institute for Fermentation, Osaka, (1992) Microorganisms, 9th edition: 229] which was sterilized by filtration followed by incubating for 15 minutes at 37° C. The cells were collected, washed with, 4CyMOM and suspended again in 4CyMOM. The cell liquid was applied on 4CyMOM agar plate and incubated for several days under a bright condition.
8: Electroporation
When a plasmid was introduced into E. coli S17-1 λ-pir, electroporation was used. A plasmid solution (2 μl) was added to 50 μl of E. coli S17-1 λ-pir melted on ice, mixed by means of pipetting, transferred to a cuvette and allowed to stand on ice for 10 minutes. Condition for the electroporation was as follows. Thus, voltage was 2.5 kV, gap between cuvette walls was 0.2 cm and condenser value was 1 μF. Immediately after the electroporation, 200 μl of an SOC medium was added thereto, mixed by means of pipetting, transferred to an Eppendorf tube and subjected to a shake culture at 37° C. for 1 hour. After that, it was sown on an LB agar plate.
9: DNA Sequence
For decoding of a base sequence of the fragment cloned on the pGEM-T Easy Vector, there were used Fluorescein-Labeled Primer RV-M and M4 (TaKaRa) and ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit With AmpliTaq DNA Polymerase, FS (Applied Biosystems) and Sequence ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Preparation of the sample was carried out according to a protocol attached to the kit while operation of the sequencer was carried out according to the manual attached to the instrument.
10: Pigment Analysis
Composition analysis of various carotenoid pigments of wild strain, variant strain and transformant strain of Rv. sulfidophilum was carried out by the following method.
The bacteria strain was incubated for one night together with stirring by a magnetic stirrer under slightly aerobic and bright condition in a Roux bottle in which 30 ml of medium was placed. After a centrifugal separation of 8,000 rpm for 15 minutes, the supernatant liquid was discarded, cell pellets were frozen at −80° C. for one night and, form the next day, the cells were freeze-dried for two nights. The resulting freeze-dried product was transferred from a centrifugal tube to a vial, 5 ml of a 7:2 (v/v) mixture of acetone and methanol was added thereto and the mixture was subjected to vortex for two minutes [S. Takaichi, et al., (2001) Photosynth. Res., 67:207-14]. The suspension was filtered and used as a sample for a high-performance liquid chromatography (HPLC).
When incubation of a bacterium strain was conducted in 10 ml of medium in a test tube under various conditions (under slightly aerobic and bright condition; under slightly aerobic and dark condition; and under anaerobic and bright condition), the incubation was done for three days. The culture liquid (1 ml) was placed in an Eppendorf tube, the pellets after centrifugal separation was frozen at −80° C. for 2 hours and then freeze-dried for one night. An appropriate amount (100 μl to 500 μl) of a mixed liquid of acetone and methanol was added to the resulting freeze-dried product, subjected to vortex for 2 minutes and then centrifuged for 5 minutes at 14,000 rpm using a portable centrifuge and the resulting supernatant liquid was used as a sample for an HPLC.
The HPLC analysis was carried out using instruments for high-performance liquid chromatography manufactured by Shimadzu, liquid-sending unit LC-10AS, autoinjector SIL-9A, detector for spectrophotometer for ultraviolet and visible regions SPD-10A and chromatopack C-R3A (Shimadzu) and μ Bondapack™ C18 125 Å 10 μm 3.9×300 mm HPLC column (Waters). Methanol was used for a liquid phase and the detection was conducted with a measuring wavelength of 481 nm. Infusing amount of the sample, flow rate of the liquid phase, recording condition for chromatopack, etc. were appropriately modified depending upon the conditions such as sample concentration.
11: RNA Extraction and RT-PCR
Extraction of RNA was carried out using RNeasy Mini Kit (QIAGEN) according to the protocol attached thereto. For dissolving the cells at that time, lysozyme was added thereto in the indicated concentration. For the removal of the contaminated DNA, DNase I (TaKaRa) was used by the method indicated in the protocol together with the buffer attached thereto. Incubation was carried out at 37° C. for 30 minutes and, after that, RNA was purified via a PCI treatment and a precipitating operation with ethanol. A reverse transcription reaction was carried out where 1 μg of purified RNA as a template using an RNA PCR Kit (AMV) Ver. 2.1 (TaKaRa). In the reverse transcription reaction, things contained in the kit, i.e. reverse transcriptase, RNase inhibitor, random nonamer, buffer, dNTP and MgCl2 were used and the reaction was conducted at 45° C. for 28 minutes. A PCR was carried out by the same manner as in the above mentioned “4: PCR” using 4 μl of the solution after the reverse transcription reaction as a template.
1. Object of the Example
The present inventors have already succeeded in constructing a CrtA-deficient pigment variant crtA−1 in which deficiency in CrtA is believed to be resulted due to insertion of kanamycin-resisting gene cassette (KmR) into crtA sequence on genome of Rv. sulfidophilum W-1S. However, although this pigment variant initially showed yellow color in color tone of culture liquid as predicted, it was found to gradually get tinged with red as the incubation was continued. With an object of obtaining basic data such as pigment composition of Rv. sulfidophilum including analysis for the above-mentioned point, analysis of pigment composition via pigment extraction and HPLC was conducted here. Incidentally, it is important to find the changes in carotenoid pigment composition using an HPLC for detailed comparison and analysis of state of expression of CrtA in various variants prepared in each stage of development of biosensors such as pigment variants and transformant strains thereof.
2. Pigment Analysis of Rv. sulfidophilum
With regard to the objects where a carotenoid pigment composition is analyzed, Rv. sulfidophilum W-1S strain and pigment variant crtA-1 were selected. Pigments were extracted from them by the above-mentioned method and each pigment peak was detected via an HPLC analysis (
In order to confirm whether presumption for each of the peaks is correct and also to specify the substance corresponding to each carotenoid derivative, analysis of each main peak was carried out using a liquid chromatographic mass spectrometer (LC-MS). The result is shown in
From the above results, it was suggested that, in Rv. sulfidophilum wild strain W-1S, accumulation was mainly noted in SO and then DMSO and then DMSE followed and, in a pigment variant crtA−1, DMSE was mainly accumulated and then SE and SO followed. The fact that SO was accumulated in the pigment variant crtA−1 was predicted by the fact that the culture liquid tended to red color as a result of a long-term incubation for several days. The above is likely to suggest that, in crtA−1 strain, CrtA activity remains although its extent was low. The possibility as such is also supported by the following fact.
In preparing the pigment variant, a 1.5-kb fragment containing crtA for which the present inventors succeeded in cloning was utilized. KmR was inserted into a single restriction enzyme site located near the center of this fragment and a homologous recombination of this sequence with the crtA region on Rv. sulfidophilum genome was conducted whereupon the preparation of the pigment variant crtA−1 was in success. However, when a DNA sequence was subjected to a sequence decoding for the 1.5-kb fragment containing crtA, it was clarified that the fragment consisted of crtA gene region of about 830 bp as well as about 290 bp which was the upstream region thereof and about 400 bp which was the downstream region thereof and it was found that the XhoI site into which KmR was inserted was present about 650 bp downstream from the crtA initiation codon. Thus, in a crtA::KmR sequence constructed on the plasmid (or on the pigment variant genome), it was thought that, about 80% from the initiation codon, crtA was perfect. Therefore, in crtA−1 strain, it was presumed that, in CrtA sequence of the wild strain consisting of 277 amino acids in total, a 215 amino acid residue moiety from its N terminal was translated and it seemed that, due to the fact that a low spheroidene oxidizing activity derived by the partial CrtA was expressed, appearance of the peak corresponding to SO in an HPLC chart in crtA−1 was able to be explained. From the result of comparison of homology of CrtA amino acid sequences of Rb. sphaeroides and Rhodobacter capsulatus (GeneBank Accession No. AJ010302 and No. X52291, respectively) to a presumed amino acid sequence of CrtA of Rv. sulfidophilum, it was not possible to find a conserved region which was presumed to be an active center and it was unable to be said that CrtA deficiency was completely induced by insertion of KmR (
As compared with the wild strain, CrtA activity in the pigment variant crtA−1 strain is greatly suppressed but, in a biosensor strain, it is likely that a low CrtA activity from the incompletely destroyed crtA on chromosome enhances the background upon measurement and greatly affects the measurement precision. Accordingly, it was thought to be necessary to construct a novel Rv. sulfidophilum pigment variant where crtA gene on genome was completely destroyed.
3. Summary
Result of this Example will be as follows.
As a result of HPLC and LC-MS analysis, analysis of carotenoid pigment compositions of Rv. sulfidophilum wild strain and pigment variant was in success.
As being presumed from the color tone, accumulation of SO was observed in crtA−1 strain and it was believed not to be appropriate to use this variant as a host for the biosensor.
From those results, in the construction of biosensor, it should be that a pigment variant where crtA gene on the genome is more completely deficient is constructed.
1. Object of the Example
In the pigment variant crtA−1, construction of CrtA deficient strain was conducted in such a manner that KmR was inserted into crtA gene on chromosome but the result was an incomplete CrtA deficiency causing accumulation of red pigment. Therefore, at this time, with an object of complete deficiency of CrtA, the region corresponding to crtA gene ORF was substituted with KmR to construct a pigment variant (
Plasmids and primers which were used or constructed in this Example are summarized in Table 3 and Table 4, respectively.
aRecognition site for restriction enzyme is in bolds.
2. Preparation of Novel Pigment Variant
For the cloning of both fragments U and D, primers crtAup1 and crtAup2 for amplification of Fragment U and primers crtAdown1 and crtAdown2 for amplification of Fragment D were designed. The primers crtAup2 and crtdown1 were designed on the basis of DNA sequence data of crtA while the primers crtAup1 and crtAdown2 were designed on the basis of DNA sequence data of Rb. sphaeroides on the database (Accession No. AJ010302). Although the crtA gene side of the fragment D contains a part of crtA gene (
Firstly, a PCR was carried out using primers crtAup1 and crtAdown2 where Rv. sulfidophilum W-1S total DNA was used as a template (
After that, a fragment UKD was prepared on the broad host region plasmid pRK415 using cloned fragment U and fragment D and also KmR.
KmR was excised from the plasmid pUC4K by a treatment with a restriction enzyme SalI and inserted into XhoI site of pGEM-U plasmid. Insertion of KmR into the plasmid was confirmed by the resistance of E. coli JM109 transformant to Km contained in the medium and by confirmation of the presence of KmR by a PCR using primers kmR1 and kmR2. The constructed plasmid was called pGEM-UK.
Further, a fragment D was excised from pGEM-D plasmid by treating with SpeI-EcoRI, made into blunt end using a T4 DNA polymerase (New England Biolabs), treated with SacI and inserted into a pRK415 plasmid which was also made into blunt end. Insertion of the fragment D was confirmed by a blue-white judgment using X-Gal and by a PCR using primers crtAdown1 and crtAdown2. In the fragment D, there are two ways in its inserting directions into the plasmid and aimed one was selected by treating with a restriction enzyme EcoRI-SacI. The constructed plasmid was called pRK-D.
A fragment UK was excised from the plasmid pGEM-UK by treatment with EcoRI, made into blunt end and inserted into PstI site of the plasmid pRK-D which was also made into blunt end. With regard to the inserting direction of the fragment UK, there are also two ways. In the resulting plasmids, the aimed one produces amplified fragment corresponding to fragment UKD by PCR using the primers crtAup1 and crtAdown2 while amplified fragment is not confirmed in another one. A PCR was carried out to a plasmid of each transformant utilizing the above difference (
Fragment UKD was transferred from the resulting plasmid pRK-UKD to a plasmid pSM3065 and, in order to construct a plasmid pHDR2, fragment UKD (about 2.6 kb) was once amplified from the plasmid pRK-UKD using newly designed primers crtAup3 and crtAdown3 and a plasmid pGEM-UKD was constructed via a TA cloning. Fragment UKD which was excised by treating the plasmid pGEM-UKD extracted from the transformant with a restriction enzyme SpeI was mixed with pSM3065 fragment which was ring-opened by treating with XbaI and ligated. The ligation reaction solution was purified by means of extracting with PCI and precipitating with ethanol and then a microbe suspension of E. coli S17-1λ-pir was added thereto followed by subjecting to electroporation so that the plasmid was introduced. As a result, colonies were observed on an LB agar plate containing Tc and Km and, from the result of confirmation of the presence of fragment UKD by an antibiotic resistance mode and a PCR, it was believed that aimed plasmid pHDR2 was constructed.
After that, the plasmid was introduced into Rv. sulfidophilum W-1S via conjugation and, after a homologous recombination, a pigment variant was prepared.
As a result of the conjugation, five kinds of colonies in reddish brown, orange, yellowish brown, yellow and white colors were observed on the plate. The colony in orange color turned reddish brown after some more incubation period. Further, when the colony in yellowish brown color was picked up and a liquid culture was continued whereupon it turned reddish brown. Those colonies in orange, yellowish brown and reddish brown colors were believed to be a single crossed strain judging from their color tone and from their Km resistance. The colony in white color is a recombinant E. coli used for the conjugation. The colony in yellow color did not become tinged with red even when picked up and a liquid culture was continued therefor and it will be highly probably an aimed double crossed pigment variant. The yellow colonies prepared at this time were three colonies and each of them was called crtA−32, crtA−33 and crtA−34 strains, respectively (
crtA gene of each of those three pigment variants was confirmed by a PCR. The primer used therefor was that which was used for crtA gene cloning. It was presumed that, from crtA gene of a wild type, amplified fragment of 1.5 kb will be observed and, from the sequence substituted with KmR fragment, amplified fragment of 1.9 kb will be prepared. As a result where the PCR was actually conducted (
After that, carotenoid pigment compositions of those three strains were analyzed. Each of the three strains was cultured for one night and freeze-dried and pigment was extracted therefrom. Operation for the pigment extraction and condition for an HPLC analysis were the same as those which were conducted before. As a result of the HPLC (
3. Summary
Result of this Example will be as follows.
Fragment UKD was inserted on a suicide vector pSM3065 and the construction of plasmid pHDR2 was in success.
When the plasmid pHDR2 was introduced into Rv. sulfidophilum W-1S strain, no reddish brown color of the wild strain was shown but a novel pigment variant showing yellow color was obtained.
As a result of confirmation by PCR, in crtA gene region of this pigment variant, a band corresponding to the thing where ORF of crtA gene was partly substituted with KmR was found and no band corresponding to a wild type crtA as noted in a single-cross pigment variant was found.
As a result of pigment composition analysis using HPLC, no significant accumulation of SO was found in the resulting novel pigment variant.
From those results, the resulting novel pigment variants crtA−32, crtA−33 and crtA−34 are able to be said to be the aimed variants as a result where a homologous double recombination took place. From the pigment composition and stable color tone thereof, those pigment variants were believed to be applicable to biosensors.
1. Object of the Example
As mentioned hereinabove already, three elements of host bacterium, reporter gene and inductive promoter sequence are essential in a biosensor. Among them, the fact that Rv. sulfidophilum pigment variant being a host bacterium for a biosensor system which is the object at this time was successfully constructed was summarized in the above Example. Now, in this Example, investigation of reporter gene and construction of crtA vector as the next steps for the construction of sensor system will be illustrated.
A sensor plasmid used for the construction of biosensor is constituted from broad host region vector, inductive promoter, reporter gene arranged at the downstream region of promoter and terminator hairpin sequence inserted for prevention of read-through derived from promoter other than inductive promoter to the reporter gene. It is an object of this Example to construct crtA vector which is a plasmid at the stage before the sensor plasmid and the stage where the inductive promoter is not inserted and to investigate its function. For this reporter gene, ORF of crtA is used while, for the terminator sequence, that on the database being shown to be of presence in gene sequence which has been already reported in Rv. sulfidophilum is used. Besides the aimed crtA vector, vector of a type having a promoter sequence of crtA is also constructed. That is to confirm whether CrtA deficiency of the pigment variant is complemented by crtA expressed from the plasmid.
Plasmids and primers used or constructed in this Example are summarized in Table 5 and Table 6, respectively.
aNudeotides in small letters show changes conducted in the sequence.
Recognition site for restriction enzyme is in bolds.
2. Construction of crtA Vector
Construction of crtA vector was carried out in accordance with the following strategy.
The terminator sequence used was designed by referring to that which is present in the downstream region of pufC gene of puf operon on genome of Rv. sulfidophilum W-1S [L. Vasilyeva, et al., (1999) Appl. Biochem. Biotechnol., 77-79:337-45; S. Masuda, et al., (1999) J. Biol. Chem., 274(16):10795-801]. On the basis of hairpin structure existing in the downstream region of pufC gene, an oligonucleotide consisting of the sequence of 5′-GGAAAGCTTGCGCCGCGCGGGCTCCTCCCCGCGCGGCGCTTTTTTTCTGCAGAA-3′ as a term1 sequence was designed (
After the crtA fragment was amplified by means of a PCR using Rv. sulfidophilum W-1S genome as a template, it was inserted onto pGEM-T Easy Vector via a TA cloning. In this PCR, the primers crtArv1 and crtArv3 were used. The fragment amplified by this PCR contained a sequence from about 300 bp upward of the crtA initiation codon to about 30 bp downward of the stop codon and that was confirmed by the fact that the pattern of the fragment obtained by the treatment of this fragment with a restriction enzyme was identical with that presumed from sequence data. Thus, crtA gene ORF, SD sequence and promoter (Pcrt) were contained in this crtA sequence. The plasmid constructed by insertion of this fragment was called pGV2.
Each of full-length and partial sequences of the crtA fragment on the plasmid pGV2 was purified and inserted into pRKΩ plasmid to construct two kinds of plasmids (
Each of those two kinds of plasmids was introduced into a pigment variant crtA−32 strain via conjugation. Colonies obtained on a 4CyMOM agar plate containing 30 μg/ml of Km and 5 μg/ml of Tc were isolated and incubated in a liquid medium containing the same concentrations of the antibiotics. The cells were recovered from the culture liquid and freeze-dried for one night and, after extraction of pigment therefrom, an HPLC analysis was carried out (
3. Summary
Result of this Example will be as follows.
It was found to be possible to utilize crtA gene as a reporter system based on a change in color tone from yellow to red to a pigment variant due to CrtA defect.
It was shown that a terminator sequence having excellent function was designed and the sequence was able to suppress the transcription from its upstream area.
Construction of pRV5Ω plasmid into which crtA ORF sequence containing Ω sequence and SD were inserted was in success. It was confirmed that, from this plasmid, no crtA expression took place including a read-through.
From those results, it is able to be said that construction of crtA vector which is the second step in the process of construction of biosensor was in success. It is likely that a sensor plasmid is able to be constructed when an inductive promoter is inserted into the crtA vector.
5. Conclusions
Contents of Example 1 will be summarized to be as follows.
1. Construction of a CrtA defective pigment variant in such a form that most of crtA gene ORF on Rv. sulfidophilum W-1S genome were substituted with a kanamycin-resisting gene cassette was in success. No accumulation of red pigment in this pigment variant was found.
2. As a result of insertion of a plasmid pRV1Ω having a crtA promoter on the upstream area of crtA gene into Rv. sulfidophilum pigment variant and analysis of pigment composition, it was found that, in the pigment variant, crtA gene of Rv. sulfidophilum was able to be utilized as a reporter gene.
1. Object of the Example
In Rv. sulfidophilum, there were few genes which have been reported up to now and clarified for their sequences and those in which participation of transcription regulation for their expression has been made clear are limited. Among the information limited as such, there is information concerning a DMS dehydrogenase of Rv. sulfidophilum.
DMS (dimethyl sulfide) is an odor substance which is utilized as a starting material for various organosulfur compounds such as pharmaceuticals and agricultural chemicals or as an odor-giving agent. In the scale of global environment, it is contained in a sulfur cycle and has been known to be produced in the ocean by phytoplankton and oxidized after being discharged into the air to give sulfuric acid. Due to its low vapor pressure, sulfuric acid becomes particles and forms condensation nuclei for clouds. The fact that sunlight is interrupted and reflected by the cloud formation has been receiving public attention from the viewpoint of suppression of global warming and, with a purpose of a search for contribution to weather variation of the earth, investigation of changes in DMS concentration in the ocean has been carried out already [M. Horinouchi, et al., (1999) Nippon Nogeikagaku Kaishi, 73(1):35-44].
When the above is taken into consideration, there will be detection of pollution by DMS in a few amount, test whether removal of DMS was properly conducted, search of changes in DMS on a global environment level, etc. as the uses for which DMS sensor is applied.
In this Example, investigation whether a promoter of a DMS dehydrogenase gene reported in Rv. sulfidophilum is suitable for utilization to biosensors is carried out, the promoter is cloned if good data are found and construction of a sensor plasmid is aimed.
Plasmids and primers which were used or constructed in this Example are summarized in Table 7 and Table 8, respectively. Other experimental materials and methods used in this Example are the same as those in Example 1.
aRecognition site for restriction enzyme is in bolds.
2. DMS Dehydrogenase Gene Operon
DMS dehydrogenase is an enzyme carrying the reaction where DMS is oxidized to give dimethyl sulfoxide and, up to now, its presence has been confirmed in Rv. sulfidophilum and an enzyme protein has been purified [S. P. Hanlon, et al., (1994) Microbiol., 140:1953-8; S. P. Hanlon, et al., (1996) Eur. J. Biochem., 239:391-6]. It has been suggested that the DMS dehydrogenase is an enzyme belonging to the same family as a dimethyl sulfoxide reductase carrying the reaction where dimethyl sulfoxide is reduced to DMS and it has been proved that expression of enzyme protein is induced by the presence of a substrate. Thus, it has been confirmed that induction of DMS dehydrogenase of Rv. sulfidophilum by DMS is able to take place on a level of protein expression. In recent years, a gene operon (ddhABDC) coding for a DMS dehydrogenase protein has been identified [C. A. McDevitt, et al., (2002) Mol. icrobiol., 44(6):1575-87] and its base sequence is also able to be utilized from databases (Accession number AF453479). In an upstream area of ddh operon, presence of a ddhS gene sequence which is thought to code for DdhS presumed to be similar in terms of N-terminal amino acid sequences to DorS protein which is one of a binary control system (DorR and DorS) of a dimethyl sulfoxide reductase system of Rb. sphaeroides has been also suggested. However, information concerning ddh operon registered in the databases is limited to the downstream area from ddhA gene initiation codon and no information has been laid-open for its promoter sequence and for more upstream area thereof. Accordingly, when a promoter (Pddh) of a ddh operon is applied to a biosensor, the following two points are necessary. They are (i) Pddh induces the transcription of downstream gene in the presence of DMS and (ii) cloning of upstream area of ddhA is conducted to make its sequence clear.
Firstly, investigation of a DMS-inductive property of Pddh was planned. Primers ddhA1 and ddhA2 were designed based on ddhA gene sequence on the database. In addition, primers rrna1 and rrna2 were designed based on an rRNA sequence information of Rv. sulfidophilum W-1S (Accession number U55277) It was confirmed that a PCR using those primers was able to amplify about 1.5-kb region contained in the ddhA gene region or about 700 bp of rRNA sequence (
Rv. sulfidophilum W-1S was incubated for 3 days in a 4CMOM medium under the condition where DMS was added, dimethyl sulfoxide was added or neither of them was added thereto and the cells were recovered from the culture liquid during a logarithmic growth phase. RNA was extracted therefrom, treated with DNase, purified and subjected to an RT-PCR. When ddhA and rRNA were used as a target in the PCR, cycles were set at 40 cycles and 20 cycles, respectively. In order to confirm that no genomic DNA was mixed therein, an RT-PCR using primers crtAup1 and crtArv3 was carried out as well. The target of this RT-PCR is the region from 600 bp upstream of crtA gene initiation codon to downstream area of stop codon of crtA ORF and it is likely that transcription of this region is not done throughout the total length. As a result of the RT-PCR (
From the above, a significant transcription induction of ddhA gene under a bright aerobic condition by addition of DMS was confirmed and it is likely that, when Pddh is used, construction of DMS biosensor is possible.
3. Cloning of Promoter Pddh
After that, Pddh which will be present in the upstream area of ddhA was cloned and its base sequence was decoded. The region is a region where its sequence has been unknown being adjacent to a ddhA gene where its sequence has been known already and, therefore, an inverse PCR [H. Ochman, et al., (1988) Genetics, 120:621-3] which is a means suitable for cloning of such a region was used. This means will be briefly mentioned as hereunder (
ORF of ddhA gene has been reported to be 2,733 bp. Target region of the primers ddhA1 and ddhA2 corresponds to a region of from +801 to +2240 when the ddhA initiation codon is defined +1 and, in the downstream area thereof, the only SalI site in ddhA ORF is present at the position of +2713. Therefore, when Rv. sulfidophilum W-1S strain genome was subjected to a SalI treatment, most of unknown sequences of upstream side of ddhA and ddhA ORF are contained in a fragment which is in a size of at least 2,700 bp and detection of the fragment will be possible using the primers ddhA1 and ddhA2. Based on that, it was decided to clone the upstream area of ddhA gene via the aforementioned inverse PCR means.
At first, genomic DNA of Rv. sulfidophilum W-1S was treated with SalI for one night, the reaction solution was subjected to a 1% agarose gel electrophoresis and four fractions of 2˜3 kb fraction, 3˜4 kb fraction, 4˜6 kb fraction and 6 kb and higher fraction were excised therefrom (
From the analysis of about 350 bp region which was succeeded in cloning, an SD sequence of ddhA was found and, as a result of analysis using a GENETYX-WIN program, two things which were presumed to be promoter sequences were found near −150. However, it was not possible to find a motif suggesting the system participating in the control of activity of promoter and a sequence similar to Dor Box [N. J. Mouncey, et al., (1998) J. Bacteriol., 180(11):2924-30] which is a bonding region of a binary control system on genome found in dimethyl sulfoxide reductase operon of Rb. sphaeroides. Although it was not possible to specify the controlling region, there will be a relatively high possibility that Pddh is contained in this region when the promoter-like region found in the fragment which was cloned at this time and a report for upstream area of ddhA which was previously conducted [C. A. McDevitt, et al., (2002) Mol. icrobiol., 44(6):1575-87] are taken into consideration and it was planned to construct a sensor plasmid in which this region was used as a Pddh region.
4. Summary
Result of this Example will be as follows.
It was found that transcription of downstream gene by a promoter Pddh of ddh gene operon coding for a DMS dehydrogenase was induced in the presence of DMS.
Cloning of about 350 bp in upstream area of ddhA gene was in success and its base sequence was made clear.
Although it was still ambiguous for a control system, a region which is thought to be Pddh was able to be found.
It is presumed that an aimed Pddh is contained in the upstream area of ddhA whose cloning was in success at this time and it is likely that, when this region is inserted into a crtA vector, construction of a sensor plasmid which is necessary for construction of a DMS biosensor is possible.
1. Object of the Example
Up to now, preparation of pigment variant, construction of crtA vector containing reporter gene and cloning of DMS-inductive promoter have been carried out as constituting elements of a biosensor. In this Example, construction of a biosensor was carried out as the final stage and its function was evaluated.
Aiming for the construction of biosensor, an inductive promoter is firstly incorporated into a crtA vector to construct a sensor plasmid and then the sensor plasmid is introduced into a pigment variant. The pigment variant prepared as such is likely to have a function which is predicted as a biosensor or, in other words, no crtA expression takes place under the condition where no DMS is added but the color tone is the same as the pigment variant and, when DMS is added, color tone of the culture liquid changes by crtA expression.
The plasmids and the primers which were used or constructed in this Example will be shown in Table 9 and Table 10, respectively.
aRecognition site for restriction enzyme is in bolds.
2. Preparation of DMS Biosensor Strain
PCR was carried out using the primers pddh1 an pddh2 where a plasmid pGEM-134 was used as a template. This targets about 320 bp from SalI site up to upstream area of SD sequence of ddhA among the upstream area of ddhA on the template plasmid. No SD sequence is contained in this sequence. It was confirmed that the amplified fragment was an aimed sequence by comparing the pattern of the fragment produced by a restriction enzymatic treatment with the sequence data of upstream area of ddhA. This fragment was subjected to a TA cloning-onto a pGEM-T Easy Vector to construct a plasmid pGPddh. This fragment was excised by an XbaI treatment and inserted into an XbaI site existing between Q of a plasmid pRV5Ω which is a crtA vector and crtA ORF to construct a sensor plasmid pSDMS (
After that, the sensor plasmid which was in success for its construction was introduced into a pigment variant via conjugation. The resulting transformant formed yellowish brown colonies. From the fact of showing resistance to Km and Tc and from the PCR, it was able to be confirmed that a sensor plasmid was introduced into the transformant. This transformant was incubated for 3 days under a bright aerobic condition without addition of DMS to analyze the pigment composition. As a result thereof (
3. Evaluation of Functions of DMS Biosensor Strain
After that, DMS was added to the constructed biosensor to evaluate its functions. Firstly, DMS was added in the initial stage of the incubation and it was checked whether the response to DMS was available.
Since DMS is hardly soluble in water, ethanol was used as a solvent for the manufacture of concentrated stocks and preparation of diluted series. DMS-containing ethanol was added to the culture liquid in an amount of 0.1% by volume and, with regard to a control, only ethanol was added in an amount of 0.1% by volume. In each sample, final concentration of the DMS added was made 3 mM, 0.3 mM, 30 μM, 3 μM or 0.3 μM and, in all samples, experiment was conducted where n=3. The incubation was carried out under a bright aerobic condition and, on the third day (logarithmic growth phase) and on the sixth day (resting phase), observation of color tone and pigment composition analysis from collected cell sample were carried out. Analysis of pigment composition was conducted via an HPLC analysis the same as before and, from the value of area of each peak shown in the resulting HPLC chart, rate of red pigments (DMSO and SO) to the sum of total carotenoid pigment (DMSO, DMSE, SO, SE and neurosporene) was calculated. For each sample, mean value and standard deviation was calculated from the above value and a graph was prepared. Color tone and rate of red pigment on the third day of the incubation are shown in
4. Summary
Result of this Example will be as follows.
It was found that a ddh operon promoter Pddh was contained in an upstream sequence of ddhA gene.
Construction of a DMS sensor plasmid was in success.
Since a pigment variant into which a DMS sensor plasmid was introduced showed a significant red pigment accumulation in the incubation under the condition where DMS was added, it is likely that construction of a DMS biosensor was in success.
Changes in color tone by a DMS response of a DMS biosensor strain reached a level being recognizable by naked eye and construction of “visual biosensor system” was in success.
5. Conclusions
Contents of Example 2 will be summarized to be as follows.
1. It was made clear that transcription of a ddh gene operon coding for a DMS dehydrogenase was induced by the presence of DMS and cloning of upstream area of ddhA containing the promoter sequence was in success.
2. Construction of “visual biosensor” accompanied by color tone change from yellow to yellowish brown responding to DMS was in success by the joint use of pigment variant, crtA gene and ddhA promoter.
1: Strains and Media Used in this Example
Strains used in this Example are shown in Table 11 while media used therein are shown in Table 12.
A 4CyMOM medium was used for incubation of Rv. sulfidophilum W-1S strain and variants and transformant strains thereof. For the incubation of E. coli JM109 strain, E. coli S17-1 strain and transformant strain for each of them, an LB medium was used. If necessary, ampicillin (Amp), kanamycin (Km) or tetracycline (Tc) was added to each medium so as to make the final concentration thereof 50 μg/ml, 30 μg/ml or 20 μg/ml, respectively. With regard to a Rv. sulfidophilum however, the amount of Tc to be added was adjusted so as to make its final concentration 5 μg/ml.
An SOC medium was used during a temporary incubation during the transformation process.
2: Extraction of Total DNA
Extraction of total DNA from Rv. sulfidophilum W-1S strain was carried out according to the protocol of the reference document [“Illustrated Bio-Experiments, (ii) Fundamental Gene Analysis” (1995) by Takahito Nishikata, published by Shujunsha].
3: PCR
Operations of a PCR were carried out using TaKaRa LA Taq™ with GC Buffer (manufactured by TaKaRa) in accordance with a protocol attached thereto. With regard to a template, each total DNA or plasmid DNA was used or a culture liquid or a solution in which colonies were suspended was used. In the latter case, 50 μl of culture liquid or a solution where colonies were suspended in 50 μl of H2O was heated at 95° C. for 5 minutes and a direct PCT was carried out using a supernatant liquid prepared by centrifugal separation as a template. A fundamental program of the PCR is shown in
4: Recovery of DNA Fragment from Gel, Ligation and Transformation
When the fragment amplified by a PCR is cloned, it was incorporated into pGEM-T Easy Vector and then introduced into E. coli JM109. The method was carried out in accordance with the protocol for pGEM-T Easy Vector System. In case a plasmid DNA was introduced into E. coli S17-1, the method therefor also followed said protocol.
The DNA fragment treated with a restriction enzyme was subjected to electrophoresis on agarose gel and then recovered by an excision from the gel. In extracting the DNA from the gel fragment, that was conducted using a dialysis tube [“A Laboratory Manual—Genetic Engineering” edited by Masahiro Muramatsu, (1988), published by Maruzen]. The liquid in the dialysis tube was recovered by paying careful attention so that gel did not come therein and the DNA was recovered by extracting with PCI (phenol-chloroform-isoamyl alcohol) [“Illustrated Bio-Experiments, (i) Fundamental Molecular Biochemical Experiments” (1995) by Takahito Nishikawa, published by Shujunsha] and by precipitating with ethanol. In the operation of precipitating with ethanol, glycogen (Nakarai Tesk) was used as a co-precipitating agent for the purpose of enhancing the recovering efficiency of DNA.
When the excised DNA fragment is subjected to ligation, it was conducted using DNA Ligation Kit Ver. 2.1 (TaKaRa).
5: Extraction of Plasmid DNA
Extraction of plasmid DNA was carried out using CONCERT Rapid Plasmid Miniprep System (GIBCO BRL), QIAprep Spin Miniprep Kit (QIAGEN) or QIAGEN Plasmid Midi Kit (QIAGEN).
6: Conjugation: Conjugation was carried out upon introduction of the plasmid DNA into Rv. sulfidophilum from Escherichia coli into which a recombinant plasmid was introduced. The method therefor is as shown below. Incidentally, all of the things used in the following operation were subjected to sterilization using an autoclave.
E. coli was subjected to a shake culture at 37° C. in 10 ml of an LB medium in a test tube while Rv. sulfidophilum was incubated under a bright condition by stirring with a stirrer in 30 ml of 4CyMOM medium in a Roux bottle. E. coli was incubated until OD660 became nearly 0.2 and Rv. sulfidophilum was incubated until OD660 became nearly 1.6 and then 170 μl and 60 μl, respectively, of each microbe liquid was mixed in an Eppendorf tube. After collecting the cells, they were suspended again in 230 μl of an LB medium and the resulting liquid was spread on an MF-Millipore Type HA Membrane Filter (Millipore) on 2% NaCl LB agar plate. After the plate was allowed to stand overnight at 32° C., the membrane was recovered into a vial. 4CyMOM (250 μl) was added into the vial and subjected to vortex whereupon the cells on the membrane were collected, washed and suspended again in 4CyMOM. The cell liquid was applied on 4Cy MOM agar plate and incubated for several days under a bright condition.
7: DNA Sequence
For decoding of a base sequence of the fragment cloned on the pGEM-T Easy Vector, there were used Fluorescein-Labeled Primer RV-M and M4 (TaKaRa) and ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit With AmpliTaq DNA Polymerase, FS (Applied Biosystems) and Sequence ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Preparation of the sample was carried out according to a protocol attached to the kit while operation of the sequencer was carried out according to the manual attached to the instrument.
8: Pigment Analysis
Composition analysis of various carotenoid pigments of wild strain, variant strain and transformant strain of Rv. sulfidophilum was carried out by the following method.
The bacteria strain was incubated for one night together with stirring by a magnetic stirrer under slightly aerobic and bright condition in a Roux bottle in which 30 ml of medium was placed. Then, 1 ml of microbe liquid was collected, and after a centrifugal separation of 14,000 rpm for 5 minutes using portable centrifuge HITACHI CENTRIFUGE SCT15B (HITACHI KOKI), the supernatant liquid was discarded, cell pellets were frozen at −80° C. for 30 minutes and the cells were freeze-dried for one night. The resulting freeze-dried product was transferred from a centrifugal tube to a vial, 150 μl of a 7:2 (v/v) mixture of acetone and methanol was added thereto and the mixture was subjected to vortex for two minutes [S. Takaichi, et al., (2001) Photosynth. Res., 67:207-14]. The suspension was filtered and used as a sample for a high-performance liquid chromatography (HPLC).
When incubation of a bacterium strain was conducted in 10 ml of medium in a test tube under various conditions (under slightly aerobic and bright condition; under slightly aerobic and dark condition; and under anaerobic and bright condition), the incubation was done for three days. Then, the culture liquid (1 ml) was placed in an Eppendorf tube, the pellets after centrifugal separation was frozen at −80° C. for 2 minutes and then freeze-dried for one night. 150 μl of a mixed liquid of acetone and methanol was added to the resulting freeze-dried product, subjected to vortex for 2 minutes and then centrifuged for 2 minutes at 14,000 rpm using a portable centrifuge and the resulting supernatant liquid was used as a sample for an HPLC.
The HPLC analysis was carried out using instruments for high-performance liquid chromatography manufactured by Shimadzu, liquid-sending unit LC-10AS, autoinjector SIL-9A, detector for spectrophotometer for ultraviolet and visible regions SPD-10A and chromatopack C-R3A (Shimadzu) and μ Bondapack™ C18 125 Å 10 μm 3.9×300 mm HPLC column (Waters). Methanol was used for a liquid phase and the detection was conducted with a measuring wavelength of 481 nm. Infusing amount of the sample, flow rate of the liquid phase, recording condition for chromatopack, etc. were appropriately modified depending upon the conditions such as sample concentration.
9: RNA Extraction and RT-PCR
Extraction of RNA was carried out using RNeasy Mini Kit (QIAGEN) according to the protocol attached thereto. For dissolving the cells at that time, lysozyme was added thereto in the indicated concentration. For the removal of the contaminated DNA, DNase I (TaKaRa) was used by the method indicated in the protocol together with the buffer attached thereto. Incubation was carried out at 37° C. for 60 minutes and, after that, RNA was purified via a PCI treatment and a precipitating operation with ethanol. A reverse transcription reaction was carried out where 1 μg of purified RNA as a template using an RNA PCR Kit (AMV) Ver. 2.1 (TaKaRa). In the reverse transcription reaction, things contained in the kit, i.e. reverse transcriptase, RNase inhibitor, random nonamer, buffer, dNTP and MgCl2 were used and the reaction was conducted at 45° C. for 28 minutes. A PCR was carried out by the same manner as in the above mentioned “3: PCR” using 4 μl of the solution after the reverse transcription reaction as a template.
10: Preparation of Probe and Southern Hybridization
Preparation of Probe was Carried Out Using an AlkPhos Direct Labelling and Detection System (Amersham) according to the manual attached thereto.
Preparation of Membrane Used for the Southern Hybridization was carried out as follows. Firstly, 10 μg of genomic DNA was used and subjected to a treatment with each restriction enzyme for 24 hours. After that, it was subjected to electrophoresis at 40 V for 14 hours using agarose gel and then stained with ethidium bromide to confirm the bands. Then blotting was conducted to a Hybond N+ (Amersham) membrane by an alkali transfer method, washed with 2×SSC and incubated at 80° C. for 2 hours to fix. The fixed membrane was subjected to hybridization with a probe for 24 hours and, after washing, a fluorescent substrate was added thereto. Measurement of fluorescence was conducted using a FluorImager 595 (Amersham).
1. Object of the Example
In order to prepare a biosensor for the detection of arsenic with high sensitivity, type of the arsenic-inductive promoter used therefor is important. With regard to that, it has been known that, particularly in bacteria, arsR which codes for control protein showing a response to arsenic in a very low concentration participates in the control of the expression of arsenic-resisting gene group ars operon [A. Carkin, et al., (1995) J. Bacteriol., 177(4):981-6] and, when it is utilized as a control region of the biosensor of the present invention, it is likely that a biosensor for the detection of arsenic in high sensitivity is able to be prepared.
Accordingly, in this Example, arsR showing a response to arsenic and being necessary for expression control of crtA will be tried to investigate from Rv. sulfidophilum.
The plasmids and the primers constructed in this Example are shown in Table 13 and Table 14, respectively.
aNucleotides in small letters show changes conducted in the sequence.
Recognition site for restriction enzyme and poly T recognition site are shaded.
2. Investigation of arsR by Southern Hybridization
Firstly, it was confirmed whether arsenic-resisting gene was present in photosynthetic bacteria including Rv. sulfidophilum by conducting a blast search analysis using the base sequence information of E. coli arsR. As a result, it was found that Rb. sphaeroides which is a closely-related species to Rv. sulfidophilum had a sequence having a homology to ars RBC with a relatively high score. From the result, it was able to be presumed that Rv. sulfidophilum will also have an arsenic-resisting gene including arsR. Therefore, cloning of arsR of Rv. sulfidophilum was attempted by a southern hybridization where E. coli arsR was used as a probe. Firstly, a PCR was carried out using primers E. coli-arsR-S1 and E. coli-arsR-A1 where genomic DNA of E. coli K-12 was used as a template. The fragments obtained as a result thereof were purified, bonded with an alkaline phosphatase by a cross-linking reaction and used as a probe which was able to be detected by fluorescence. Hybridization of the probes was conducted at each of the temperatures of 50° C., 55° C. and 60° C. As a result, although non-specific band was able to be detected, no signal believed to be arsR was unable to be obtained when hybridization temperature was 50° C. When hybridization temperature was 55° C. and 60° C., no signal in the strength of positive control was able to be obtained in any of both cases. From the above, it was suggested that Rv. sulfidophilum had no sequence showing a homology to E. coli arsR (
3. Evaluation of Promoter Activity of E. coli Pars in Rv. sulfidophilum
Taking the result of the above experiment into consideration, it is believed to be preferred for the construction of arsenic biosensors to use Pars and arsR of E. coli in which functions and sequences have been fully made clear.
Control of expression by arsR is inhibition of transcription by the bond of ArsR protein to the ArsR-bonding site contained in Pars. This bond is due to a physical interaction between DNA and protein and, even in the case where microorganism species in which Pars and arsR are expressed are different, it is believed that the same bonding property is available. A problem here is whether Pars derived from E. coli is recognized by an RNA polymerase of Rv. sulfidophilum whereby initiation of transcription takes place. In order to confirm that, it was decided to construct crtA vector in which Pars derived from E. coli was incorporated (
Firstly, a PCR was carried out using primers E. coli-Pars-S1 and E. coli-Pars-A1 in which genomic DNA of E. coli K-12 was used as a template. After that, the fragments were subjected to a TA cloning on pGEM-T Easy Vector to construct a plasmid pGPars. The fragments were excised by an XbaI treatment and inserted into an XbaI site existing between Ω of a plasmid pRV5Ω which is a crtA vector and crtA ORF to construct a sensor plasmid pRV5ΩPars (
Then each of plasmids pRV5ΩPars, pRV1Ω and pRV5Ω which were successfully constructed was introduced into a pigment variant via conjugation and incubated for 3 days under a bright aerobic condition to give colonies. Each of the colonies was picked up and subjected to liquid culture for 3 days under a bright aerobic condition and the culture liquid was subjected to observation for its color tone and to analysis of the pigment composition by an HPLC. As a result, the culture liquid was in red color in the pigment variant into which the plasmid pRV5ΩPars was introduced and it is able to be expected the accumulation of SO took place by incorporation of Pars (
4. Summary
Result of this Example will be as follows.
It was suggested that no arsR was present in Rv. sulfidophilum.
It was made clear that the promoter Pars of ars operon of E. coli was recognized by an RNA polymerase of Rv. sulfidophilum and functioned as a promoter.
From the above results, it was expected that, when Pars and arsR of E. coli were used as arsenic-inductive promoters, it was possible to construct an arsenic-detecting biosensor.
1. Object of the Example
From the results of Example 3-A, it was expected that, in the construction of arsenic-detecting biosensors, use of Pars and arsR of E. coli as arsenic-inductive promoters is effective. Accordingly, in this Example, construction of sensor plasmids using them was attempted. That which was constructed in this Example is a sensor plasmid where a sequence from Pars to immediately before the initiation codon of arsB (The sequence includes sequences of Pars and arsR and corresponds to the sequence of SEQ ID No. 29. Incidentally, the bases from the first to the 97th ones in SEQ ID No. 29 correspond to Pars and those from the 98th to the 451st ones therein correspond to arsR) was incorporated into upstream area of crtA and that was constructed with such an object that the same control as ars operon of a wild type is conducted in crtA as well (
The plasmids and the primers constructed in this Example are shown in Table 15 and Table 16, respectively.
aNucleotides in small letters show changes conducted in the sequence.
Recognition site for restriction enzyme and poly T recognition site are shaded.
2. Construction of Arsenic-Detecting Biosensor Plasmid
Firstly, a PCR was carried out by a combination of a primer E. coli-Pars-S1 and a primer E. coli-arsR-A2 using genomic DNA of E. coli K-12 as a template. The resulting amplified sequence corresponds to a sequence (SEQ ID No. 29) from Pars to immediately before initiation codon of arsB. The fragment was subjected to a TA cloning onto pGEM-T easy vector to construct a plasmid pGASK. The fragment was excised by an XbaI treatment and inserted into an XbaI site existing between Ω of plasmid pRV5Ω which is a crtA vector and crtA ORF to construct a sensor plasmid pSENS-AS (
After that, the sensor plasmid which was in success for its construction was introduced into a pigment variant by conjugation to give an arsenic-detecting biosensor SENS-AS strain. Confirmation of introduction of the sensor plasmid was conducted by the fact of showing a resistance of Km to Tet and from the result of the PCR.
3. Evaluation of Function of Arsenic-Detecting Biosensor
After that, arsenic was added to the constructed biosensor strain and the responding property was evaluated. At that time, arsenious acid (hereinafter, referred to as As2O3) was used as a trivalent arsenic. Addition of As2O3 was carried out so as to make the final concentration 0.0006 ppm, 0.0060 ppm, 0.0600 ppm, 0.6000 ppm or 6.0000 ppm and, in all samples, the experiment was conducted where n=3. After 24 hours from the start of the incubation, an observation of color tone and a pigment composition analysis from the collected cell sample were conducted. Analysis of the pigment composition was conducted via an HPLC and rate of red pigments (DMSO and SO) to the sum of DMSO, DMSE, SO and SE was calculated from an area value of each peak shown in the resulting HPLC chart. Further, mean value and standard deviation were calculated for each sample and graphs were prepared. As a result, color tone changed from yellow to red when As2O3 was added to the SENS-AS strain and the color tone change as such was able to be confirmed by addition of As2O3 within a concentration range from 0.0060 ppm to 0.6000 ppm (
Further, an RNA extraction and an RT-PCR were carried out for a sample of after 24 hours from addition of 0.06 ppm of As2O3 and changes in transcribed amount of mRNA to As2O3 were checked whereby the responding property was evaluated. PCR where a sample after the RT reaction was used as a template was carried out for crtA using a primer crtA-RT-S1 and a primer crtA-RT-A1; for arsR using a primer arsR-RT-S1 and a primer arsR-RT-A1; and for rRNA using a primer rRNA-RT-S1 and a primer rRNA-RT-A1. From the result thereof, it was able to be confirmed that transcription induction of crtA took place by addition of As2O3 and it was able to be presumed that expression control which was just the same as in the construct of a biosensor plasmid took place (
It was also evaluated that how long time was needed until the culture liquid showed red color by addition of As2O3. Culture liquids to which As2O3 was and was not added were prepared, carotenoid pigment was extracted from each of the samples after 0, 3, 6, 12, 24 and 48 hour(s) and analyzed by an HPLC and, from the area value of each peak shown in the resulting HPLC chart, rate of red pigments (DMSO and SO) to the total sum of DMSO, DMSE, SO and SE was calculated. Mean value and standard deviation for each sample were also calculated and graphs were prepared. As a result, it was found that the rate of red pigment increased from the stage after 6 hours from addition of As2O3 and that the increase proceeded until 24 hours thereafter and, finally, accumulation of about 55% of red pigment was able to be confirmed. On the other hand, in case no arsenic was added, about 30% of red pigment was able to be confirmed until the stage after 6 hours from initiation of the measurement and, after that, changes were rarely noted. From those results, it was found that observation of color tone after 24 hours from addition of As2O3 was the shortest time and, moreover, color tone change was most easily understood as compared with the control to which no As2O3 was added (
4. Specificity of Arsenic Biosensor
From the experiments up to now, it has been made clear that a biosensor SENS-AS strain shows a responding property to arsenic but its specificity is ambiguous. Therefore, specificity to metal of the SENS-AS strain was investigated by adding a sample containing metal other than arsenic to the SENS-AS strain and checking the responding property to those samples. With regard to the metal other than arsenic, there were used antimony and bismuth which have been reported to inherently show a responding property to ArsR and also cobalt, cadmium, nickel, zinc and lead where responding property in a protein having the similar sequence to metal-bonding site of ArsR has been confirmed already [L. S. Busenlehner, et al., (2003) FEMS Microbiol. Rev., 27:131-43]. The responding property was also tested for a solution in which iron, manganese, copper and aluminum which are abundantly contained in tap water were mixed. The experiment was carried out by the same method as in the detection of arsenic, rate of red pigments (DMSO and SO) to the total sum of DMSO, DMSE, SO and SE was calculated from the area value of each peak shown in the chart prepared as a result of an HPLC analysis, mean value and standard deviation for each sample were calculated and graphs were prepared. As a result, response was confirmed in bismuth within a concentration range of 0.6000 ppm to 6.0000 ppm but no response was found to other metals (
From the above results, it was now made clear that the arsenic-detecting biosensor SENS-AS strain responded arsenic and bismuth. However, in order to respond to bismuth, a bismuth concentration of as high as 100-fold as compared with arsenic was necessary. Accordingly, the SENS-AS strain had an apparent higher specificity to arsenic than to bismuth and was found to be effective for the detection of arsenic.
5. Summary
Result of this Example will be as follows.
E. coli Pars and arsR showed a responding ability to arsenic in Rv. sulfidophilum and was clarified to function as an arsenic-inductive promoter.
By the use of E. coli Pars and arsR, and crt A of Rv. sulfidophilum, construction of an arsenic biosensor SENS-AS strain was in success.
It was made clear that the SENS-AS strain responded to arsenic within a concentration range of 0.0006 ppm to 6.0000 ppm. Further, to arsenic within a concentration range of 0.0060 ppm to 0.6000 ppm, visual detection of arsenic by color tone change of the biosensor strain from yellow color to red color was possible.
The SENS-AS strain showed a responding property to bismuth besides arsenic but, with regard to the threshold concentration for the detection, it was found that arsenic was the lowest and that specificity to arsenic was the highest.
As mentioned above, construction of an arsenic-detecting biosensor showing a responding property of arsenic was in success in this Example. Such a biosensor has a high detecting sensitivity for arsenic and the specificity is also high and, therefore, it has a property of being fully able to be utilized in a screening of contamination of arsenic in drinking water and is very useful.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-243163 | Aug 2004 | JP | national |
| 2005-136762 | May 2005 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP05/15224 | 8/22/2005 | WO | 2/20/2007 |
