阅读说明：本技术 一种生物传感器及其制备方法和应用 (Biosensor and preparation method and application thereof ) 是由 周明 付卫雷 于 2021-05-28 设计创作，主要内容包括：本申请涉及一种生物传感器及其制备方法和应用。所述生物传感器包括同时表达血红素氧化酶-1、藻胆体核蛋白亚基ApcF2和血红素跨膜转运蛋白的工程化细胞。本申请的生物传感器能够同时表达血红素氧化酶-1、藻胆体核蛋白亚基ApcF2和血红素跨膜转运蛋白,并低表达血红素,能够有效响应胞外血红素并产生荧光,且自身血红素背景荧光低,可有效进行血红素和/或血液检测。(The application relates to a biosensor and a preparation method and application thereof. The biosensor comprises an engineered cell which simultaneously expresses heme oxygenase-1, phycobilisome nucleoprotein subunit ApcF2 and heme transmembrane transporter. The biosensor can simultaneously express heme oxidase-1, phycobilisome nucleoprotein subunit ApcF2 and heme transmembrane transport protein, can express heme at low level, can effectively respond to extracellular heme and generate fluorescence, has low heme background fluorescence, and can effectively detect heme and/or blood.)

1. A biosensor comprising an engineered cell that simultaneously expresses heme oxidase-1, phycobilisoma nucleoprotein subunit ApcF2, and a heme transmembrane transporter.

2. The biosensor of claim 1, wherein the engineered cell lacks a heme synthase gene.

3. The biosensor of claim 2, wherein the heme synthase gene comprises hemF.

4. The biosensor of any one of claims 1-3, wherein the phycobilisome nucleoprotein subunit ApcF2 comprises a fusion protein of any one or at least two of BDFP1.1, BDFP1.2, BDFP1.3, BDFP1.4, BDFP1.5, BDFP1.6, BDFP1.7, BDFP1.8 or BDFP 1.9.

5. The biosensor of any of claims 1-4, wherein the heme transmembrane transporter comprises transmembrane transporter ChuA.

6. The biosensor of any of claims 1-5, wherein the engineered cells comprise engineered E.

7. The biosensor of any one of claims 1-6, wherein the engineered cell contains a gene encoding heme oxidase-1, a gene encoding phycobilisome nucleoprotein subunit ApcF2, and a gene encoding heme transmembrane transporter;

optionally, the heme oxygenase-1 comprises the amino acid sequence shown in SEQ ID NO. 11;

optionally, the gene encoding the heme transmembrane transporter comprises the nucleic acid sequence shown in SEQ ID NO 12.

8. The biosensor of any one of claims 1-7, wherein the biosensor comprises an engineered E.coli comprising a gene encoding heme oxidase-1, a gene encoding phycobilisome nucleoprotein subunit ApcF2BDFP1.6, and a gene encoding transmembrane transporter ChuA, and lacking heme synthase gene hemF.

9. A method for preparing the biosensor according to any one of claims 1 to 8, comprising:

knocking out a heme synthetase gene of a host cell, and introducing an encoding gene of heme oxidase-1, an encoding gene of phycobilisome nucleoprotein subunit ApcF2 and an encoding gene of heme transmembrane transport protein into the host cell to obtain the biosensor.

10. The method of claim 9, wherein the host cell comprises escherichia coli.

11. The method of claim 9 or 10, wherein the introducing comprises any one of electrical transduction, a viral vector system, a non-viral vector system, or direct gene injection.

12. A composition comprising the biosensor of any one of claims 1-8;

optionally, the composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, electronic circuit or diluent.

13. Use of a biosensor as claimed in any one of claims 1 to 8 or a composition as claimed in claim 12 for the detection of heme and/or blood.

Technical Field

The application belongs to the technical field of genetic engineering, relates to a biosensor and a preparation method and application thereof, and particularly relates to a biosensor sensitive to heme and a preparation method and application thereof.

Background

Microorganisms in the human body affect the health of humans and can also be used to detect or treat certain diseases. In recent years, artificially modified microorganisms have been widely used in the diagnosis and treatment of diseases.

Synthetic biology is a highly efficient method for artificially modifying microorganisms, and mainly uses basic DNA and RNA elements to construct a gene loop (genecirles) capable of responding to some small molecules, outputting quantifiable signals such as fluorescent signals, thereby endowing the microorganism engineering bacteria with new functions, constructs related gastrointestinal microorganisms into molecular sensors (related engineering bacteria), and then enables the related microorganism engineering bacteria to colonize in a host by means of orally taking the molecular sensors, and when the host suffers from diseases, the molecular sensors can respond to the diseases and convert the diseases into output signals, thereby realizing detection of the diseases.

According to research reports, a gene loop capable of responding to tetrathionate is constructed in a strain by using escherichia coli (NGF-1) in a mouse body, so that the escherichia coli molecular sensor can monitor intestinal inflammation by detecting tetrathionate formed during intestinal inflammation, and the molecular sensor engineering bacteria can monitor inflammation in the intestinal tract of the mouse in 6 months.

Heme (Heme) and globin are combined to form hemoglobin, which is an important component in erythrocytes, and thus, Heme can be used as a marker for blood detection. In E.coli, heme synthesis starts with 5-aminolevulinic acid (5-ALA), first, the 5-ALA precursor is catalyzed by three steps of enzyme, HemB, HemC and HemD, in turn, and through intermediates: porphobilinogen (PBG) and Uroporphyrinogen precursor (pre-uromorphinogen) to produce a first ring tetrapyrrole Uroporphyrinogen III (Uroporphyrinogen III), which is then successively catalyzed by HemE, HemF (or HemN), HemG and HemH enzymes through intermediates: coproporphyrinogen III (coproporphyrin III), protoporphyrinogen IX (protogenin IX, PROTOGEN IX) and protoporphyrin IX (protorphyrin IX, PROTO) are converted into heme, wherein coproporphyrinogen III is catalyzed by either oxygen-dependent oxidase (HemF) or oxygen-independent oxidase (HemN) during oxidation to give protoporphyrinogen IX.

Research reports have combined with artificially designed escherichia coli Nissle 1917 strain and capsule endoscopy to develop a method capable of detecting intestinal bleeding, which uses heme as an input signal, which can induce the expression of downstream luciferase gene and generate light signal by decomposing related luciferin, converts the light signal into an electrical signal through a small electronic circuit, and transmits the electrical signal to a computer or a mobile phone in a wireless transmission manner, so that the engineered bacterium and the small electronic circuit are packaged in a small capsule, and a patient can detect whether intestinal bleeding occurs after taking the capsule orally, however, due to system diversity, heterologous expression of reporter genes, or transfer of promoters and transcription factors to a heterologous system, biosensors of such a mode may have problems in specificity (see: mime M, Nadeau P, hayward A, et al, an ingestible bacterial-electronic system to monitor university chemical health [ J ] Science,2018,360(6391): 915.).

In conclusion, the efficient and high-specificity blood detection sensor is provided, and has important significance for the field of blood detection.

Disclosure of Invention

The biosensor can emit far-red light FR or near-infrared light NIR after being excited by a light source with a specific wavelength in the presence of heme, and has high specificity and sensitivity, so that heme and blood can be detected.

In a first aspect, the present application provides a biosensor comprising an engineered cell simultaneously expressing heme oxidase-1, phycobilisome nucleoprotein subunit ApcF2, and a heme transmembrane transporter.

In the biosensor of this application, heme transmembrane transport protein can shift heme to the cell, heme oxidase-1 (Ho1) becomes Biliverdin (BV) with heme oxidation, phycobilisome nucleoprotein subunit ApcF2 can be based on enzymatic reaction, autocatalysis covalent binding biliverdin, and the combination back, through the light source excitation of specific wavelength, can send far-infrared FR or near infrared NIR to detection heme has been realized.

Preferably, the engineered cell lacks a heme synthase gene.

Preferably, the hemE synthase genes include hemB, hemC, hemD, hemE, hemF, hemG, hemH, and hemN.

In the application, the heme synthase gene in the engineered cell is knocked out, and the heme synthesized by the engineered cell is weakened, so that the background interference of heme in the engineered cell is reduced.

Preferably, the phycobilisome nucleoprotein subunit ApcF2 is a group of fluorescent proteins which are derived from phycomycetes PCC7203 and can emit far-red light (about 670nm) and near-infrared light (about 710nm), and comprises wild-type proteins ApcF2 (such as SEQ ID NO:1) and mutant series proteins BDFP1.1, BDFP1.2, BDFP1.3, BDFP1.4, BDFP1.5, BDFP1.6, BDFP1.7, BDFP1.8 and BDFP1.9, wherein the amino acid sequences are respectively shown as SEQ ID NO: 2-10.

SEQ ID NO:1：

MQDKLTSVAKNCDLTGSSLNREVVETLKEFLADGEKRVQVAGVIGSNAAEIVKTAVSLLFQEYPELVSPGGNAYTTRRYNMYVRDMNYFLRYCSYAIVAGDASVLDERLLAGLRDTFNSLGIPLGPTARSIQLMKNIVKEKLVTAGMTNITFVDEPFDYVVREISETEI。

SEQ ID NO:2：

MANREVVETLKEFLADGEKRVQVAGVIGTNAAEVVKTAVSLLFQEYPELVSPGGCAYTTRRYNMCVRDMNYFLRMCSYAIVAGDASVLDERLLAGLRDTFNSLGIPLGPTARSIQLMKKIVKEKLVTAGMTNITFVDEPFDYIAREISETEI。

SEQ ID NO:3：

MANREVVETLKELLADGEKRVQVAGVIGTNAAEVVKTAVSLLFQEYPELVSPGGCAYTTRRYNMCVRDMNYFLRMCSYAIVAGGASVLDERLLAGFRDTFNSLGIPLCPTARSIQLMKKIVKEKLATAGMTNIAFVDEPFDYIAREISETEI。

SEQ ID NO:4：

MANREVVETLEEFLADGEKRVQVAGVIGTNAAEVVKAAVSLLFQEYPELVSPGGCAYTTRRYNMCVRDMNYFLRMCSYAIVAGDASVLDERLLAGLRDTFNSCGIPLGPTARSIQLMKKIVKEKLVTAGMTNITFVDEPFDYIAREISETEI。

SEQ ID NO:5：

ADGEKRVQVAGVIGTNAAEVVKTAVSLLFQEYPELVSPGGCAYTTRRYNMCVRDMNYFLRMCSYAIVAGDASVLDGRLLAGLRDTFNSLGIPLGPTARGIQLMKKIVKEKLVTAGMTNITFVDEPFDYIVRGISETEI。

SEQ ID NO:6：

ADGEKRVQVAGVIGTNAAEVVKTAVSLLFQEYPELVSPGGCAYTTRRYNMCVRDMNYFLRMCSYAIVAGGASVLDERMLAGLRDTFNSLGIPLGPTARSIQLMKKIVKEKLVTAGMTNITFVDEPFDYIARVISETEI。

SEQ ID NO:7：

MANREVVETLKELLADGEKRVQVAGVIGTNAAEVVKTAVSLLFQEYPELVSPGGCAYTTRRYNMCVRDMNYFLRMCSYAIVAGGASVLDGRMLAGFRDTFNSLGIPLCPAARGIQLMKKIVKEKLATAGMTNIAFVDEPFDYIARVISETEI。

SEQ ID NO:8：

MANREVVETLKELLADGEKRVQVAGVIGTNAAEVVKTAVSLLFQEYPELVSPGGCAYTTRRYNMCVRDMNYFLRMCSYAIVAGGASVLDGRMLAGLRDTFNSLGIPLGPTARGIQLMKKIVKEKLATAGMTNIAFVDEPFDYIARVISETEI。

SEQ ID NO:9：

MANREVVETLKELLADGEKRVQVAGVIGTNAAEVVKTAVSLLFQEYPELVSPGGCAYTTRRYNKCVRDMNYFLRMCSYAIVAGGASVLDGRMLAGLRDTFNSLGIPLGPVARGIQLMKKIVKEKLATAGMTNIAFVDEPFDYIARVISETEI。

SEQ ID NO:10：

MANREVRETQKEQQADGEKRRQVAGVIGTNAAEVVKTAVSLLFQEYPELVSPGGCAYTTRRYNKCVRDMNYFLRMCSYAIVAGGASVLDGRMLAGLRDTFNSLGIPLGPVARGIQLMKKIVKEKLATAGMTNIAFVDEPFDYIARVISETEI。

According to the application, any one or at least two fusion proteins of BDFP1.1, BDFP1.2, BDFP1.3, BDFP1.4, BDFP1.5, BDFP1.6, BDFP1.7, BDFP1.8 or BDFP1.9 can achieve similar effects in the biosensor of the application.

Preferably, the heme transmembrane transporter comprises the transmembrane transporter ChuA.

Preferably, the engineered cells comprise engineered escherichia coli.

Preferably, the engineered cell contains a gene encoding heme oxidase-1, a gene encoding phycobilisome nucleoprotein subunit ApcF2, and a gene encoding heme transmembrane transporter.

Preferably, the heme oxygenase-1 comprises the amino acid sequence shown in SEQ ID NO 11.

Preferably, the gene encoding the heme transmembrane transporter comprises the nucleic acid sequence shown in SEQ ID NO 12.

SEQ ID NO:11：

MSSNLANKLRVGTKKAHTMAENVGFVKCFLKGVVEKSSYRKLVANFYYVYSAMEEEMEKHSQHPIVSKINFSQLNRKQTLEQDLSYYYGANWREQIQLSPAGEAYVQRIREISATEPELLIAHSYTRYLGDLSGGQILKNIAVTAMNLNDGQGTAFYEFADISDEKAFKAKYRQTLDELAIDEATGDRIVDEANAAFGMNMKMFQELEGNLIKAIGMMLFNTLTRKRTRGATELATAE。

SEQ ID NO:12：

atgagccgtcctcagtttaccagcctgcgtctgagcctgctggcgttagcagttagcgcgaccctgcctacctttgcctttgcgacggaaaccatgaccgttaccgcaaccgggaatgcccgcagttcatttgaggcaccgatgatggttagcgttattgataccagtgcaccggaaaatcagaccgccaccagtgccaccgacctgttacgtcatgttccgggtattacactggatggtaccggtcgcacaaatggtcaggatgttaatatgcgtgggtatgatcatagaggggtgttagttctggttgatggtgttagacaaggtactgataccggtcatctgaacggtacgtttctggatcctgcactgattaaaagagttgaaattgtacgtggtccgagcgcattactgtatggtagtggtgctctgggtggggttatttcctatgatacagttgatgcaaaagacctgctgcaggaaggtcagagcagcggttttagagtttttggtacaggtggaaccggtgatcatagtttaggtctgggtgcgagtgcatttggtcgtaccgaaaatctggatggtattgttgcctggagcagtcgtgatcgtggtgatctgcggcagagtaatggggagacagcaccgaatgatgagagtattaataatatgctggccaaaggtacgtggcagattgatagcgcacagagtctgagcgggttagttagatattataacaacgatgcacgtgaaccgaaaaatccgcagaccgttggtgcatccgagagctctaatccgatggttgatcgcagcaccattcagcgtgatgcacagttaagctataagctggccccacaaggtaatgattggttaaacgcagatgcaaaaatttattggtccgaagttcgtatcaatgcgcagaataccggaagcagcggtgaatatcgtgaacagattaccaaaggtgcacgtttagaaaaccgcagcacattattcgcggatagctttgcaagccatctgctgacctatggcggtgaatactatagacaggaacagcatccgggtggggcaacaacaggttttccgcaggcaaaaatcgattttagctctggttggctgcaggatgaaatcacactgcgggacctgccgattacccttctgggtggtaccagatatgatagttatagaggcagcagcgatggttacaaagatgttgatgcagataaatggagtagcagagcaggaatgaccattaatccgaccaattggttaatgctgtttgggagctatgcacaggcattccgggcacctacaatgggggaaatgtataatgatagtaaacatttcagtatcgggagattttacaccaactactgggttccgaatccgaatttacgtccggaaaccaatgaaacccaagaatatggtttcggcctgcgttttgatgacctgatgctgtcaaatgatgcattagagtttaaggcaagctattttgataccaaagcaaaagattacatcagtaccaccgttgacttcgcagcagcaaccacaatgtcatataatgttcctaacgcaaaaatctggggttgggatgttatgaccaaatataccacagatctgtttagcctggatgttgcatataatcgtacccgtggaaaagataccgacaccggtgaatacattagcagcattaacccggatacagttacctcaaccctgaatattcccattgcacattctggatttagcgttggttgggttggtacctttgcagatcgtagcacacacattagcagcagctatagcaagcagcctggatatggggttaatgatttttatgttagttaccagggtcagcaggccctgaaaggtatgaccacaaccctggtgttaggtaatgcatttgataaggaatattggagcccgcaaggtattccgcaggatggtcgtaatggtaaaatttttgtgagctatcagtggtaa。

Preferably, the biosensor comprises engineered escherichia coli, wherein the engineered escherichia coli contains a coding gene of Heme oxidase-1, a coding gene of phycobilisome nucleoprotein subunit ApcF2BDFP1.6 and a coding gene of transmembrane transporter chuA, and a Heme synthase gene hemF is deleted, the detection principle of the biosensor is shown in fig. 1, the transmembrane transporter chuA can transfer Heme (Heme) into cells, Heme oxidase-1 (Ho1) oxidizes Heme into Biliverdin (BV), phycobilisome nucleoprotein subunit ApcF2(BDFP1.6) can self-catalyze and covalently bond biliverdin based on enzymatic reaction, after bonding, excitation by a light source with a specific wavelength can emit far-infrared FR or near-infrared NIR, so that detection of Heme is realized, and in addition, knockout of hemF (delta hemF) can weaken the synthesis of engineered cells per se, thereby reducing the background interference of hemoglobin in the engineered cell.

In a second aspect, the present application provides a method for preparing the biosensor of the first aspect, the method comprising:

knocking out a heme synthetase gene of a host cell, and introducing an encoding gene of heme oxidase-1, an encoding gene of phycobilisome nucleoprotein subunit ApcF2 and an encoding gene of heme transmembrane transport protein into the host cell to obtain the biosensor.

Preferably, the gene knockout is performed using a lambda-red recombination system as described herein.

Preferably, the host cell comprises escherichia coli.

Preferably, the method of introduction comprises any one of electrical transduction, viral vector systems, non-viral vector systems or direct gene injection.

Preferably, the gene is introduced into the host cell by a plasmid vector in the present application.

Preferably, the plasmid vector comprises pacycuet.

In a third aspect, the present application provides a composition comprising a biosensor as described in the first aspect.

Preferably, the composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, electronic circuit or diluent.

In a fourth aspect, the present application provides the use of a biosensor as described in the first aspect or a composition as described in the third aspect for detecting hemoglobin and/or blood.

Compared with the prior art, the method has the following beneficial effects:

(1) the biosensor can simultaneously express heme oxidase-1, phycobilisome nucleoprotein subunit ApcF2 and heme transmembrane transport protein, can express heme at low level, can effectively respond to extracellular heme and generate fluorescence, has low heme background fluorescence, and can effectively detect heme and/or blood;

(2) the biosensor is simple to prepare, low in cost, capable of effectively detecting internal bleeding and wide in development period prospect.

Drawings

FIG. 1 is a schematic diagram of a biosensor according to the present application;

FIG. 2 is a plot of fluorescence intensity of a biosensor in accordance with the present application reacted with blood, with error bars indicating standard deviation SD from 3 independent sample data statistics;

FIG. 3 is a plot of fluorescence intensity of the reaction of the biosensor with hemoglobin according to the present application, with error bars indicating the standard deviation SD from 3 independent sample data statistics;

FIG. 4 is a plot of fluorescence intensity of a biosensor reaction with buffer of the present application, error bars indicate standard deviation SD from 3 independent sample data statistics;

FIG. 5 is a plot of fluorescence intensity of the reaction of the biosensor of the present application with biliverdin, error bars indicating standard deviation SD from 3 independent sample data statistics;

FIG. 6 is a drawing of the culture solution No. 1 in test example 1;

FIG. 7 is a drawing of a culture solution No. 2 in test example 1;

FIG. 8 is a drawing of culture solution No. 3 in test example 1;

FIG. 9 is a drawing of culture solution No. 4 in test example 1;

FIG. 10 is a drawing of culture solution No. 5 in test example 1;

FIG. 11 is a graph of fluorescence intensity for different engineered cells;

FIG. 12 is a dose response graph of fluorescence intensity of a biosensor with blood;

FIG. 13 is a graph of the dose response of the fluorescence intensity of the biosensor with hemoglobin;

FIG. 14 is a flow chart of the zebrafish in vivo test;

FIG. 15 is an image of a zebra fish;

FIG. 16 is a fluorescent intensity chart of zebra fish.

Detailed Description

To further illustrate the technical means adopted by the present application and the effects thereof, the present application is further described below with reference to the following embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the application and not restrictive of the application.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1

In this example, recombinant plasmids were constructed, and the genetic experiment was performed according to molecular cloning, and the primer sequences are shown in Table 1.

The gene sequence (SEQ ID NO:12) encoding the transmembrane transporter chuA was synthesized by Wuhan Tianyihui Biotech, Inc., and inserted into plasmid pACYCDuet (Novagen, Inc.) through restriction enzyme sites NcoI and XhoI to give recombinant plasmid pACYCDuet-chuA, which has chloramphenicol resistance.

Gene sequences of heme oxidase-1 (using primers P3 and P4) and phycobilisome nucleoprotein subunit ApcF2 (using primers P5 and P6) were respectively amplified from plasmids pACYCDuet-ho1 (containing heme oxidase-1 gene ho1 and ho1 gene sequences derived from all1897(GeneBank) and algal species PCC7120) and pET28a-bdfp1.6, wherein the amino acid sequence of heme oxidase-1 is SEQ ID NO:11, the amino acid sequence of phycobilisome nucleoprotein subunit ApcF2 is SEQ ID NO:7, and then heme oxidase-1 gene ho1 is inserted into the first Open Reading Frame (ORFs) of vector plasmid pCDFDuet using restriction enzyme cleavage sites NcoI and PstI; the phycobilisome nucleoprotein subunit ApcF2 gene bdfp1.6 was inserted into the second Open Reading Frame (ORFs) of the vector plasmid pCDFDuet (Novagen) by using restriction enzyme sites BglII and XhoI, resulting in a recombinant plasmid pCDFDuet-ho1-bdfp1.6 with streptomycin resistance.

TABLE 1

Example 2

This example performed a hemF knockout of the E.coli heme synthase gene.

Plasmid pKD4 is used as a template to amplify hemF and genes at two sides of the hemF, a kanamycin gene and an FRT site, wherein the hemF gene sequence is shown as SEQ ID NO. 13, the hemF upstream region sequence is shown as SEQ ID NO. 14, the hemF downstream region sequence is shown as SEQ ID NO. 15, the kanamycin gene sequence is shown as SEQ ID NO. 16, and the FRT site sequence is shown as SEQ ID NO. 17.

SEQ ID NO:13：

atgaaacccgacgcacaccaggttaaacagtttctgctcaaccttcaggatacgatttgtcagcagctgaccgccgtcgatggcgcagaatttgtcgaagatagttggcagcgcgaagctggcggcggcgggcgtagtcgggtgttgcgtaatggtggtgttttcgaacaggcaggcgtcaacttttcgcatgtccacggtgaggcgatgcctgcttccgccaccgctcatcgcccggaacttgccgggcgcagtttcgaggcgatgggcgtttcactggtagtgcatccgcataacccgtatgttcccaccagccacgcgaatgtgcggttttttattgccgaaaaaccgggtgccgatcccgtctggtggtttggcggcggcttcgatttaacccctttctatggttttgaagaagacgccattcactggcaccgcaccgcccgtgacctgtgcctgccatttggtgaagacgtttatccccgttacaaaaagtggtgcgacgattacttctacctcaaacatcgcaacgaacagcgcggtattggcgggctgttctttgatgatctgaacacgccagatttcgaccactgttttgcctttatgcaggcggtaggcaaaggctacaccgacgcttatttaccaattgtagagcgacgtaaagcgatggcctacggcgagcgcgagcgcaattttcagctctaccgtcgcggtcgttatgtcgagttcaatctggtctgggatcgcggcacgctgtttggcctgcaaactggcgggcgcaccgagtctatcctgatgtcaatgccgccactggtacgctgggaatatgattatcagccaaaagatggcagcccagaagcggcgttaagtgagtttattaaggtcagggattgggtgtaa。

SEQ ID NO:14：

cgttccattttgcgtaatcatgggattgatgcgcgtttaacgcgttctggcgatacgtttatcccactttacgatcgcgttgaaatcgcccataaacatggcgcagatctgtttatgtcaattcatgccgatggctttaccaacccgaaagctgccggtgcttcggtatttgccctctctaaccgtggggcaagtagcgcaatggcgaaatacctgtctgaacgcgaaaaccgcgccgatgaagttgccggtaaaaaggcgactgacaaggatcacctattgcaacaagtgctgtttgatctggtgcaaacagataccattaaaaatagtctgacgctcggctcgcatattctgaagaagattaagccggtgcataaactgcacagccgcaacaccgaacaagcggcatttgtggtgttgaaatcaccgtcggttccttcggtgctggtggaaacctcgtttatcaccaacccggaagaagaacggctgttaggcacggcggcgtttcgtcagaaaatcgccacagcgattgctgaaggcgtgatcagttatttccactggttcgacaaccagaaagcacattcgaaaaagcgataagtt。

SEQ ID NO:15：

ctccctcacccccactcccgcatccgctgatgcagcgtcagtgacggcttctcggaaaacagctgctggtaatccgtggcaaattgccccagatgccagaatccccactgcatggcggcgtcttttaccgtcatactttgcgaccacggacttatcagttcgcggcgtacggcgttcaggcgaatgcgtttcagccacgcgttcgggccaatgcctaaaatagcgtgaaacgcgttttgtagcgtgcggcggctgacatgcagttgattacacaaatccagcaccgtcaccggttcggacatgttttccagcacatattcacgggcgcgggaaagcaatcgacggtaactctgatgactgatgctttccgccgtcaccattggttgcgcttcttccagcatggcccccatcgccattagcaaattatcccccagcacttttcgcactgcaggctgatggagattttccggattctcgcaaaacgtcgccagcgcctgttggacaaagccccacagcgcggctttatgctgctctttcacttccagcgccgactggttacgcaacatatgtaatacccgatccgggttatgcaaaaagtta。

SEQ ID NO:16：

atgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctga。

SEQ ID NO:17：

gaagttcctatactttctagagaataggaacttcggaataggaact。

Plasmid pKD46 was transformed into E.coli BL21, which harbored ampicillin resistance gene, lambda-red recombinase gene and arabinose promoter, and the transformed E.coli was cultured in LB medium containing ampicillin (50g/mL) when OD was measured₆₀₀When the concentration reaches 0.3, arabinose with the final concentration of 2% is added to induce escherichia coli to express lambda-red recombinase, then gene fragments containing hemF and the genes of the two side regions thereof, the kanamycin gene and the FRT site are transformed into the escherichia coli containing the lambda-red recombinase, an LB plate containing kanamycin (30g/mL) is used for screening a strain with successful knockout, the strain is further verified by PCR experiments, the strain is cultured overnight at 42 ℃, and the plasmid pKD46 is removed, so that the hemF-knocked escherichia coli is obtained.

Example 3

This example provides a biosensor, which is an engineered escherichia coli, containing a gene encoding heme oxidase-1, a gene encoding phycobilisome nucleoprotein subunit ApcF2BDFP1.6, and a gene encoding transmembrane transporter chuA, and lacking heme synthase gene hemF.

The recombinant plasmids pACYCDuet-chuA and pCDFDuet-ho1-bdfp1.6 prepared in example 1 were introduced into hemF-knocked-out E.coli prepared in example 2 to obtain an engineered E.coli, which was cultured in TB medium containing kanamycin (30g/mL), chloramphenicol (40g/mL) and streptomycin (50g/mL) to be OD of E.coli₆₀₀When the protein expression reaches 0.7, 1mM IPTG is added to induce the protein expression, the protein expression is induced at 16 ℃ for 15h to enable the protein to co-express ChuA, BDFP1.6 and HO1, a 50mL centrifuge tube is used, the centrifugation is carried out at 16 ℃ and 5000 Xg for 5min, and the cells are collected after being washed twice by distilled water.

In addition, in order to determine whether the biosensor (engineered E.coli) reacted with hemoglobin, hemoglobin was added to the medium at a final concentration of 20. mu.M while IPTG was added, and the resultant was induced at 16 ℃ for 15 hours, and then fluorescence of E.coli was detected by a fluorescence spectrometer.

Test example 1

This test example examined the affinity of the biosensor prepared in example 3 for hemoglobin or blood.

Taking 4 culture flasks, respectively culturing the engineered Escherichia coli in TB medium containing kanamycin (30g/mL), chloramphenicol (40g/mL) and streptomycin (50g/mL) to obtain OD of Escherichia coli₆₀₀When reaching 0.7, protein expression was induced by adding 1mM IPTG each, inducing at 16 ℃ for 16h, followed by addition of 10. mu.M heme, 1000ppm blood, and buffer (10. mu.M heme dissolved in 100mM NaHCO)₃Medium) and 10 μ M biliverdin, and then monitoring fluorescence every 20 minutes at 37 ℃, the results are shown in fig. 2-5, fig. 2 and 3 are respectively test result graphs of adding blood and heme, the fluorescence intensity is continuously enhanced and reaches a higher level, which indicates that the biosensor prepared by the application can effectively respond to heme or blood and generate high-intensity fluorescence, fig. 4 and 5 are respectively test results of adding buffer solution and biliverdin, the fluorescence intensity is lower and has no obvious change, which indicates that the biosensor prepared by the application has lower background interference, and thus can effectively detect.

Taking 5 culture flasks numbered 1-5, inoculating Escherichia coli which is knocked out hemF but only introduced with pCDFDuet-ho1-bdfp1.6 plasmid into No. 1, adding IPTG and heme into the culture medium, and culturing; no. 2 inoculation of the biosensor prepared in example 3, and IPTG and heme were added to the medium for culture; no. 3 inoculated the biosensor prepared in example 3, and only IPTG was added to the culture medium for culture; no. 4 Escherichia coli to which no hemF knockout had been added but plasmids pCDFDuet-ho1-bdfp1.6 and pACYCDuet-chuA were introduced was cultured by adding IPTG alone to the medium; no. 5 Escherichia coli to which no hemF knockout had been added but plasmids pCDFDuet-ho1-bdfp1.6 and pACYCDuet-chuA were introduced was cultured by adding IPTG and heme to the medium; the fluorescence of each group of culture solution is detected, and the result is shown in fig. 6-11, the fluorescence intensity of the culture solution No. 1 is very low, which indicates that the cell which can not express the heme transmembrane transport protein can not react with heme; the fluorescence intensity of the No. 2 culture solution is higher, which shows that the biosensor prepared by the method can effectively respond to the heme; the signal intensity of the culture solution No. 3 is very low, which shows that the background interference of the biosensor prepared by the application is low; the fluorescence intensity of culture solutions No. 4 and No. 5 is higher than that of culture solution No. 2, which indicates that strong background fluorescence can be generated without knockout of hemF, and detection is influenced.

For determining K for binding of hemoglobin or blood to the biosensor of the present application_DThe values (dissociation constants) of the titration test of the biosensor prepared in example 3 with different concentrations of hemoglobin or blood specifically include: the biosensors were separately cultured using TB medium containing kanamycin (30g/mL), chloramphenicol (40g/mL) and streptomycin (50g/mL) to treat OD of E.coli₆₀₀When the concentration reached 0.7, 1mM IPTG was added to each of them to induce protein expression, followed by induction at 16 ℃ for 16 hours, then the cells were divided into 50 parts, 12 parts were randomly added with 10ppm, 30ppm, 40ppm, 70ppm, 100ppm, 200ppm, 300ppm, 400ppm, 500ppm, 600ppm, 1000ppm and 2000ppm of blood, respectively, and 13 parts were randomly added with 0.3. mu.M, 0.5. mu.M, 0.7. mu.M, 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 7. mu.M, 20. mu.M, 40. mu.M, 70. mu.M and 100. mu.M of hemoglobin, and the above experiments were carried out for 100min and 180min, followed by measuring fluorescence intensity, and the above experiments were carried out for 1 technical repetition, respectively.

The relationship between fluorescence intensity and dose can be fitted using the Scatchard equation (1)) for the single site binding model, and the results are shown in FIGS. 12 and 13.

F＝(Fmax[c])/(K_D+[c]) (1)

Wherein "F" represents fluorescence, "[ c ]]"indicates the concentration of hemoglobin or blood, K_DIndicates the dissociation constant.

As can be seen from FIGS. 12 and 13, the dissociation constant K_DValues of 108ppm (blood), 2.85 μ M (hemoglobin) were achieved, indicating that the biosensor of the present application reacts efficiently with blood or hemoglobin and possesses high specificity.

Test example 2

The experimental example verifies the effectiveness of the biosensor in zebra fish, the process is shown in fig. 14, and all experiments using zebra fish pass the approval of the scientific ethics committee of the university of agriculture in china.

The experimental selection of AB strain zebra fish (purchased from institute of aquatic organisms, academy of Chinese sciences) was carried out at 28 ℃ with 14 hours of light cycle and 10 hours of dark cycle, and the zebra fish was fed with fairy shrimp, 40 zebra fish were randomly selected, anesthetized with 3-aminobenzoic acid ethyl ester methanesulfonic acid (MS-222, 100mg/L), and then 4 × 10 zebra fish were injected with a microinjector⁶The biosensor (engineered escherichia coli) prepared in example 3 of CFU was injected into the abdomen of zebrafish, 20 of which were randomly selected 4 days later, aspirin (20mg/mL) was injected into the abdomen in the same manner, and the remaining 20 were injected with 10 μ L of sterile water as a control, wherein aspirin (aspirin) is an inflammatory drug that causes gastrointestinal bleeding under high concentration conditions (considering insolubility of aspirin in water, aspirin was first pulverized and then prepared into a suspension).

After 4 days zebrafish were imaged using the device QPIX420(Molecular Devices Company), excitation wavelength was 628nm, emission wavelength was 692nm, fluorescence images were corrected and analyzed using imagej (national Institutes of health)), images and statistics were processed using Origin 9.0(Origin lab), during the experiment, 3 replicates of each experiment were used to calculate the mean fluorescence intensity of engineered e.coli, to ensure that a P-value of <0.05 reached significance level, and a two-tailed student's t-test was used to calculate P-value, all error bars represent the standard deviation SD (n 3, number of experiments), the results are shown in fig. 15 and 16.

FIG. 15 is an imaging diagram of zebrafish, which is an imaging diagram of a white light channel and an imaging diagram of a red light channel, respectively, it can be seen that aspirin-injected zebrafish has significant fluorescence, and it can be seen from FIG. 16 that the fluorescence value of aspirin-injected zebrafish is significantly higher than that of a control group (p <0.01, two-tailed student's t test; N ═ 20) after the fluorescence value of aspirin-injected zebrafish is unified by using ImageJ calculation, which indicates that the biosensor of the present application can effectively perform in vivo detection.

To sum up, the biosensor of this application, can high-efficient and heme reaction, and possess the high specificity, in addition, self background fluorescence is lower, consequently can effectively carry out heme detection to can effectively detect internal hemorrhage.

The applicant states that the detailed method of the present application is illustrated by the above examples, but the present application is not limited to the above detailed method, that is, the present application does not mean that the present application has to rely on the above detailed method for implementation. It should be understood by those skilled in the art that any modification, equivalent substitution of each raw material, addition of auxiliary components, selection of specific modes and the like, of the product of the present application falls within the scope and disclosure of the present application.

SEQUENCE LISTING

<110> university of agriculture in Huazhong

GUANGZHOU TEBSUN BIO-TECH DEVELOPMENT Co.,Ltd.

<120> biosensor and preparation method and application thereof

<130> BY21DX1761FGPC-CN

<160> 23

<170> PatentIn version 3.5

<210> 1

<211> 169

<212> PRT

<213> Synechococcus sp 7203 (Chroococcidiopsis sp. PCC 7203)

<400> 1

Met Gln Asp Lys Leu Thr Ser Val Ala Lys Asn Cys Asp Leu Thr Gly

1 5 10 15

Ser Ser Leu Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala

20 25 30

Asp Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Ser Asn Ala

35 40 45

Ala Glu Ile Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro

50 55 60

Glu Leu Val Ser Pro Gly Gly Asn Ala Tyr Thr Thr Arg Arg Tyr Asn

65 70 75 80

Met Tyr Val Arg Asp Met Asn Tyr Phe Leu Arg Tyr Cys Ser Tyr Ala

85 90 95

Ile Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly

100 105 110

Leu Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala

115 120 125

Arg Ser Ile Gln Leu Met Lys Asn Ile Val Lys Glu Lys Leu Val Thr

130 135 140

Ala Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Val

145 150 155 160

Val Arg Glu Ile Ser Glu Thr Glu Ile

165

<210> 2

<211> 152

<212> PRT

<213> Artificial sequence

<220>

<223> BDFP1.1

<400> 2

Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp

1 5 10 15

Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala

20 25 30

Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu

35 40 45

Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met

50 55 60

Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile

65 70 75 80

Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Leu

85 90 95

Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg

100 105 110

Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala

115 120 125

Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala

130 135 140

Arg Glu Ile Ser Glu Thr Glu Ile

145 150

<210> 3

<211> 152

<212> PRT

<213> Artificial sequence

<220>

<223> BDFP1.2

<400> 3

Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp

1 5 10 15

Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala

20 25 30

Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu

35 40 45

Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met

50 55 60

Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile

65 70 75 80

Val Ala Gly Gly Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Phe

85 90 95

Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Thr Ala Arg

100 105 110

Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala

115 120 125

Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala

130 135 140

Arg Glu Ile Ser Glu Thr Glu Ile

145 150

<210> 4

<211> 152

<212> PRT

<213> Artificial sequence

<220>

<223> BDFP1.3

<400> 4

Met Ala Asn Arg Glu Val Val Glu Thr Leu Glu Glu Phe Leu Ala Asp

1 5 10 15

Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala

20 25 30

Glu Val Val Lys Ala Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu

35 40 45

Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met

50 55 60

Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile

65 70 75 80

Val Ala Gly Asp Ala Ser Val Leu Asp Glu Arg Leu Leu Ala Gly Leu

85 90 95

Arg Asp Thr Phe Asn Ser Cys Gly Ile Pro Leu Gly Pro Thr Ala Arg

100 105 110

Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala

115 120 125

Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala

130 135 140

Arg Glu Ile Ser Glu Thr Glu Ile

145 150

<210> 5

<211> 138

<212> PRT

<213> Artificial sequence

<220>

<223> BDFP1.4

<400> 5

Ala Asp Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn

1 5 10 15

Ala Ala Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr

20 25 30

Pro Glu Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr

35 40 45

Asn Met Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr

50 55 60

Ala Ile Val Ala Gly Asp Ala Ser Val Leu Asp Gly Arg Leu Leu Ala

65 70 75 80

Gly Leu Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr

85 90 95

Ala Arg Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val

100 105 110

Thr Ala Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr

115 120 125

Ile Val Arg Gly Ile Ser Glu Thr Glu Ile

130 135

<210> 6

<211> 138

<212> PRT

<213> Artificial sequence

<220>

<223> BDFP1.5

<400> 6

Ala Asp Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn

1 5 10 15

Ala Ala Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr

20 25 30

Pro Glu Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr

35 40 45

Asn Met Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr

50 55 60

Ala Ile Val Ala Gly Gly Ala Ser Val Leu Asp Glu Arg Met Leu Ala

65 70 75 80

Gly Leu Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr

85 90 95

Ala Arg Ser Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val

100 105 110

Thr Ala Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr

115 120 125

Ile Ala Arg Val Ile Ser Glu Thr Glu Ile

130 135

<210> 7

<211> 152

<212> PRT

<213> Artificial sequence

<220>

<223> BDFP1.6

<400> 7

Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp

1 5 10 15

Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala

20 25 30

Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu

35 40 45

Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met

50 55 60

Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile

65 70 75 80

Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe

85 90 95

Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg

100 105 110

Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala

115 120 125

Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala

130 135 140

Arg Val Ile Ser Glu Thr Glu Ile

145 150

<210> 8

<211> 152

<212> PRT

<213> Artificial sequence

<220>

<223> BDFP1.7

<400> 8

Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp

1 5 10 15

Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala

20 25 30

Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu

35 40 45

Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met

50 55 60

Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile

65 70 75 80

Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu

85 90 95

Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg

100 105 110

Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala

115 120 125

Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala

130 135 140

Arg Val Ile Ser Glu Thr Glu Ile

145 150

<210> 9

<211> 152

<212> PRT

<213> Artificial sequence

<220>

<223> BDFP1.8

<400> 9

Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp

1 5 10 15

Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala

20 25 30

Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu

35 40 45

Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Lys

50 55 60

Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile

65 70 75 80

Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu

85 90 95

Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg

100 105 110

Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala

115 120 125

Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala

130 135 140

Arg Val Ile Ser Glu Thr Glu Ile

145 150

<210> 10

<211> 152

<212> PRT

<213> Artificial sequence

<220>

<223> BDFP1.9

<400> 10

Met Ala Asn Arg Glu Val Arg Glu Thr Gln Lys Glu Gln Gln Ala Asp

1 5 10 15

Gly Glu Lys Arg Arg Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala

20 25 30

Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu

35 40 45

Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Lys

50 55 60

Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile

65 70 75 80

Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu

85 90 95

Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg

100 105 110

Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala

115 120 125

Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala

130 135 140

Arg Val Ile Ser Glu Thr Glu Ile

145 150

<210> 11

<211> 238

<212> PRT

<213> houttuynia PCC7120 (Anabaena sp. PCC7120)

<400> 11

Met Ser Ser Asn Leu Ala Asn Lys Leu Arg Val Gly Thr Lys Lys Ala

1 5 10 15

His Thr Met Ala Glu Asn Val Gly Phe Val Lys Cys Phe Leu Lys Gly

20 25 30

Val Val Glu Lys Ser Ser Tyr Arg Lys Leu Val Ala Asn Phe Tyr Tyr

35 40 45

Val Tyr Ser Ala Met Glu Glu Glu Met Glu Lys His Ser Gln His Pro

50 55 60

Ile Val Ser Lys Ile Asn Phe Ser Gln Leu Asn Arg Lys Gln Thr Leu

65 70 75 80

Glu Gln Asp Leu Ser Tyr Tyr Tyr Gly Ala Asn Trp Arg Glu Gln Ile

85 90 95

Gln Leu Ser Pro Ala Gly Glu Ala Tyr Val Gln Arg Ile Arg Glu Ile

100 105 110

Ser Ala Thr Glu Pro Glu Leu Leu Ile Ala His Ser Tyr Thr Arg Tyr

115 120 125

Leu Gly Asp Leu Ser Gly Gly Gln Ile Leu Lys Asn Ile Ala Val Thr

130 135 140

Ala Met Asn Leu Asn Asp Gly Gln Gly Thr Ala Phe Tyr Glu Phe Ala

145 150 155 160

Asp Ile Ser Asp Glu Lys Ala Phe Lys Ala Lys Tyr Arg Gln Thr Leu

165 170 175

Asp Glu Leu Ala Ile Asp Glu Ala Thr Gly Asp Arg Ile Val Asp Glu

180 185 190

Ala Asn Ala Ala Phe Gly Met Asn Met Lys Met Phe Gln Glu Leu Glu

195 200 205

Gly Asn Leu Ile Lys Ala Ile Gly Met Met Leu Phe Asn Thr Leu Thr

210 215 220

Arg Lys Arg Thr Arg Gly Ala Thr Glu Leu Ala Thr Ala Glu

225 230 235

<210> 12

<211> 1983

<212> DNA

<213> Artificial sequence

<220>

<223> heme transmembrane transporter

<400> 12

atgagccgtc ctcagtttac cagcctgcgt ctgagcctgc tggcgttagc agttagcgcg 60

accctgccta cctttgcctt tgcgacggaa accatgaccg ttaccgcaac cgggaatgcc 120

cgcagttcat ttgaggcacc gatgatggtt agcgttattg ataccagtgc accggaaaat 180

cagaccgcca ccagtgccac cgacctgtta cgtcatgttc cgggtattac actggatggt 240

accggtcgca caaatggtca ggatgttaat atgcgtgggt atgatcatag aggggtgtta 300

gttctggttg atggtgttag acaaggtact gataccggtc atctgaacgg tacgtttctg 360

gatcctgcac tgattaaaag agttgaaatt gtacgtggtc cgagcgcatt actgtatggt 420

agtggtgctc tgggtggggt tatttcctat gatacagttg atgcaaaaga cctgctgcag 480

gaaggtcaga gcagcggttt tagagttttt ggtacaggtg gaaccggtga tcatagttta 540

ggtctgggtg cgagtgcatt tggtcgtacc gaaaatctgg atggtattgt tgcctggagc 600

agtcgtgatc gtggtgatct gcggcagagt aatggggaga cagcaccgaa tgatgagagt 660

attaataata tgctggccaa aggtacgtgg cagattgata gcgcacagag tctgagcggg 720

ttagttagat attataacaa cgatgcacgt gaaccgaaaa atccgcagac cgttggtgca 780

tccgagagct ctaatccgat ggttgatcgc agcaccattc agcgtgatgc acagttaagc 840

tataagctgg ccccacaagg taatgattgg ttaaacgcag atgcaaaaat ttattggtcc 900

gaagttcgta tcaatgcgca gaataccgga agcagcggtg aatatcgtga acagattacc 960

aaaggtgcac gtttagaaaa ccgcagcaca ttattcgcgg atagctttgc aagccatctg 1020

ctgacctatg gcggtgaata ctatagacag gaacagcatc cgggtggggc aacaacaggt 1080

tttccgcagg caaaaatcga ttttagctct ggttggctgc aggatgaaat cacactgcgg 1140

gacctgccga ttacccttct gggtggtacc agatatgata gttatagagg cagcagcgat 1200

ggttacaaag atgttgatgc agataaatgg agtagcagag caggaatgac cattaatccg 1260

accaattggt taatgctgtt tgggagctat gcacaggcat tccgggcacc tacaatgggg 1320

gaaatgtata atgatagtaa acatttcagt atcgggagat tttacaccaa ctactgggtt 1380

ccgaatccga atttacgtcc ggaaaccaat gaaacccaag aatatggttt cggcctgcgt 1440

tttgatgacc tgatgctgtc aaatgatgca ttagagttta aggcaagcta ttttgatacc 1500

aaagcaaaag attacatcag taccaccgtt gacttcgcag cagcaaccac aatgtcatat 1560

aatgttccta acgcaaaaat ctggggttgg gatgttatga ccaaatatac cacagatctg 1620

tttagcctgg atgttgcata taatcgtacc cgtggaaaag ataccgacac cggtgaatac 1680

attagcagca ttaacccgga tacagttacc tcaaccctga atattcccat tgcacattct 1740

ggatttagcg ttggttgggt tggtaccttt gcagatcgta gcacacacat tagcagcagc 1800

tatagcaagc agcctggata tggggttaat gatttttatg ttagttacca gggtcagcag 1860

gccctgaaag gtatgaccac aaccctggtg ttaggtaatg catttgataa ggaatattgg 1920

agcccgcaag gtattccgca ggatggtcgt aatggtaaaa tttttgtgag ctatcagtgg 1980

taa 1983

<210> 13

<211> 900

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 13

atgaaacccg acgcacacca ggttaaacag tttctgctca accttcagga tacgatttgt 60

cagcagctga ccgccgtcga tggcgcagaa tttgtcgaag atagttggca gcgcgaagct 120

ggcggcggcg ggcgtagtcg ggtgttgcgt aatggtggtg ttttcgaaca ggcaggcgtc 180

aacttttcgc atgtccacgg tgaggcgatg cctgcttccg ccaccgctca tcgcccggaa 240

cttgccgggc gcagtttcga ggcgatgggc gtttcactgg tagtgcatcc gcataacccg 300

tatgttccca ccagccacgc gaatgtgcgg ttttttattg ccgaaaaacc gggtgccgat 360

cccgtctggt ggtttggcgg cggcttcgat ttaacccctt tctatggttt tgaagaagac 420

gccattcact ggcaccgcac cgcccgtgac ctgtgcctgc catttggtga agacgtttat 480

ccccgttaca aaaagtggtg cgacgattac ttctacctca aacatcgcaa cgaacagcgc 540

ggtattggcg ggctgttctt tgatgatctg aacacgccag atttcgacca ctgttttgcc 600

tttatgcagg cggtaggcaa aggctacacc gacgcttatt taccaattgt agagcgacgt 660

aaagcgatgg cctacggcga gcgcgagcgc aattttcagc tctaccgtcg cggtcgttat 720

gtcgagttca atctggtctg ggatcgcggc acgctgtttg gcctgcaaac tggcgggcgc 780

accgagtcta tcctgatgtc aatgccgcca ctggtacgct gggaatatga ttatcagcca 840

aaagatggca gcccagaagc ggcgttaagt gagtttatta aggtcaggga ttgggtgtaa 900

<210> 14

<211> 600

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 14

cgttccattt tgcgtaatca tgggattgat gcgcgtttaa cgcgttctgg cgatacgttt 60

atcccacttt acgatcgcgt tgaaatcgcc cataaacatg gcgcagatct gtttatgtca 120

attcatgccg atggctttac caacccgaaa gctgccggtg cttcggtatt tgccctctct 180

aaccgtgggg caagtagcgc aatggcgaaa tacctgtctg aacgcgaaaa ccgcgccgat 240

gaagttgccg gtaaaaaggc gactgacaag gatcacctat tgcaacaagt gctgtttgat 300

ctggtgcaaa cagataccat taaaaatagt ctgacgctcg gctcgcatat tctgaagaag 360

attaagccgg tgcataaact gcacagccgc aacaccgaac aagcggcatt tgtggtgttg 420

aaatcaccgt cggttccttc ggtgctggtg gaaacctcgt ttatcaccaa cccggaagaa 480

gaacggctgt taggcacggc ggcgtttcgt cagaaaatcg ccacagcgat tgctgaaggc 540

gtgatcagtt atttccactg gttcgacaac cagaaagcac attcgaaaaa gcgataagtt 600

<210> 15

<211> 600

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 15

ctccctcacc cccactcccg catccgctga tgcagcgtca gtgacggctt ctcggaaaac 60

agctgctggt aatccgtggc aaattgcccc agatgccaga atccccactg catggcggcg 120

tcttttaccg tcatactttg cgaccacgga cttatcagtt cgcggcgtac ggcgttcagg 180

cgaatgcgtt tcagccacgc gttcgggcca atgcctaaaa tagcgtgaaa cgcgttttgt 240

agcgtgcggc ggctgacatg cagttgatta cacaaatcca gcaccgtcac cggttcggac 300

atgttttcca gcacatattc acgggcgcgg gaaagcaatc gacggtaact ctgatgactg 360

atgctttccg ccgtcaccat tggttgcgct tcttccagca tggcccccat cgccattagc 420

aaattatccc ccagcacttt tcgcactgca ggctgatgga gattttccgg attctcgcaa 480

aacgtcgcca gcgcctgttg gacaaagccc cacagcgcgg ctttatgctg ctctttcact 540

tccagcgccg actggttacg caacatatgt aatacccgat ccgggttatg caaaaagtta 600

<210> 16

<211> 795

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 16

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210> 17

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> FRT site sequence

<400> 17

gaagttccta tactttctag agaataggaa cttcggaata ggaact 46

<210> 18

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> P1

<400> 18

ggcatccctc tgggtcccac tgcccggggc 30

<210> 19

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> P2

<400> 19

gagggagttg aatgtatccc gcagcccggc 30

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> P3

<400> 20

cctggcgggt cggcctatac aacaa 25

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> P4

<400> 21

ggacaccaac tctgggtact cctga 25

<210> 22

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> P5

<400> 22

gacctcaact acttcctcag gatgtgt 27

<210> 23

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> P6

<400> 23

cctcacacac atgttgtagc gccttgt 27