Corynebacterium glutamicum membrane protein Ncgl2775, surface display system and construction method thereof
阅读说明：本技术 谷氨酸棒杆菌膜蛋白Ncgl2775及其表面展示系统和构建方法 (Corynebacterium glutamicum membrane protein Ncgl2775, surface display system and construction method thereof ) 是由 郑穗平 林珂瑞 韩双艳 林影 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种谷氨酸棒杆菌膜蛋白Ncgl2775及其表面展示系统和构建方法。该谷氨酸棒杆菌膜蛋白Ncgl2775的氨基酸序列如SEQ ID NO:1所示,其在谷氨酸棒杆菌中表达量较高,因此可将其用于构建高展示效率的谷氨酸棒杆菌表面展示系统。本发明中还成功构建一种谷氨酸棒杆菌细胞表面展示系统,是以所述谷氨酸棒杆菌膜蛋白Ncgl2775为锚定蛋白,将靶蛋白固定在谷氨酸棒杆菌细胞表面构成,提高了谷氨酸棒杆菌表面展示系统的内源锚定蛋白的展示效率。(The invention discloses a corynebacterium glutamicum membrane protein Ncgl2775, a surface display system and a construction method thereof. The amino acid sequence of the corynebacterium glutamicum membrane protein Ncgl2775 is shown in SEQ ID NO. 1, and the expression level of the protein is high in corynebacterium glutamicum, so that the protein can be used for constructing a surface display system of corynebacterium glutamicum with high display efficiency. The invention also successfully constructs a corynebacterium glutamicum cell surface display system, which is formed by fixing target protein on the surface of the corynebacterium glutamicum cell by taking the corynebacterium glutamicum membrane protein Ncgl2775 as the anchoring protein, so that the display efficiency of endogenous anchoring protein of the corynebacterium glutamicum surface display system is improved.)
1. A corynebacterium glutamicum membrane protein Ncgl2775, characterized in that: the amino acid sequence is shown as SEQ ID NO. 1.
2. A gene encoding the membrane protein Ncgl2775 of corynebacterium glutamicum of claim 1, wherein: the nucleotide sequence is shown as SEQ ID NO. 2.
3. Use of the corynebacterium glutamicum membrane protein Ncgl2775 of claim 1 as an anchoring protein in a surface display system.
4. A corynebacterium glutamicum cell surface display system, comprising: the protein of Corynebacterium glutamicum Ncgl2775 according to claim 1, which is an anchor protein, is immobilized on the cell surface of Corynebacterium glutamicum.
5. The C.glutamicum cell surface display system of claim 4, wherein: the target protein is any one of fluorescent protein or amylase.
6. The C.glutamicum cell surface display system of claim 5, wherein: the target protein is enhanced green fluorescent protein, red fluorescent protein or alpha-amylase.
7. The method for constructing a C.glutamicum cell surface display system of any one of claims 4 to 6, comprising the steps of:
(1) cloning a gene encoding the membrane protein Ncgl2775 of C.glutamicum according to claim 1 into an expression cassette of an expression vector, to obtain a surface display expression vector with the membrane protein Ncgl2775 of C.glutamicum as an anchor protein;
(2) cloning a gene sequence of a target protein to the upstream of the gene sequence of the surface display expression vector of the corynebacterium glutamicum membrane protein Ncgl2775 obtained in the step (1), and forming a fusion gene with the gene of the corynebacterium glutamicum membrane protein Ncgl 2775;
(3) transforming the corynebacterium glutamicum, and then screening positive transformants according to the screening markers on the expression vectors to obtain a corynebacterium glutamicum cell surface display system.
8. The method for constructing a cell surface display system of Corynebacterium glutamicum as set forth in claim 7, comprising the steps of:
(i) constructing a recombinant plasmid by homologous recombination of a gene encoding the membrane protein Ncgl2775 of Corynebacterium glutamicum of claim 1, a gene encoding a target protein and an expression vector;
(ii) (ii) transforming the recombinant plasmid obtained in the step (i) into corynebacterium glutamicum, and selecting a positive transformant to obtain a corynebacterium glutamicum cell surface display system;
(ii) the target protein in step (i) is any one of fluorescent protein or amylase; further enhancing green fluorescent protein, red fluorescent protein or alpha-amylase;
the nucleotide sequence of the enhanced green fluorescent protein is shown as SEQ ID NO. 5;
the nucleotide sequence of the red fluorescent protein is shown as SEQ ID NO. 17;
the nucleotide sequence of the alpha-amylase is shown as SEQ ID NO. 21;
the expression vector in the step (i) is a corynebacterium glutamicum expression vector pEC-XK99 e.
9. Use of a membrane protein of Corynebacterium glutamicum Ncgl2775 according to claim 1, of a gene coding for a membrane protein of Corynebacterium glutamicum Ncgl2775 according to claim 1, or of a cell surface display system of Corynebacterium glutamicum according to any of claims 4 to 6, for the preparation of an amylase.
10. Use according to claim 9, characterized in that: the amylase is alpha-amylase.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a corynebacterium glutamicum membrane protein Ncgl2775, a surface display system thereof and a construction method thereof.
Background
The microbial cell surface display technology is a novel genetic engineering technology for displaying a target fragment on the surface of a microorganism in the form of fusion protein by utilizing a genetic engineering means through a polypeptide fragment (or a protein structural domain), in particular to a technology for fixing protein or polypeptide on the surface of a cell through anchoring protein, wherein the displayed polypeptide or protein can keep a relatively independent spatial structure and biological activity. The microbial cell surface display system comprises a host bacterium, an anchor protein and a target protein, and a connecting (linker) sequence is added between the anchor protein and the target protein. The microbial cell surface display has wide application prospects in the aspects of polypeptide separation, whole-cell catalysts, whole-cell adsorbents, vaccine and antibody production, protein library screening, biosensors, bioremediation and the like.
Currently, phage (Bacteriophages), saccharomyces cerevisiae (saccharomyces cerevisiae), gram-negative bacteria (escherichia coli), gram-positive bacteria (Corynebacterium glutamicum), Bacillus subtilis (Bacillus subtilis), and the like are the main host bacteria applied to a microorganism surface display system. The dockerin used for surface display generally has the following characteristics: (1) is anchored on the cell surface more firmly; (2) can be effectively fused with a target protein sequence without influencing the structure and the function of the target protein. The dockerin proteins of gram-positive bacterial display systems are mainly: (1) membrane associated proteins (with transmembrane domains or lipoproteins); (2) a cell wall-associated protein (having a C-terminal Leu-Pro-X-Thr-Gly (LPXTG) motif or Cell Wall Binding Domain (CWBD)).
The corynebacterium glutamicum has the advantages of strong robustness, low extracellular protease activity, wide natural carbon source substrate and the like, and has very wide application prospect in the aspect of microbial cell surface display. Currently, there are few available anchoring proteins for the display system of C.glutamicum, of only three types: foreign proteins (PgsA), mycoylated proteins (Ncgl1337, porin series proteins PorB, PorC) and membrane proteins (Ncgl1221, also known as the mechanical tunnel protein mscg). These ankyrins have now successfully displayed a variety of enzyme proteins, such as amylases, glucanases, glucosidases, cellulase complexes, etc., on the surface of C.glutamicum. To increase the versatility of the cell surface display technology of C.glutamicum, it is necessary to develop new and efficient anchoring motifs.
Choi et al (Choi J W, Yim S, Jeong K J. development of a potential protein display form in Corynebacterium glutamicum using a mycolic acid layer protein, NCgl1337, as an anchoring motif [ J ]. Biotechnology Journal,2017:1700509.) reported the use of Corynebacterium glutamicum mycolic acid layer protein, Ncgl1337, as an anchor protein for cell surface display of foreign proteins. Yao et al (Display of alpha-amylase on the surface of Corynebacterium glutamicum cells by using NCgl1221 as the anchoring protein, and production of glutamate from stage. electrodes of microbiology.2009) reported the cell surface Display of foreign proteins using Corynebacterium glutamicum membrane protein NCgl1221 as the anchor protein. However, the existing Corynebacterium glutamicum has fewer types and numbers of anchoring proteins, and the display system is single.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a corynebacterium glutamicum membrane protein Ncgl 2775.
Another object of the present invention is to provide a gene encoding the membrane protein Ncgl2775 of Corynebacterium glutamicum.
Still another object of the present invention is to provide a C.glutamicum cell surface display system.
The invention also aims to provide a construction method of the corynebacterium glutamicum cell surface display system.
The purpose of the invention is realized by the following technical scheme:
a Corynebacterium glutamicum membrane protein Ncgl2775, the amino acid sequence of which is shown in SEQ ID NO. 1.
The corynebacterium glutamicum membrane protein consists of 309 amino acids and is about 32.62 KDa.
The nucleotide sequence of the gene for coding the corynebacterium glutamicum membrane protein Ncgl2775 is shown in SEQ ID NO. 2.
The application of the corynebacterium glutamicum membrane protein Ncgl2775 as an anchoring protein in a surface display system.
The surface display system is a Corynebacterium glutamicum cell surface display system.
The corynebacterium glutamicum membrane protein Ncgl2775 can be used for constructing a corynebacterium glutamicum cell surface display system, and the corynebacterium glutamicum cell surface display system is formed by fixing a target protein on the surface of a corynebacterium glutamicum cell by taking the corynebacterium glutamicum membrane protein Ncgl2775 as an anchor protein.
A Corynebacterium glutamicum cell surface display system is formed by fixing target protein on the surface of a Corynebacterium glutamicum cell by taking the Corynebacterium glutamicum membrane protein Ncgl2775 as an anchoring protein.
The target protein is any one of fluorescent protein or amylase; preferably Enhanced Green Fluorescent Protein (EGFP), red fluorescent protein (mCherry) or alpha-amylase (alpha-amylase).
The Corynebacterium glutamicum is preferably Corynebacterium glutamicum ATCC 13032.
The construction method of the corynebacterium glutamicum cell surface display system comprises the following steps:
(1) cloning the gene coding the corynebacterium glutamicum membrane protein Ncgl2775 into an expression cassette of an expression vector to obtain a surface display expression vector taking the corynebacterium glutamicum membrane protein Ncgl2775 as an anchoring protein;
(2) cloning a gene sequence of a target protein to the upstream of the gene sequence of the surface display expression vector of the corynebacterium glutamicum membrane protein Ncgl2775 obtained in the step (1), and forming a fusion gene with the gene of the corynebacterium glutamicum membrane protein Ncgl 2775;
(3) transforming corynebacterium glutamicum, and then screening positive transformants according to the screening markers on the expression vectors to obtain the corynebacterium glutamicum cell surface display system.
The construction method of the corynebacterium glutamicum cell surface display system specifically comprises the following steps:
(i) constructing a recombinant plasmid by a homologous recombination mode of a gene for coding the corynebacterium glutamicum membrane protein Ncgl2775, a gene for target protein and an expression vector;
(ii) and (e) transforming the recombinant plasmid obtained in the step (i) into corynebacterium glutamicum, and selecting a positive transformant to obtain the corynebacterium glutamicum cell surface display system.
The gene sequence encoding the C.glutamicum membrane protein Ncgl2775 described in step (i) is shown in SEQ ID NO: 2.
(ii) the target protein in step (i) is any one of fluorescent protein or amylase; preferably Enhanced Green Fluorescent Protein (EGFP), red fluorescent protein (mCherry) or alpha-amylase (alpha-amylase); more preferably an alpha-amylase.
The nucleotide sequence of the Enhanced Green Fluorescent Protein (EGFP) is shown in SEQ ID NO: 5.
The nucleotide sequence of the red fluorescent protein (mCherry) is shown as SEQ ID NO: 17.
The nucleotide sequence of the alpha-amylase (amyE gene) is shown as SEQ ID NO: 21.
The expression vector in step (i) is a Corynebacterium glutamicum expression vector which is conventional in the art and has kanamycin resistance; preferably, the expression vector pEC-XK99e is a Corynebacterium glutamicum expression vector (in the case of pEC-XK99e, after the surface display expression vector is constructed, the gene sequence of the target protein is ligated to the downstream of the Corynebacterium glutamicum protein gene sequence of the surface display expression vector by homologous recombination cloning).
The Corynebacterium glutamicum described in step (ii) is preferably Corynebacterium glutamicum ATCC 13032.
The corynebacterium glutamicum membrane protein Ncgl2775, the gene for coding the corynebacterium glutamicum membrane protein Ncgl2775 or the application of a corynebacterium glutamicum cell surface display system in preparing amylase.
The amylase is preferably an alpha-amylase.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides a corynebacterium glutamicum membrane protein Ncgl2775, which is an endogenous protein of corynebacterium glutamicum, has higher expression level in corynebacterium glutamicum, has higher display efficiency compared with endogenous anchor proteins Ncgl1221 protein and Ncgl1337 protein which are used for a surface display system of corynebacterium glutamicum, and can be used for constructing the surface display system of corynebacterium glutamicum with high display efficiency.
(2) The surface display system of the corynebacterium glutamicum is formed by fixing target proteins (such as fluorescent protein or amylase) on the cell surface of the corynebacterium glutamicum by taking corynebacterium glutamicum membrane protein Ncgl2775 as an anchor protein, so that the display efficiency of endogenous anchor proteins of the surface display system of the corynebacterium glutamicum is improved.
Drawings
FIG. 1 is a diagram showing the results of flow cytometry detection of CG/Ncgl2775-EGFP and negative control bacteria CG/EGFP and positive control bacteria CG/Ncgl1221-EGFP and CG/Ncgl 1337-EGFP.
FIG. 2 is a graph showing the results of confocal laser microscopy of CG/Ncgl 2775-EGFP.
FIG. 3 is a diagram showing the results of flow cytometry detection of CG/Ncgl2775-mCherry, negative control bacterium ATCC13032, and positive control bacteria CG/Ncgl1221-mCherry, CG/Ncgl 1337-mCherry.
FIG. 4 is a graph showing the results of confocal laser microscopy of CG/Ncgl 2775-mCherry.
FIG. 5 is a diagram showing the amylase activity of the recombinant strain (in the drawing, WT: Corynebacterium glutamicum ATCC 13032; NC: negative control strain CG/pEC-XK99 e; PC 1: positive control strain CG/Ncgl 1221-Amy; PC 2: positive control strain CG/Ncgl 1337-Amy; Ncgl 2775: recombinant strain CG/Ncgl 2775-Amy).
FIG. 6 is a graph showing the growth of recombinant strain CG/Ncgl2775-Amy and control strains ATCC13032, CG/pEC-XK99 e; wherein A is a growth curve of the strain when glucose is used as a unique carbon source; b is the growth curve of the strain when starch is used as a unique carbon source.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The following examples are given without reference to specific experimental conditions, and are generally in accordance with conventional experimental conditions. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
Interpretation of terms
LB plate: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of NaCl and 20g/L of agar.
LBH plate: 2.5g/L of yeast powder, 5g/L of peptone, 5g/L of NaCl, 18.5g/L of Brain Heart Infusion (Bacto Brain Heart Infusion), 91g/L of sorbitol and 20g/L of agar.
BHISG medium: 37g/L of brain-heart infusion, 9.1g/L of sorbitol and 10g/L of glucose.
10 × PBS (phosphate buffered saline): 40g NaCl, 1g KCl, 7.1g Na2HPO4、1.2g KH2PO4The above reagents were dissolved in 400ml of water, the pH was adjusted to 7.4, and the volume was adjusted to 500 ml).
1 × PBS: 10 × PBS dilution 10 times.
MOPS buffer solution: 10ml of 0.5M 3- (N-morpholinyl) propanesulfonic acid (MOPS) (pH 6.9) and diluted 10-fold.
Example 1: construction of surface display vector pEC-EGFP of Corynebacterium glutamicum membrane protein Ncgl2775
(1) Cloning of the membrane protein Ncgl2775 Gene from Corynebacterium glutamicum
Amplification primers were designed based on the gene sequence (SEQ ID NO:2) of the C.glutamicum membrane protein Ncgl2775 (amino acid sequence shown in SEQ ID NO: 1), the target protein EGFP gene and the sequence characteristics on the C.glutamicum plasmid pEC-XK99e (purchased from Novagen):
P1：5'-atgAAAGGAGGCCCTTCAGATGAGGAAAACCATCACCGTTAT-3'(SEQ ID NO:3)；
P2：5'-CTTATCGTCATCATCCTTGTAATCTCCGTTGTCGATGAGGTTGGTC-3'(SEQ ID NO:4)。
the gene sequence of protein Ncgl2775 was amplified by PCR using the genomic DNA of corynebacterium glutamicum ATCC13032 as template and P1 and P2 as primers under the following amplification conditions: pre-denaturation at 94 ℃ for 5 min; another 30 cycles of: denaturation at 94 deg.C for 30 seconds, Tm-5 deg.C (Tm is melting temperature; the same applies below), annealing for 30 seconds, and extension at 68 deg.C for 1 minute; and final extension at 68 ℃ for 10 min.
(2) Cloning of target protein EGFP Gene
According to the sequence characteristics of target protein EGFP gene (SEQ ID NO:5) and Corynebacterium glutamicum plasmid pEC-XK99e, an amplification primer is designed:
P3：5'-GATTACAAGGATGATGACGATAAGATGGTGAGCAAGGGCGAGGA-3'(SEQ ID NO:6)；
P4：5'-TCGTCATCATCCTTGTAATCTTACTTGTACAGCTCGTCCATG-3'(SEQ ID NO:7)。
the target protein EGFP gene is amplified by a PCR method by taking the genome DNA of Corynebacterium glutamicum ATCC13032 as a template and P3 and P4 as primers, and the amplification conditions are as follows: pre-denaturation at 94 ℃ for 5 min; another 30 cycles of: denaturation at 94 ℃ for 30 seconds, annealing at Tm-5 ℃ for 30 seconds, and extension at 68 ℃ for 1 minute; and final extension at 68 ℃ for 10 min.
(3) Cloning of vector pEC-XK99e
According to the gene sequence (SEQ ID NO:2) of the corynebacterium glutamicum membrane protein Ncgl2775, the target protein EGFP gene and the sequence characteristics on the corynebacterium glutamicum plasmid pEC-XK99e, amplification primers were designed:
P5：5'-GATTACAAGGATGATGACGATAAGggctgttttggcg-3'(SEQ ID NO:8)；
P6：5'-CTGAAGGGCCTCCTTTcatggtctgtttcctgtgtg-3'(SEQ ID NO:9)。
the gene sequence of the vector pEC-XK99e is amplified by a PCR method by taking pEC-XK99e as a template and P5 and P6 as primers, and the amplification conditions are as follows: pre-denaturation at 94 ℃ for 5 min; another 30 cycles of: denaturation at 94 ℃ for 30 seconds, annealing at Tm-5 ℃ for 30 seconds, and extension at 68 ℃ for 2 minutes; and final extension at 68 ℃ for 10 min.
(4) Construction of vector pEC/Ncgl2775-FLAG-EGFP
The PCR product of the membrane protein Ncgl2775 gene of Corynebacterium glutamicum obtained in step (1), the PCR product of the EGFP green fluorescent protein gene obtained in step (2) and the PCR product of the Corynebacterium glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination (this step uses the Seamless Assembly cloning kit), and the ligation system was transformed into the E.coli host Top10 (purchased from Novagen). Transformants were selected on LB plates containing 50mg/L kanamycin, positive transformants were picked using identifying primers P7 and P8, plasmids were extracted, identified and sequenced, which indicated that the gene sequence of the protein Ncgl2775 was correctly transformed and that the FLAG tag was upstream of the gene of said protein Ncgl 2775.
The primer sequences involved are as follows:
P7：5'-CACTGCATAATTCGTGTCGCTCAAGGCGCACTCC-3'(SEQ ID NO:10)；
P8：5'-GTCGCCGTCCAGCTCGACCAGGATG-3'(SEQ ID NO:11)。
(5) construction and identification of recombinant Corynebacterium glutamicum surface display system CG/Ncgl2775-EGFP
Transforming the plasmid pEC/Ncgl2775-FLAG-EGFP obtained in the step (4) into Corynebacterium glutamicum ATCC13032 by an electrotransformation method, picking up positive transformants on LBH plates, performing PCR amplification by using P7 and P8 as primers, and confirming that the gene sequence of the plasmid pEC/Ncgl2775-FLAG-EGFP is transformed into Corynebacterium glutamicum ATCC13032, wherein the obtained recombinant bacterium is named as CG/Ncgl 2775-EGFP.
(6) Construction and identification of negative control Strain CG/EGFP and Positive control Strain CG/Ncgl1221-EGFP, CG/Ncgl1337-EGFP of recombinant Corynebacterium glutamicum surface display System CG/Ncgl2775-EGFP
Construction of negative control Strain CG/EGFP: EGFP (SEQ ID NO:5) is taken as a template, and P9 and P4 are taken as primers, and the gene sequence of the EGFP is amplified by a PCR method; the gene sequence of pEC-XK99e is amplified by a PCR method by taking a corynebacterium glutamicum plasmid pEC-XK99e as a template and P5 and P6 as primers; then, the PCR product of the EGFP gene and the PCR product of the expression plasmid pEC-XK99e of corynebacterium glutamicum are subjected to Gibson homologous recombination and connected, and finally a negative control strain CG/EGFP is constructed, wherein the specific preparation and identification methods are shown in the steps (1) to (5);
② construction of a positive control strain CG/Ncgl 1221-EGFP: the gene sequence of Ncgl1221 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P10 and P11 as primers; then, the PCR product of the Ncgl1221 gene, the PCR product of the EGFP gene obtained in the step (2) and the PCR product of the Corynebacterium glutamicum expression plasmid pEC-XK99e obtained in the step (3) are subjected to Gibson homologous recombination and connected, and finally, a positive control strain CG/Ncgl1221-EGFP is constructed, wherein the identification method is shown in the steps (1) to (5);
construction of Positive control Strain CG/Ncgl 1337-EGFP: the gene sequence of Ncgl1337 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P12 and P13 as primers; then, the PCR product of the Ncgl1337 gene, the PCR product of the EGFP gene obtained in step (2) and the PCR product of the C.glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination, and finally a positive control strain CG/Ncgl1337-EGFP was constructed, the identification method being as in steps (1) to (5) above;
the primer sequences involved are as follows:
P9：5'-catgAAAGGAGGCCCTTCAGATGGATTACAAGGATGATGACGATAAGATGGTGAGCAAGGGCGAG-3'(SEQ ID NO:12)；
P10：5'-catgAAAGGAGGCCCTTCAGATGATTTTAGGCGTACCCATTCAATATTTG-3'(SEQ ID NO:13)；
P11：5'-CTTATCGTCATCATCCTTGTAATCAGGGGTGGACGTCGGCGCAACTGTC-3'(SEQ ID NO:14)；
P12：5'-catgAAAGGAGGCCCTTCAGATGGCTCAGCGAAAACTGGCCTC-3'(SEQ ID NO:15)；
P13：5'-CTTATCGTCATCATCCTTGTAATCGGCGTTTACTCGATCTCGCAGGATC-3'(SEQ ID NO:16)。
(7) flow cytometry analysis and confocal microscope analysis of recombinant corynebacterium glutamicum surface display system CG/Ncgl2775-EGFP
The recombinant strain CG/Ncgl2775-EGFP was inoculated into 5ml of BHISG medium containing 0.5mM IPTG (isopropyl beta-D-1-thiogalactoside), induced and cultured at 30 ℃ and 220rpm for 24 hours, resuspended after centrifugation, and resuspended after incubation with anti-FLAG monoclonal antibody and goat anti-mouse IgG antibody labeled with Alexa Fluor647 (both antibodies were purchased from Muyunyun). Finally, fluorescence intensity was measured using a C6 Plus flow cytometer. Meanwhile, CG/EGFP was used as a negative control, and CG/Ncgl1221-EGFP and CG/Ncgl1337-EGFP were used as positive controls.
The flow cytometry results are shown in FIG. 1 (M in FIG. 1 represents Comp-FL-A:: average value of APC-A _ Area): the results show that the fluorescence of the recombinant strain CG/Ncgl2775-EGFP is greatly shifted compared with the negative control strains CG/EGFP, the positive control strains CG/Ncgl1221-EGFP and CG/Ncgl1337-EGFP, and the protein Ncgl2775-EGFP fusion protein is successfully expressed on the cell surface of the recombinant strain CG/Ncgl 2775-EGFP.
Subsequently, the sample was centrifuged and resuspended, then coated on a microscope slide, and finally observed by a confocal microscope, which revealed that red fluorescence of the secondary antibody was displayed on the surface of the recombinant bacterium CG/Ncgl2775-EGFP (FIG. 2) (in FIG. 2: EGFP indicates fluorescence excited by EGFP fluorescent protein; Alexa Fluor647 indicates fluorescence excited by Alexa Fluor647 antibody), and it was confirmed again that the cell surface of the recombinant bacterium CG/Ncgl2775-EGFP successfully expressed the fusion protein of Corynebacterium glutamicum protein Ncgl 2775-EGFP.
Example 2: construction of surface display vector pEC-Ncgl2775-mCherry for Corynebacterium glutamicum membrane protein Ncgl2775
(1) Cloning of the membrane protein Ncgl2775 Gene from Corynebacterium glutamicum
Amplification primers P1 and P2 were designed based on the gene sequence of C.glutamicum membrane protein Ncgl2775 (SEQ ID NO:2), the target protein mCherry gene and the sequence characteristics of C.glutamicum plasmid pEC-XK99e, and the gene sequence of C.glutamicum membrane protein Ncgl2775 was obtained by PCR amplification, as described in example 1, step (1).
(2) Cloning of target protein mCherry Gene
Amplification primers P14 and P15 were designed based on the gene sequence of Corynebacterium glutamicum membrane protein Ncgl2775 (SEQ ID NO:2), the target protein mCherry gene (SEQ ID NO:17) and the sequence characteristics of Corynebacterium glutamicum plasmid pEC-XK99e, and the target protein mCherry gene was obtained by PCR amplification, as described in example 1.
P14：5'-GATTACAAGGATGATGACGATAAGATGGTTTCCAAGGGCGAGGAGGAC-3'
(SEQ ID NO:18)；
P15：5'-cagccCTTATCGTCATCATCCTTGTAATCTTACTTGTAGAGTTCGTCCATG-3'(SEQ ID NO:19)。
(3) Cloning of vector pEC-XK99e
Amplification primers P5 and P6 were designed based on the gene sequence of C.glutamicum membrane protein Ncgl2775 (SEQ ID NO:2), the target protein mCherry gene and the sequence characteristics of C.glutamicum plasmid pEC-XK99e, and the gene sequence of vector pEC-XK99e was amplified by PCR, as described in step (3) of example 1.
(4) Construction of vector pEC/Ncgl2775-mCherry
The PCR product of the membrane protein Ncgl2775 gene of Corynebacterium glutamicum obtained in step (1), the PCR product of the mCheerry gene obtained in step (2) and the PCR product of the Corynebacterium glutamicum expression plasmid pEC-XK99e obtained in step (3) are subjected to Gibson homologous recombination and connected (the method is the same as in example 1), and the connected system is transformed into an Escherichia coli host Top 10. Transformants were selected on LB plates containing 50mg/L kanamycin, positive transformants were picked using identifying primers P7 and P16, plasmids were extracted, identified and sequenced, resulting in recombinant C.glutamicum surface display expression plasmid pEC/Ncgl2775-mCherry with a FLAG tag upstream of the gene for the protein Ncgl 2775. The primer sequences involved are as follows:
P16：5'-CTTGTAGGTGGTCTTAACCTCAGCGTCGTAGTGACCG-3'(SEQ ID NO:20)。
(5) construction and identification of recombinant Corynebacterium glutamicum surface display system pEC/Ncgl2775-mCherry
Transforming the plasmid pEC/Ncgl2775-mCherry obtained in the step (4) into Corynebacterium glutamicum ATCC13032 by an electrotransfer method, picking up positive transformants on an LBH plate, carrying out PCR amplification by using P7 and P11 as primers, and as a result, proving that the gene sequence of the plasmid pEC/Ncgl 2775-mChery is transformed into Corynebacterium glutamicum ATCC13032, and the obtained recombinant bacterium is named as CG/Ncgl 2775-mChery.
(6) Construction and identification of negative control Strain CG/pEC-XK99e and Positive control Strain CG/Ncgl1221-mCherry, CG/Ncgl1337-mCherry of recombinant Corynebacterium glutamicum surface display System CG/Ncgl2775-mCherry
Construction of a positive control strain CG/Ncgl 1221-mCherry: the gene sequence of Ncgl1221 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P10 and P11 as primers; then, the PCR product of the Ncgl1221 gene, the PCR product of the mCherry gene obtained in step (2) and the PCR product of the C.glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination, and finally a positive control strain CG/Ncgl1221-mCherry was constructed, the identification method being as described in example 1.
② construction of a positive control strain CG/Ncgl 1337-mCherry: the gene sequence of Ncgl1337 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P12 and P13 as primers; then, the PCR product of Ncgl1337 gene, the PCR product of mCherry gene obtained in step (2) and the PCR product of Corynebacterium glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination, and finally, a positive control strain CG/Ncgl1337-mCherry was constructed, the identification method of which is described in example 1.
(7) Flow cytometry analysis and confocal microscopy analysis of recombinant Corynebacterium glutamicum CG/Ncgl2775-mCherry and control strains
The recombinant strain CG/Ncgl2775-mCherry and the control strain are inoculated into 5ml of BHISG culture medium containing 0.5mM IPTG, induced and cultured for 24 hours at 30 ℃ and 220rpm, resuspended after centrifugation, and added with anti-FLAG monoclonal antibody and Alexa Fluor 488-labeled goat anti-mouse IgG antibody (the antibodies are all purchased from Muyunyun day) for incubation and then resuspended. Finally, fluorescence intensity was measured using a C6 Plus flow cytometer. Meanwhile, the strain ATCC13032 was used as a negative control, and CG/Ncgl1221-mCherry and CG/Ncgl1337-mCherry were used as positive controls.
The results are shown in FIG. 3 (M in FIG. 3 denotes Comp-FL-A:: average value of FITC-A _ Area): the results show that the fluorescence of the recombinant strain CG/Ncgl2775-mCherry is greatly shifted compared with the negative control strain ATCC13032, the positive control strain CG/Ncgl1221-mCherry and the CG/Ncgl1337-mCherry, and indicate that the corynebacterium glutamicum protein Ncgl2775-mCherry fusion protein is successfully expressed on the cell surface of the recombinant strain CG/Ncgl 2775-mCherry.
Subsequently, the sample was centrifuged and resuspended, then coated on a microscope slide, and finally observed by confocal microscopy, which revealed that the green fluorescence of the secondary antibody was displayed on the surface of recombinant bacterium CG/Ncgl2775-mCherry (FIG. 4) (in FIG. 4: mCherry represents the fluorescence excited by mCherry fluorescent protein; Alexa Fluor647 represents the fluorescence excited by Alexa Fluor647 antibody), and it was confirmed again that the cell surface of recombinant bacterium CG/Ncgl2775-mCherry successfully expressed the fusion protein of Corynebacterium glutamicum protein Ncgl 2775-mCherry.
Example 3: construction of surface display vector pEC-Amy of Corynebacterium glutamicum membrane protein Ncgl2775
(1) Cloning of the membrane protein Ncgl2775 Gene from Corynebacterium glutamicum
Amplification primers were designed based on the gene sequence of C.glutamicum membrane protein Ncgl2775 (SEQ ID NO:2), and the gene sequence of C.glutamicum protein Ncgl2775 was obtained by PCR amplification, as described in example 1, step (1).
(2) Cloning of the target protein alpha-amylase Gene
Amplification primers P17 and P18 were designed based on the gene amyE (derived from Bacillus subtilis 168; SEQ ID NO: 21) of the target protein alpha-amylase (alpha-amylase EC 3.2.1.1), and the gene sequence of the target protein alpha-amylase was amplified by PCR using the genome of Bacillus subtilis 168 as a template, as prepared in example 1:
P17：5'-GATTACAAGGATGATGACGATAAGATGTTTGCAAAACGATTCAAAACCTCTTTACTGCC-3'(SEQ ID NO:22)；
P18：5'-CTTATCGTCATCATCCTTGTAATCTCAATGGGGAAGAGAACCGCTTAAGCCCG-3'(SEQ ID NO:23)。
(3) cloning of vector pEC-XK99e
The gene sequence of the vector pEC-XK99e was amplified by PCR method using amplification primers P5 and P6 designed based on the sequence characteristics of plasmid pEC-XK99e of Corynebacterium glutamicum, see step (3) of example 1.
(4) Construction of vector pEC/Ncgl2775-Amy
The PCR product of the membrane protein Ncgl2775 gene of Corynebacterium glutamicum obtained in step (1), the PCR product of the alpha-amylase gene obtained in step (2) and the PCR product of the expression plasmid pEC-XK99e of Corynebacterium glutamicum obtained in step (3) are subjected to Gibson homologous recombination and ligation (the method is the same as in example 1), so as to obtain the surface display expression plasmid pEC/Ncgl2775-Amy of recombinant Corynebacterium glutamicum, and the obtained pEC/Ncgl2775-Amy plasmid is transformed into an Escherichia coli host Top 10. Transformants were selected on LB plates containing 50mg/L kanamycin, positive transformants were picked, plasmids were extracted, identified and sequenced using identifying primers P7 and P19, which indicated that the gene sequence of the protein Ncgl2775 was correctly transformed and that the FLAG tag was upstream of the gene of said protein Ncgl 2775. The primer sequences involved are as follows:
P19：5'-GAACGACCAATTCCATGCATGAAGAATGGTTCCGC-3'(SEQ ID NO:24)。
(5) construction of recombinant Corynebacterium glutamicum surface display System CG/Ncgl2775-Amy
The plasmid pEC/Ncgl2775-Amy obtained in step (4) was transformed into Corynebacterium glutamicum ATCC13032 by the electrotransformation method, positive transformants were picked up on LBH plates, PCR amplification was performed using P7 and P14 as primers, and the results confirmed that the gene sequence of the plasmid pEC/Ncgl2775-Amy was transformed into Corynebacterium glutamicum ATCC13032, and the resulting recombinant strain was named CG/Ncgl 2775-Amy.
(6) Construction and identification of Positive control strains CG/Ncgl1221-Amy, CG/Ncgl1337-Amy and negative control strains CG/pEC-XK99e of the recombinant Corynebacterium glutamicum surface display System Ncgl2775-Amy
Construction of a Positive control Strain CG/Ncgl1221-Amy (PC 1): the gene sequence of Ncgl1221 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P10 and P11 as primers; then, the PCR product of the Ncgl1221 gene, the PCR product of the Amy gene obtained in step (2), and the PCR product of the Corynebacterium glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination, and finally a positive control strain CG/Ncgl1221-Amy was constructed, and the identification method was described in example 1.
② construction of a positive control strain CG/Ncgl1337-Amy (PC 2): the gene sequence of Ncgl1337 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P12 and P13 as primers; then, the PCR product of the Ncgl1337 gene, the PCR product of the Amy gene obtained in step (2), and the PCR product of the Corynebacterium glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination, and finally a positive control strain CG/Ncgl1337-Amy was constructed, as identified in example 1.
Construction of negative control Strain CG/pEC-XK99 e: the vector pEC-XK99e was directly transformed into Corynebacterium glutamicum ATCC13032, and finally a negative control strain CG/pEC-XK99e was constructed, the identification method being described in example 1.
(7) Fermentation of recombinant Corynebacterium glutamicum CG/Ncgl2775-Amy and determination of amylase Activity
CG/Ncgl2775-Amy was inoculated into 5mL of BHISG medium for overnight culture, and then transferred to 30mL of BHISG medium containing 0.5mM IPTG (resistant strain supplemented with 25. mu.g/mL kanamycin) and induced and cultured at 30 ℃ and 220rpm for 24 hours to obtain a cell fermentation broth. The determination method of amylase activity adopts EnzChekTMAn amylase detection kit (cat # E33651). The reaction substrate comprises cell suspension, supernatant and cell fermentation liquor, wherein the supernatant and the cell suspension are obtained by centrifuging the cell fermentation liquor, the cell suspension needs to be washed by MOPS buffer solution for 3 times and then resuspended, and the centrifugation condition is 6000 rpm. One unit of enzyme activity (U/ml) is defined as the amount of enzyme required to release 1 mg of maltose from starch at 20 ℃ in 3 minutes at pH 6.9. Corynebacterium glutamicum ATCC13032, (WT) and CG/pEC-XK99e (NC) were used as negative controls, and CG/Ncgl1221-Amy (PC1) and CG/Ncgl1337-Amy (PC2) were used as positive controls.
The results of the amylase activity assay are shown in FIG. 5: the results show that the enzyme activity of the cell suspension of CG/Ncgl2775-Amy after 24h fermentation reaches 0.40U/ml, which is higher than that of the two positive control bacteria CG/Ncgl1221-Amy (0.13U/ml) and CG/Ncgl1337-Amy (0.24U/ml). It was shown that the α -amylase was successfully and efficiently displayed on the cell surface of Corynebacterium glutamicum in active form by the Ncgl2775 protein.
(8) Determination of the growth Curve of the recombinant Corynebacterium glutamicum CG/Ncgl2775-Amy
The recombinant strain CG/Ncgl2775-Amy selected from the plate was inoculated into BHIS (resistant strain supplemented with kanamycin to a final concentration of 25. mu.g/mL) medium and cultured overnight at 30 ℃ and 220 rpm. The medium was inoculated with an appropriate volume of bacteria using glucose or soluble starch ((Tianjin Dacron chemical Co., Ltd.; CAS No:9005-84-9)) (4%, w/w) solution as the sole carbon source, and then cultured in 12-well plates (resistant strains supplemented with kanamycin to a final concentration of 25. mu.g/mL). The initial OD600nm was about 0.3, and the culture conditions were 30 ℃ and 280 rpm. The OD600 optical density was measured every 4 hours for 32 hours.
ATCC13032, CG/pEC-XK99e and CG/Ncgl2775-Amy strains have similar growth curves in glucose medium (FIG. 6). The cells of the negative controls ATCC13032 and CG/pEC-XK99e grew poorly in starch medium because they were unable to utilize starch. In contrast, the recombinant strain CG/Ncgl2775-Amy grew better in starch medium and grew towards similar trends in starch and glucose media, including the time point of entry into stationary phase and the maximum OD600 value. This indicates that the alpha-amylase from CG/Ncgl2775-Amy was successfully displayed in active form on the cell surface of C.glutamicum and that it has good starch utilization.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of southern China's science
<120> Corynebacterium glutamicum membrane protein Ncgl2775, and surface display system and construction method thereof
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 309
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Corynebacterium glutamicum membrane protein Ncgl2775
<400> 1
Met Arg Lys Thr Ile Thr Val Ile Ala Val Leu Ile Val Leu Ala Leu
1 5 10 15
Ile Gly Val Gly Ile Val Gln Tyr Val Asn Thr Ser Asp Asp Ser Asp
20 25 30
Phe Ile Gly Gln Pro Gly Glu Pro Thr Gly Thr Glu Thr Thr Glu Pro
35 40 45
Pro Val Gln Pro Asp Trp Cys Pro Ala Val Glu Val Ile Ala Ala Pro
50 55 60
Gly Thr Trp Glu Ser Ala Ala Asn Asp Asp Pro Ile Asn Pro Thr Ala
65 70 75 80
Asn Pro Leu Ser Phe Met Leu Ser Ile Thr Gln Pro Leu Gln Glu Arg
85 90 95
Tyr Ser Ala Asp Asp Val Lys Val Trp Thr Leu Pro Tyr Thr Ala Gln
100 105 110
Phe Arg Asn Ile Asn Ser Gln Asn Glu Met Ser Tyr Asp Asp Ser Arg
115 120 125
Asn Glu Gly Thr Ala Lys Met Asn Glu Glu Leu Ile Asn Thr His Asn
130 135 140
Glu Cys Pro Ala Thr Glu Phe Ile Ile Val Gly Phe Ser Gln Gly Ala
145 150 155 160
Val Ile Ala Gly Asp Val Ala Ala Gln Ile Gly Ser Glu Gln Gly Val
165 170 175
Ile Pro Ala Asp Ser Val Arg Gly Val Ala Leu Ile Ala Asp Gly Arg
180 185 190
Arg Glu Pro Gly Val Gly Gln Phe Pro Gly Thr Phe Val Asp Gly Ile
195 200 205
Gly Ala Glu Val Thr Leu Gln Pro Leu Asn Leu Leu Val Gln Pro Ile
210 215 220
Val Pro Gly Ala Thr Met Arg Gly Gly Arg Ala Gly Gly Phe Gly Val
225 230 235 240
Leu Asn Asp Arg Val Gln Asp Ile Cys Ala Pro Asn Asp Ala Ile Cys
245 250 255
Asp Ala Pro Val Asn Val Gly Asn Ala Leu Asp Arg Ala Leu Ala Met
260 265 270
Val Ser Ala Asn Gly Val His Ala Leu Tyr Ala Thr Asn Pro Asp Val
275 280 285
Phe Pro Gly Thr Thr Thr Asn Ala Trp Val Val Asp Trp Ala Thr Asn
290 295 300
Leu Ile Asp Asn Gly
305
<210> 2
<211> 933
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Gene sequence of Corynebacterium glutamicum membrane protein Ncgl2775
<400> 2
atgaggaaaa ccatcaccgt tattgctgta ttgatcgtcc tcgccttaat cggcgtgggc 60
atcgtgcagt atgtgaacac atccgatgac tcagatttca ttggccagcc tggcgagcca 120
accggtaccg aaaccacgga accaccggtt caacctgatt ggtgccctgc ggtagaagtc 180
attgccgcgc cgggtacgtg ggagtcggct gctaatgatg atccgatcaa cccgaccgct 240
aatccgctgt cattcatgtt gagcatcact cagccactgc aggagcgtta ttctgcggat 300
gacgtcaagg tgtggacgct gccgtacact gcgcagttcc gcaacatcaa ctcgcaaaat 360
gagatgtcct atgatgattc gcgcaatgaa ggcaccgcga agatgaatga ggaactgatc 420
aacactcaca atgagtgccc tgccacggag ttcatcatcg ttggtttctc ccagggtgcg 480
gtcattgcgg gcgatgtggc tgctcagatc ggttcagagc aaggtgttat tccagctgac 540
agcgtcaggg gtgtcgccct gatcgctgac ggtcgccggg agcctggtgt gggccagttc 600
ccaggcacgt ttgtggatgg catcggcgcg gaggttactc tgcagccttt gaacttgctg 660
gtgcagccga ttgttccggg cgcaaccatg cgtggcgggc gcgcgggcgg tttcggtgtg 720
ctcaacgacc gggtgcagga tatttgtgct ccaaatgatg cgatctgtga tgctccggtg 780
aatgtcggca acgcccttga tcgtgcgttg gccatggtct ccgccaacgg tgtgcacgcg 840
ctctacgcca ccaatccgga tgttttccca ggcacaacca ccaatgcgtg ggttgtggat 900
tgggcgacca acctcatcga caacggataa 930
<210> 3
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P1
<400> 3
atgaaaggag gcccttcaga tgaggaaaac catcaccgtt at 42
<210> 4
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P2
<400> 4
cttatcgtca tcatccttgt aatctccgtt gtcgatgagg ttggtc 46
<210> 5
<211> 720
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> EGFP Gene
<400> 5
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 742
<210> 6
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P3
<400> 6
gattacaagg atgatgacga taagatggtg agcaagggcg agga 44
<210> 7
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P4
<400> 7
tcgtcatcat ccttgtaatc ttacttgtac agctcgtcca tg 42
<210> 8
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P5
<400> 8
gattacaagg atgatgacga taagggctgt tttggcg 37
<210> 9
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P6
<400> 9
ctgaagggcc tcctttcatg gtctgtttcc tgtgtg 36
<210> 10
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P7
<400> 10
cactgcataa ttcgtgtcgc tcaaggcgca ctcc 34
<210> 11
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P8
<400> 11
gtcgccgtcc agctcgacca ggatg 25
<210> 12
<211> 65
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P9
<400> 12
catgaaagga ggcccttcag atggattaca aggatgatga cgataagatg gtgagcaagg 60
gcgag 67
<210> 13
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P10
<400> 13
catgaaagga ggcccttcag atgattttag gcgtacccat tcaatatttg 50
<210> 14
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P11
<400> 14
cttatcgtca tcatccttgt aatcaggggt ggacgtcggc gcaactgtc 49
<210> 15
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P12
<400> 15
catgaaagga ggcccttcag atggctcagc gaaaactggc ctc 43
<210> 16
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P13
<400> 16
cttatcgtca tcatccttgt aatcggcgtt tactcgatct cgcaggatc 49
<210> 17
<211> 711
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> mCherry gene
<400> 17
atggtttcca agggcgagga ggacaacatg gcaatcatca aggaattcat gcgcttcaag 60
gttcacatgg agggctccgt caacggtcac gagttcgaaa tcgagggcga gggcgaaggt 120
cgtccatacg agggcaccca gaccgctaag ctcaaggtta ctaagggcgg tccactgcct 180
ttcgcatggg acatcctctc cccacagttc atgtacggct ctaaggctta cgttaagcac 240
ccagcagata tccctgacta cctgaagctt tccttcccag agggcttcaa gtgggaacgc 300
gtcatgaact tcgaggacgg tggcgttgtt accgtcaccc aggattcctc cctccaggac 360
ggcgagttca tctacaaggt gaagctgcgt ggtaccaact tcccatctga cggccctgtt 420
atgcagaaga agactatggg ctgggaagct tcctccgagc gcatgtaccc agaggatggt 480
gcactcaagg gcgaaatcaa gcagcgtctg aagcttaagg acggcggtca ctacgacgct 540
gaggttaaga ccacctacaa ggcaaagaag ccagtccagc tccctggcgc ttacaacgtt 600
aacatcaagc tggatatcac ctcccacaac gaggactaca ctatcgttga acagtacgag 660
cgcgcagagg gccgtcactc taccggtggc atggacgaac tctacaagta a 711
<210> 18
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P14
<400> 18
gattacaagg atgatgacga taagatggtt tccaagggcg aggaggac 48
<210> 19
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P15
<400> 19
cagcccttat cgtcatcatc cttgtaatct tacttgtaga gttcgtccat g 51
<210> 20
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P16
<400> 20
cttgtaggtg gtcttaacct cagcgtcgta gtgaccg 37
<210> 21
<211> 1980
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> amyE Gene
<400> 21
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat 120
gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa ttggtcgttc 180
aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc cattcagaca 240
tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc gaactggtac 300
tggctgtatc agccgacatc gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa 360
tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc 420
atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag tattccaaac 480
tggacacatg gaaacacaca aattaaaaac tggtctgatc gatgggatgt cacgcagaat 540
tcattgctcg ggctgtatga ctggaataca caaaatacac aagtacagtc ctatctgaaa 600
cggttcttag acagggcatt gaatgacggg gcagacggtt ttcgatttga tgccgccaaa 660
catatagagc ttccagatga tggcagttac ggcagtcaat tttggccgaa tatcacaaat 720
acatctgcag agttccaata cggagaaatc ctgcaggata gtgcctccag agatgctgca 780
tatgcgaatt atatggatgt gacagcgtct aactatgggc attccataag gtccgcttta 840
aagaatcgta atctgggcgt gtcgaatatc tcccactatg catctgatgt gtctgcggac 900
aagctagtga catgggtaga gtcgcatgat acgtatgcca atgatgatga agagtcgaca 960
tggatgagcg atgatgatat ccgtttaggc tgggcggtga tagcttctcg ttcaggcagt 1020
acgcctcttt tcttttccag acctgaggga ggcggaaatg gtgtgaggtt cccggggaaa 1080
agccaaatag gcgatcgcgg gagtgcttta tttgaagatc aggctatcac tgcggtcaat 1140
agatttcaca atgtgatggc tggacagcct gaggaactct cgaacccgaa tggaaacaac 1200
cagatattta tgaatcagcg cggctcacat ggcgttgtgc tggcaaatgc aggttcatcc 1260
tctgtctcta tcaatacggc aacaaaattg cctgatggca ggtatgacaa taaagctgga 1320
gcgggttcat ttcaagtgaa cgatggtaaa ctgacaggca cgatcaatgc caggtctgta 1380
gctgtgcttt atcctgatga tattgcaaaa gcgcctcatg ttttccttga gaattacaaa 1440
acaggtgtaa cacattcttt caatgatcaa ctgacgatta ccttgcgtgc agatgcgaat 1500
acaacaaaag ccgtttatca aatcaataat ggaccagaga cggcgtttaa ggatggagat 1560
caattcacaa tcggaaaagg agatccattt ggcaaaacat acaccatcat gttaaaagga 1620
acgaacagtg atggtgtaac gaggaccgag aaatacagtt ttgttaaaag agatccagcg 1680
tcggccaaaa ccatcggcta tcaaaatccg aatcattgga gccaggtaaa tgcttatatc 1740
tataaacatg atgggagccg agtaattgaa ttgaccggat cttggcctgg aaaaccaatg 1800
actaaaaatg cagacggaat ttacacgctg acgctgcctg cggacacgga tacaaccaac 1860
gcaaaagtga tttttaataa tggcagcgcc caagtgcccg gtcagaatca gcctggcttt 1920
gattacgtgc taaatggttt atataatgac tcgggcttaa gcggttctct tccccattga 1980
<210> 22
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P17
<400> 22
gattacaagg atgatgacga taagatgttt gcaaaacgat tcaaaacctc tttactgcc 59
<210> 23
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P18
<400> 23
cttatcgtca tcatccttgt aatctcaatg gggaagagaa ccgcttaagc ccg 53
<210> 24
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> P19
<400> 24
gaacgaccaa ttccatgcat gaagaatggt tccgc 35