HYBRID EXPRESSION VECTORS, THEIR CONSTRUCTION AND USES

Abstract: The present invention provides a hybrid expression plasmid with a hybrid trp / lac ( tac ) promoter and operator region, proximally downstream of which is located a unique Eco RI restriction site. A unique Bam HI restriction site is located downstream of the Eco RI site, the tac promoter and operator region being capable of expressing DNA inserted between the Eco RI site and the Bam HI site. The plasmid further includes a selectable phenotypic marker and a functional replicon for E. coli .

Description: The present invention relates to hybrid expression vectors, their construction and uses. Expression vectors (e.g., plasmids) for the expression of inserted DNA in microorganisms (e.g., E. coli ) are known in the art, and are commercially available from several sources. Such vectors typically include a promotor and operator region including transcription and translation control signals, downstream from which region is located one or more restriction endonuclease sites which are preferably unique to the vector. The restriction site or sites are positioned downstream of the promotor and operator region to provide a location for insertion into the vector of a DNA segment of interest for expression of the DNA under the control of the promotor and operator region. In attempting to insert DNA segments into expression vectors for expression of the DNA, certain difficulties may be encountered. For example, if the promotor or other crucial region of the vector (e.g., origin of replication, phenotypic marker, etc.) is located between two unique but different restriction endonuclease sites of the vector, and a DNA segment of interest is bounded by the same two restriction sites, cutting the vector at the two unique restriction sites for insertion of the DNA segment removes the promotor region (or other critical region) from the vector and destroys the usefulness of the vector as an expression vector. Loss of the promotor (or other critical region) can be avoided with such a vector by modifying the DNA segment of interest and inserting the segment into a single unique restriction site of the vector located downstream of the promoter region. One method of modifying the DNA segment of interest involves altering the DNA segment so that the segment is bounded by identical restriction sites which match a unique restriction site downstream of the promoter region of the vector. However, difficulties are encountered when inserting a DNA segment bounded by identical restriction sites into a unique matching restriction site of a vector. One problem is that the DNA segment of interest can be inserted into the vector in two orientations, only one orientation of which can result in the desired expression product coded for by the DNA segment. Another problem is that after opening the vector at the unique restriction site, the vector can recircularize before insertion of the DNA segment of interest takes place. In accordance with the present invention, there is provided the plasmid designated pML3 (as present in E. coli strain JM103 (pML3) deposited with the American Type Culture Collection, Rockville, Maryland as ATCC. No. 53377). Such a hybrid plasmid comprises a hybrid trp/lac ( tac ) promoter and operation region including transcription and translation control signals, proximally downstream of which region is located a unique Eco RI restriction site, downstream of which Eco RI site is located a unique Bam HI restriction site. The plasmid further comprises a selectable phenotypic marker and a functional replicon for E. coli , the promoter and operator region being capable of expressing DNA inserted between the Eco RI site and the Bam HI site. The invention further relates to microorganisms transformed by such a hybrid plasmid, a method for expressing DNA utilizing such a hybrid plasmid, and a method for constructing such a hybrid plasmid. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of plasmid starting material which can be used to construct a vector according to the present invention. FIG. 2 is a schematic diagram showing deletion of an Eco RI site from the plasmid shown in FIG. 1. FIG. 3 is a schematic diagram illustrating restriction site conversion of the plasmid shown in FIG. 1 after removal of the Eco RI site. DESCRIPTION OF THE PREFERRED EMBODIMENTS A suitable starting material for construction of a hybrid expression plasmid according to the present invention is plasmid pDR540, which is commercially available from PL Biochemicals, Inc., Milwaukee, Wisconsin. See Fig. 1. While the invention will be further described with reference to plasmid pDR540 as the starting material, it is to be understood that any suitable plasmid possessing similar characteristics as pDR540 can be used as a starting material. Plasmid pDR540 is a multicopy expression vector derived from plasmid pK0-1 (McKenney, K. et al, Gene Amplification and Analysis , Vol. II (Chirikjian, J.G. and Papas, T., ed.) Elsevier, North Holland, p. 383 (1981)). Plasmid pDR540 includes a trp/lac ( tac ) promotor and operator region proximally downstream from which is located a Bam HI site unique to the plasmid (deBoer, H. et al., Proc. Natl. Acad. Sci. USA , 80 :21 (1983) and Russel, D.R. and Bennett, G.N., Gene , 20 :321 (1982). The unique Bam HI site of pDR540 is in position for expression of DNA inserted therein under the control of the hybrid tac promotor and operator region. Plasmid pDR540 includes an expressible nucleotide sequence coding for Gal K downstream of the unique Bam HI site, the Gal K sequence being under the control of the tac promoter. Plasmid pDR540 has a unique Eco RI site upstream of the tac promoter, and a selectable phenotypic maker in the form of an expressible nucleotide sequence coding for amp r , the amp r sequence being upstream of the Eco RI site and the tac promoter. The amp r sequence has an opposite direction of transcription relative to the Gal K sequence. Plasmid pDR540 also includes a functional replicon for E. coli , permitting replication of the plasmid in E. coli independent of chromosomal replication. Plasmid pDR540 is not particularly well suited for the insertion and expression of DNA segments bounded by an Eco RI site and a Bam HI site (such as a growth hormone-releasing factor (GRF) gene bounded by a 5′ Eco RI site and a 3′ Bam HI site). Endonuclease digestion of pDR540 with Eco RI and Bam HI cuts the hybrid tac promoter region from the plasmid and destroys the plasmid's usefulness as an expression vector. A hybrid plasmid according to the present invention can be constructed using plasmid pDR540 by first removing the unique Eco RI restriction site located upstream of the hybrid tac promoter. See Fig. 2. According to one embodiment, the Eco RI site is removed from pDR540 by linearizing the plasmid with restriction endonuclease Eco RI, filling in the resultant sticky ends (Klenow fragment) to form a flush (blunt) ended molecule, and recircularizing the plasmid by bluntend ligation to form pDR540 (- Eco RI). In a second embodiment, the unique Eco RI site of pDR540 is removed by linearizing the plasmid with Eco RI, chewing away the sticky ends with an exonuclease to produce a flush-ended molecule, and recircularizing the plasmid by blunt-end ligation to form pDR540 (- Eco RI). Following the removal of the Eco RI site from pDR540, the Bam HI site downstream of the hybrid tac promoter is modified to form an Eco RI- Bam HI site. This can be accomplished by inserting a suitable DNA segment into the Bam HI site of pDR540 (- Eco RI). A suitable DNA segment for insertion into the Bam HI site of pDR540 (- Eco RI) is bounded on its 5′ end by a cut Sau 3A restriction site, and bounded on its 3′ end by a cut Bam HI restriction site. The segment also includes an intact and unique Eco RI restriction site which preferably is adjacent to or in close proximity to the 5′ cut Sau 3A site. The 5′ end of the Sau 3A- Bam HI segment has a sticky end which is complementary to the 3′ end of Bam HI linearized pDR540 (- Eco RI). However, a Sau 3A restriction site cannot be recognized and cut by Bam HI restriction endonuclease. Thus, by inserting the Sau 3A- Bam HI segment into the Bam HI site of pDR540 (- Eco RI), a Sau 3A site is created at the 3′ portion of the Bam HI site of pDR540 (- Eco RI) which is not recognized by Bam HI. However, the Bam HI site at the 5′ portion of the Bam HI site of pDR540 (- Eco RI) remains intact. Insertion of the Sau 3A- Bam HI segment into the Bam HI site of pDR540 (- Eco RI) provides the plasmid with a unique Eco RI restriction site proximally downstream from the tac promoter, and with a unique Bam HI site located downstream of the inserted Eco RI site. A suitable Sau 3A- Bam HI segment can be constructed using a GRF gene (supra) bounded 5′ by an Eco RI site and bounded 3′ by a Bam HI. See Fig. 3. A pair of Eco RI- Sau 3A convertors having the sequence AATTCGATCG are ligated to the 5′ end of an Eco RI-cut GRF gene. The convertor DNA then is cut with Sau 3A and the 3′ end of the GRF segment is cut with Bam HI. The Sau 3A- Bam HI GRF segment is then inserted into the Bam HI site of pDR540 (- Eco RI) to form plasmid pML3. Plasmid pML3 has a unique inserted Eco RI site 36 base pairs downstream of the tac promoter, and a unique Bam HI site downstream of the inserted Eco RI site. Plasmid pML3 has been used to transform E. coli strain JM103, a lac I Q host (Δ ( lac i pro ), thi , str A, sup E, end A, sbc B, hsd R, F′[ tra D36, pro AB, lac i Q , ZΔM15]), the transformed strain being designated Bacterium JM103 (pML3) and deposited with American Type Culture Collection, Rockville, Maryland as ATCC No. 53377. E. coli cells transformed by pML 3 can be cultivated to express the GRF gene. Furthermore, any DNA segment which is flanked by Eco RI and Bam HI can be inserted into Eco RI/ Bam HI-cut pML3 according to the present invention. Such insertions are unidirectional, unambiguous and take place without recircularization of the plasmid. A DNA segment so inserted is located proximally downstream of the hybrid tac promoter for optimal expression of the inserted DNA by transformed E. coli microorganisms which are cultivated under protein-producing conditions (e.g., induction by IPTG or X-gal). The invention is further illustrated by the following examples which are not intended to be limiting. Example I Elimination of the EcoRI site of Plasmid pDR540 (Fig. 2) Plasmid pDR540 ( supra ) was linearized with restriction endonuclease Eco RI (10,000 units/ml; Bethesda Research Laboratories) according to manufacturer's instructions to produce linearized DNA molecules have recessed (sticky) ends. The Eco RI site of Eco RI linearized pDR540 was removed by each of methods (a), (b) and (c) below. (a) Polymerization of Recessed Ends of EcoRI linearized pDR540 1 µg of Eco RI linearized pDR540 DNA with recessed ends was mixed with 5 µ l of the 10 x nick translation buffer (0.5 M Tris-HCl pH 7.2, 0.1 M MgSO4, 1 mM DTT (Dithiothreitol, ultra pure, International Biotechnologies Inc.), 0.5 mg/ml BSA (bovine serum albumin, Sigma), 2 nM of each of dATP and dTTP (the deoxynucleotides necessary to fill in the recessed 3′ ends resulting from Eco RI digestion), 1 unit/µg DNA Klenow fragment ( E. coli DNA polymerase I Klenow, 4500 units/ml, Pharmacia), and sterile water, to a volume of 50 µl 0.5M EDTA. The flush-ended DNA was extracted with phenol/chloroform, and precipitated by making mix 2M in acetate and adding 2 volumes of isopropanol, incubating the mixture at room temperature for 10 min., spinning the mixture in microfuge for 10 min., air-drying the precipitated pellet, and resuspending the pelletted DNA in water. (b) Mung Bean Nuclease Digestion of EcoRI Linearized pDR540 1 µg of Eco RI linearized pDR540 DNA with recessed ends was mixed with 25 mM NH₄OAc pH 5.0, and 2 units of mung bean nuclease (Mung bean nuclease 100,000 units/ml P-L Biochemicals) for 30 min. at 30°C. The reaction was stopped by adding 110 mM K phosphate pH 70, 100 mM NaCl, and heating at 72°C for 15 min., followed by chilling to 0°C. The flush-ended DNA was then ethanol precipitated, microfuged to form a pellet, which was air-dried, and the pellet was resuspended in water. (c) Sl Nuclease Digestion of EcoRI Linearized pDR540 1 µg of Eco RI linearized pDR540 was incubated in 0.03 M Na acetate, pH 4.6, 0.3 M NaCl, 4.5 mM ZnCl, and 5 units S1 nuclease (S1 nuclease, 250,000 units/ml, BRL) at 22°C for 30 min. followed by 15 min. at 10°C. The digestion was stopped by addition of Tris base to 0.1 M, EDTA to 25 mM. The flush-ended DNA was then ehtanol precipitated, microfuged to form a pellet which was air-dried, and the pellet was resuspended in water. (d) Blunt End Ligation 1µg of the resuspended flush-ended DNA in water from procedures (a), (b) and (c) above were individually mixed with 10x ligase buffer (0.3 M Tris-HCl, pH 8.0, 40 mM MgCl₂, 10 mM EDTA, 0.1 M DTT, 0.5 mg/ml BSA), 0.5 mM ATP, 5 units DNA ligase/µg DNA ( E. coli T4 DNA ligase, 3000/ml units; P-L Biochemicals), and sterile water to obtain final DNA concentrations of 0.5-1 µg DNA/20 µl mix. The ligation mixtures were incubated overnight at 14°C. The mixtures were adjusted to 0.25 M in Na acetate, and the recircularized DNA was precipitated from each mixture with 2.5 volumes EtOH. The precipitated pellets of recircularized DNA from procedures a, b and c were then individually resuspended in water. (e) Transformation DNA suspensions from procedures (a), (b) and (c) above which had been recircularized according to procedure (d) above were individually digested with Eco RI which site was to be deleted from the DNA, to eliminate any plasmids still containing the site. The digestion mixes were individually isopropanol precipitated by making them 20 mM in EDTA, 2 M in ammonium acetate, adding 2 volumes isopropanol, and incubating at room temperature for 10 min. The mixtures were spun at room temperature for 5 min., air dried, and resuspended in small volumes of TE (1 µg DNA/10 µl TE:10 mM Tris-HCl pH 7.9, 1 mM EDTA). E. coli cells were transformed with the samples, colonies were harvested, and plasmid DNA was screened for deletion of the Eco RI restriction endonuclease site by restriction mapping. Example II Confirmation of deletion of the EcoRI site from pDR540 Flush-ended DNA from Example I(a) which had been blunt-end ligated as in Example I(d) was sequenced according to the procedure of Maxam and Gilbert, Proc. Natl. Acad. Sci. USA , 74 :560 (1977) and the sequence was confirmed by the dideoxy sequencing procedure of Sanger et al., Proc. Acad. Sci. USA , 74 : 5403 (1977). The data confirmed the deletion of the Eco RI site by polymerase I Klenow fragment (Example I(a)). However, instead of the expected sequence with the doubling of the Eco RI sticky ends, the polymerase had degraded one nucleotide from one end to give the sequence -GAATAATTC- instead of the expected -GAATTAATTC-. This deletion does not affect the functioning of the plasmid since it is not in a reading frame and it is not in an otherwise important part of the plasmid. The plasmid is designated pDR540 (- Eco RI). Example III Conversion of BamHI site of pDR540 (-EcoRI) to an EcoRI-BamHI site (Fig. 3) Plasmid pUC9 Vieira et al., Gene , 19 :259 (1982) and commercially available from Pharmacia P-L Biochemicals, containing a nucleotide sequence coding for GRF ( supra ) bounded by a 5′ Eco RI site and a 3′ Bam HI site was cut out of pUC9 by digestion with Eco RI and Bam HI endonucleases under the following conditions: 1 µg of pUC9 DNA in high salt buffer (10 mM Tris-HCl (pH 8.0), 100 mN NaCl, 10 mM MgCl₂, 1 mM β-mercaptoethanol, 100 µg BSA/ml) was mixed with 1 unit Eco RI and 1 unit Bam HI. The mixture was incubated one hour at 37°C, and the cut Eco RI- Bam HI GRF segment was isolated by gel electrophoresis and electroelution. Dephosphorylated Eco RI- Sau 3A convertors having the sequence AATTCGATCG were phosphorylated as follows: 1 µg dephosphorylated convertor DNA was mixed with 10 µl 10 x kinase buffer I (0.5 M Tris-Cl (pH 7.6), 0.1 M MgCl₂, 50 mM dithiothreitol, 1 mM spermidine, 1 mM EDTA), 50 pmoles (200 µ Ci) [γ-³²P] ATP (sp. act. = 3,000 in/mmole), 10 units T4 polynucleotide kinase and H₂O to 50 µl. The mixture was incubated at 37°C for 30 minutes. To the incubated mixture was added 2 µl of 0.5 M EDTA, convertor DNA was extracted once from the mixture with phenol/chloroform, and the convertor DNA was precipitated with ethanol. The ethanol precipitated DNA was redissolved in 50 µl of TE (pH 7.9), and phosphorylated convertor DNA was separated from the redissolveed DNA by centrifugation through small columns of Sephadex G-50. The Eco RI- Bam HI cut GRF DNA was mixed with phosphorylated convertor DNA to ligate the convertors to the GRF DNA under the conditions set forth in Example I(d) except that 1 unit DNA ligase/µg DNA was present in the reaction mixture. The GRF gene with convertor attached was digested with Sau 3A and Bam HI enzymes, under the same conditions set forth above with respect to the Eco RI/ Bam HI double digest of pUC9, but with Sau 3A enzyme substituted for Eco RI enzyme, to give a GRF gene segment bounded by a 5′ cut Sau3A site and a 3′ cut Bam HI site, with an intact Eco RI site adjacent the Sau 3A site (termed Sau 3A- Bam HI GRF segment). Plasmid pDR540 (- Eco RI) (Example II, supra ) was linearized with 1 unit Bam HI enzyme/µg DNA (per manufacturer's instructions), and the linearized DNA was dephosphorylated as follows: Bam HI linearized plasma was extracted once with phenol/chloroform and precipitated with ethanol. The precipitated DNA was dissolved in a minimum volume of 10 mM Tris-Cl (pH 8.0), to which was then added 5 µl 10x CIP (calf intestional alkaline phosphatase) buffer (0.5 M Tris-Cl (pH 9.0), 10 mM MgCl₂, 1 mM ZmCl₂, 10 mM spermidine), 0.01 unit CIP and H₂O to 48 µl. The mixture was incubated at 37°C for 30 minutes, a second aliquot of 0.01 unit CIP was added to the mixture, and the incubation was continued an additional 30 minutes. To the incubated mixture was then added 40 µl H₂O, 10 µl 10x STE (100 mM Tris-Cl (pH 8.0), 1 M NaCl, 10 mM EDTA) and 5 µl 10% SDS. This mixture was then incubated at 68°C for 15 minutes. Dephosphorylated DNA was extracted twice with phenol/chloroform and twice with chloroform, then precipitated with ethanol. 1 µg of Bam HI cut dephosphorylated pDR540 (- Eco RI) was mixed with 1 µg Sau 3A- Bam HI GRF segment and 2 units DNA ligase to ligate the segments and form plasmid pML3. The ligation conditions were otherwise as set forth above for ligation of convertor DNA to Eco RI cut GRF DNA. Plasmid pML3 has a unique inserted Eco RI site 35 base pairs downstream of the tac promoter and a unique Bam HI site downstream of the inserted Eco RI site. E. coli strain JM103 has been transformed by plasmid pML3 and deposited with American Type Culture Collection, Rockville, Maryland as ATCC No. 53377.

Claims: Claims for the following Contracting States : BE, CH, DE, FR, GB, GR, IT, LI, LU, NL, SE The plasmid designated pML3 (as present in E. coli strain JM103 (pML3) deposited with the American Type Culture Collection, Rockville, Maryland as ATCC. No. 53377). A microorganism transformed by the plasmid as claimed in claim 1. A method for expressing a DNA sequence comprising inserting said DNA sequence between the Eco RI site and the Bam HI site of a plasmid as claimed in claim 1, transforming a microorganism with said plasmid and cultivating the microorganism so as to express said DNA sequence. A method for expressing a GRF gene comprising cultivating a microorganism transformed with the plasmid of claim 1 so as to express the GRF gene therein present A method for constructing a hybrid plasmid as claimed in claim 1 characterised in that a plasmid comprising a hybrid trp / lac ( tac ) promoter and operator region including transcription and translation control signals, proximally downstream of which region is located a unique Bam HI restriction site, the plasmid further comprising a selectable phenotypic marker and a functional replicon for E.coli , is treated to insert into said Bam HI site a DNA sequence bounded by a 5' cut Sau 3A restriction site and a 3' cut Bam HI restriction site and including a unique Eco RI restriction site. A method as claimed in claim 5 comprising: (a) linearizing a plasmid with restriction endonuclease Eco RI to produce sticky ends, the plasmid having a unique Eco RI restriction site located upstream of a hybrid trp / lac ( tac ) promoter and operator region including transcription and translation control signals, a unique Bam HI restriction site proximally downstream of said region, this region being capable of expressing DNA inserted into the Bam HI site, the plasmid further comprising a selectable phenotypic marker and a functional replicon for E. coli ; (b) blunting the sticky ends to provide a linearized plasmid with flush ends; (c) blunt-end ligating the linearized plasmid of step (b) to provide a circularized plasmid without an Eco RI site; and (d) inserting into the Bam HI site of the circularized plasmid of step (c) a DNA segment bounded by a 5' cut Sau 3A restriction site and a 3' cut Bam HI restriction site and including a unique Eco RI restriction site. A method as claimed in claim 5 or claim 6, wherein the starting plasmid and the final hybrid plasmid further comprise an expressible nucleotide sequence coding for Gal K downstream of said Bam HI site, the Gal K sequence being under the control of said promoter and operator region. A method as claimed in claim 6, wherein the selectable phenotypic marker of the starting plasmid and the final hybrid plasmid is an expressible nucleotide sequence coding for amp r , said amp r sequence being upstream of said promoter and having an opposite direction of transcription from the Gal K sequence. A method as claimed in any one of claims 5 to 8, wherein the Eco RI site of the final hybrid plasmid is about 36 base pairs downstream of said promoter. A method as claimed in claim 9 wherein the final hybrid plasmid is pML3 (present in E. coli strain JM103 (pML3), deposited with the American Type Culture Collection, Rockville, Maryland as ATCC No. 53377). Claims for the following Contracting States : AT, ES A method of constructing a hybrid plasmid for expressing a DNA sequence characterised in that a plasmid comprising a hybrid trp / lac promoter and operator region including transcription and translation control signals, proximally downstream of which region is located a unique Bam HI restriction site, the plasmid further comprising a selectable phenotypic marker and a functional replicon for E.coli , is treated to insert into said Bam HI site a DNA sequence bounded by a 5' cut Sau 3A restriction site and a 3' cut Bam HI restriction site and including a unique Eco RI site so as to provide a hybrid plasmid designated pML3 (as present in E.coli strain JM103 (pML3), deposited with the American Type Culture Collection, Rockville, Maryland as ATCC No. 53377), and having a unique Eco RI restriction site proximally downstream of said promoter and a unique Bam HI site downstream of said Eco RI site, said promoter and operator region being capable of expressing DNA inserted between the Eco RI and Bam HI sites. A method as claimed in claim 1 comprising: (a) linearizing a plasmid with restriction endonuclease Eco RI to produce sticky ends, the plasmid having a unique Eco RI restriction site located upstream of a hybrid trp / lac ( tac ) promoter and operator region including transcription and translation control signals, a unique Bam HI restriction site proximally downstream of said region, this region being capable of expressing DNA inserted into the Bam HI site, the plasmid further comprising a selectable phenotypic marker and a functional replicon for E. coli ; (b) blunting the sticky ends to provide a linearized plasmid with flush ends; (c) blunt-end ligating the linearized plasmid of step (b) to provide a circularized plasmid without an Eco RI site; and (d) inserting into the Bam HI site of the circularized plasmid of step (c) a DNA segment bounded by a 5' cut Sau 3A restriction site and a 3' cut Bam HI restriction site and including a unique Eco RI restriction site. A method as claimed in claim 1 or claim 2, wherein the starting plasmid and the final hybrid plasmid further comprise an expressible nucleotide sequence coding for Gal K downstream of said Bam HI site, the Gal K sequence being under the control of said promoter and operator region. A method as claimed in claim 3, wherein the selectable phenotypic marker of the starting plasmid and the final hybrid plasmid is an expressible nucleotide sequence coding for amp r , said amp r sequence being upstream of said promoter and having an opposite direction of transcription from the Gal K sequence. A method as claimed in any one of claims 1 to 4 wherein the Eco RI site of the final hybrid plasmid is about 36 base pairs downstream of said promoter. A method as claimed in any one of claims 1 to 5 wherein the starting plasmid and the final hybrid plasmid are multicopy plasmids. A microorganism transformed by the plasmid pML3 and designated E. coli strain JM103 (pML3), deposited with the American Type Culture Collection, Rockville, Maryland as ATCC No. 53377. A method for expressing a DNA sequence comprising inserting said DNA sequence between the Eco RI site and the Bam HI site of a plasmid obtainable by a method as claimed in any one of claims 1 to 6, transforming a microorganism with said plasmid and cultivating the microorganism so as to express said DNA sequence. A method for expressing a GRF gene comprising cultivating a microorganism transformed with the plasmid designated pML3 (as deposited in E. coli strain JM103 (pML3) with the American Type Culture Collection, Rockville, Maryland as ATCC No. 53377) so as to express the GRF gene therein present.

Inventors: MACCECCHINI, MARIA-LUISA
Applicants: INTERNATIONAL MINERALS AND CHEMICAL CORPORATION
Application year: 1987
Main classification: C12N15
IPC codes: C12N15, C07K14, C12R1, C12P21, C12N1