Improved plasmid multi-site directed mutagenesis method based on polymerase chain reaction (PCR)

Abstract

The invention discloses an improved plasmid site-directed mutagenesis method based on PCR (Polymerase Chain Reaction). The method comprises the following steps: introducing at least two mutations on a plasmid DNA (Deoxyribose Nucleic Acid) fragment through site-directed mutagenesis; wherein one mutation is expected mutation, and the other mutation changes the function of the mutant plasmid, so that the function of the mutant plasmid is different from that of a template plasmid. The change in function makes it possible to reduce the detention of unintegrated template DNA even without digesting the wild-type template with DpnI.

Description

[0001] This application contains a Sequence Listing submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file was created on February 26, 2024, is named Zhu-SDM.xml, and is 45,000 bytes in size. BACKGROUND

[0002] Site-directed mutagenesis (SDM) is a technique to change DNA sequences in a directed, non-random fashion, including substitutions, deletions, insertions, or combinations of these changes. In SDM, a DNA primer with the desired mutation is first synthesized, and the rest of the primer is complementary to the template DNA around the mutation site, so it can hybridize to the DNA in the target gene. The mutation can be a single base change (point mutation), multiple base changes, deletion, or insertion. Then, a DNA polymerase is used to extend the single-stranded primer to replicate the rest of the gene. The replicated gene contains the mutation site, which can then be introduced into a host cell using a vector and cloned. Mutants can be screened by DNA sequencing to confirm that they contain the desired mutation. Plasmids are commonly used targets for site-directed mutagenesis.

[0003] A recent paper (1) summarizes the development of PCR-based SDM, as shown in Table 1 below: Table 1: PCR-based SDM

[0004] The above methods can be used in combination, for example, in the C method, the primers are not complementary, and two or more pairs of primers can be used to phosphorylate and ligate the PCR products, and then cell transformation. More than two pairs of primers can also be used in the E method.

[0005] If the starting template plasmid is, for example, fully methylated N6A, the restriction enzyme DpnI (which digests methylated DNA at Gm6ATC sites) can be used to conveniently remove the methylated template DNA without affecting the unmethylated DNA product produced by PCR. This use of DpnI is a key step in the above-mentioned B, C, D, and E mutagenesis methods, especially in the B and E methods. However, it is known that DpnI digests hemimethylated DNA (i.e., the product of the first round of PCR) very slowly (2). The remaining template remains unchanged (methylated wild type). In addition to the problem of digesting hemimethylated DNA, DpnI activity decreases during storage and has poor compatibility with certain polymerase buffers. These factors can significantly increase the amount of wild-type DNA in the final mixture.

[0006] Thus, a more efficient method is needed to reduce the amount of wild-type DNA in the final mixture so that the final mixture contains almost only the PCR generated unmethylated DNA product. SUMMARY

[0007] In the present invention, at least one functional switch (e.g., addition of a selectable marker) and at least one addition mutation are simultaneously achieved in two PCR product recombination stages on plasmid DNA by site-directed mutagenesis ("SDM") and PCR using primer pairs directed to the functional switch and the addition mutation. After PCR, one (or more) segment(s) of the plasmid DNA contains the mutation of the functional switch and one (or more) segment(s) of the plasmid DNA contains the addition mutation. PCR of the different plasmid DNA segments is performed in different reactions using different primer pairs, followed by recombination of the PCR products, and then transformation of the target cells with the PCR products. Recombination can be achieved by ligation or mixing of the PCR products. After recombination, the transformed cells are screened for the functional change. Two examples of the fuGFP fluorescent on-off switch are described in detail below.

[0008] The present invention is particularly useful for finding polynucleotides with specific mutations in plasmids, including immediately applicable mutations such as protein engineering mutations, restriction enzyme mutations, or mutations that encode a protein of interest. At least the addition of a functional switch with a mutation is described in more detail below, where both the functional switch and the mutation are added by site-directed mutagenesis techniques. This technique can be readily applied to either bacterial or eukaryotic cells to make large quantities of DNA that encodes any protein of interest or has a sequence of interest itself. For more background information on site-directed mutagenesis techniques, see U.S. Patent No. 5,556,747 (incorporated herein by reference). BRIEF DESCRIPTION OF DRAWINGS

[0009] FIGS. 1A-1C show the various stages of ampicillin resistance restoration. FIG. 1A shows the structure of the starting template plasmid. FIG. IB shows the PCR reaction using ampicillin resistance gene primers. FIG. 1C shows the structure of the assembled mutant plasmid.

[0010] FIGS. 2A-2C show the various stages of simultaneous site-directed mutagenesis and alteration of ampicillin resistance. Figure 1 A shows the starting template plasmid. FIG. IB shows the PCR reactions using two different pairs of primers, one from site-directed mutagenesis and one from the ampicillin resistance gene; the dashed and dotted lines represent the two independent PCR products; Figure 1 C shows the mutant plasmid with the site-directed mutation and the ampicillin resistance restoration gene.

[0011] Figures 3A-3D show the plasmids used in mutagenesis experiments, where the asterisk in Figure 3B indicates the serine residue at amino acid position 68 in NHfuGFP. Figure 1A shows the structure of plasmid pKaLNHfuGFPWT. Figure IB shows the structure of plasmid pALNHfuGFPG68S. Figure 1C shows the structure of plasmid pKALNHfuGFPG68S. Figure ID shows the structure of plasmid pALNHfuGFP wild type.

[0012] Figure 4A shows the products obtained after mutagenesis of plasmid pKaLNHfuGFPWT using PCR, where the products were run on an agarose gel after the first and second rounds of PCR.

[0013] Figure 4B shows a photograph of an agar plate of mutagenized colonies obtained after transformation of cells with the products of Figure 4A.

[0014] Figure 5A shows the mutagenized products obtained after PCR amplification of plasmid pKaLNHfuGFPG68S, where the products of both PCR reactions were run on an agarose gel.

[0015] Figure 5B shows a photograph of an agar plate of mutagenized colonies obtained after transformation of cells with the products of Figure 5A. DETAILED DESCRIPTION

[0016] The present application utilizes site-directed mutagenesis to change the function of the mutant produced by PCR amplification from a template plasmid, and to change one or more additional sites on the mutant. In this way, after transformation of cells with the mutant plasmid, the mutant / PCR product can be removed or distinguished from the template plasmid by screening for the change in function in the transformed cells, without the need for Dpnl digestion. The two PCR reactions (or more, for example, using additional primer pairs to introduce more mutations) described herein, followed by screening, can be used to isolate large numbers of target mutant DNAs.

[0017] For example, using the site-directed mutagenesis technique in two separate PCR reactions, one PCR reaction using primers for the original site-directed mutagenesis ("SDM") and the other PCR reaction using primers directed to a specific sequence region that can change function, i.e., add a selectable marker to the product. The mutagenesis methods described herein are applicable to the C, D, or E type mutagenesis in Table 1 above. Table 2 lists some of the products of this mutagenesis method and their properties and how these properties can be used for selection. Using the techniques of the present application, multiple functional changes can be introduced into the PCR product by using primers with additional functional changes or by using the same primers but introducing an additional differential sequence that represents an additional functional change using the template plasmid DNA as a template.

[0018] Table 2: Functional changes made simultaneously with SDM

[0019] The cells transformed in Table 2 can be any cell that has the ability to be transformed and to clone. More specifically, the plasmid (template or mutant) can be expressed in any suitable host system, including bacterial, yeast, fungal, baculovirus, plant, or mammalian host cells. For bacterial host cells, promoters suitable for directing transcription of the nucleic acid constructs of the present disclosure include the E. coli lactose operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levan sucrase gene (sacB), the B. licheniformis alpha-amylase gene (amyL), the B. stearothermophilus maltogenic amylase gene (amyM), the B. amyloliquefaciens alpha-amylase gene (amyQ), the B. licheniformis penicillinase gene (penP), the B. subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., (1978) Nucleic Acids Res. 5:3727-3731) as well as the tac promoter (DeBoer et al., (1983) Proc. Natl. Acad. Sci. USA 80:21-25).

[0020] For filamentous fungal host cells, promoters which find use in directing transcription of the nucleic acid constructs of the present disclosure include promoters derived from Aspergillus niger TAKA amylase, Aspergillus awini aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awini glucoamylase (glaA), Aspergillus awini lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Trichoderma reesei pantain (WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof.

[0021] In yeast host cells, useful promoters include those from the gene encoding yeast enolase (ENO-l), yeast galactokinase (GALl), alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and yeast 3-phosphoglycerate kinase. Additional promoters which find use in yeast host cells are described in Romanos et al., 1992, Yeast 8:423-488, “Expression of foreign genes in yeast: a review.”

[0022] For baculovirus expression, insect cell lines derived from lepidopteran insects, such as Spodoptera frugiperda, are typically used as hosts. Gene expression is controlled by strong promoters, such as pPolh.

[0023] Plant expression vectors are based on the Ti plasmid of Agrobacterium tumefaciens, or on the tobacco mosaic virus (TMV), potato virus X, or cowpea mosaic virus. The cauliflower mosaic virus (CaMV) 35S promoter is a commonly used constitutive promoter in plant expression vectors.

[0024] For mammalian expression, cultured mammalian cell lines (e.g., Chinese hamster ovary cells (CHO), COS cells, and human cell lines (e.g., HEK and HeLa cells)) can be used to express the labeled restriction enzymes. Examples of mammalian expression vectors include adenovirus vectors, pSV and pCMV series plasmid vectors, vaccinia virus, and retroviral vectors, and baculovirus. Promoters from cytomegalovirus (CMV) and SV40 are commonly used in mammalian expression vectors to drive gene expression. Non-viral promoters, such as the elongation factor (EF)-1 promoter, have also been found.

[0025] The control sequence can also be a suitable transcription terminator sequence, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice can be used.

[0026] For example, typical transcription terminators in filamentous fungal host cells can be derived from genes of Aspergillus oryzae TAKA amylase, Aspergillus niger glucosylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease.

[0027] Typical terminators for yeast host cells can be obtained from the genes of *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Terminators for insect, plant, and mammalian host cells are also well known.

[0028] Control sequences can also be suitable guide sequences, i.e., untranslated regions of mRNA that are crucial for translation in the host cell. Guide sequences are operatively linked to the 5' end of the nucleic acid sequence. Any guide sequence that functions in the target host cell can be used. Typical guide sequences for filamentous fungal host cells are derived from the *Aspergillus oryzae* TAKA amylase gene and the *Aspergillus nidus* triose phosphate isomerase gene. Suitable guide sequences for yeast host cells are derived from the *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* 3-phosphoglycerate kinase, *Saccharomyces cerevisiae* α-factor, and *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP) genes.

[0029] The control sequence can also be a polyadenylated sequence, which is operatively linked to the 3' end of a nucleic acid sequence and recognized by the host cell during transcription as a signal to add polyadenylated residues to the transcribed mRNA. This invention can use any polyadenylated sequence that is functional in a selected host cell. Exemplary polyadenylated sequences for filamentous fungal host cells can be derived from the genes of *Aspergillus oryzae* TAKA amylase, *Aspergillus niger* glucosyl amylase, *Aspergillus nidus* o-aminobenzoic acid synthase, *Fusarium oxysporum* trypsin-like protease, and *Aspergillus niger* α-glucosidase.

[0030] The control sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the N-terminus of the polypeptide and guides the encoded polypeptide to the cellular secretion pathway. The 5' end of the coding sequence may naturally contain a signal peptide coding region, which is naturally linked to the coding region encoding the secreted polypeptide within the translation reading frame. Alternatively, the 5' end of the coding sequence may contain a foreign signal peptide coding region. When the coding sequence itself does not contain a signal peptide coding region, it may be necessary to introduce this foreign signal peptide coding region.

[0031] Alternatively, the coding region of the natural signal peptide can be directly replaced with the coding region of the exogenous signal peptide to enhance peptide secretion. However, any signal peptide coding region that can guide the expressed peptide into the secretory pathway of the target host cell can be used.

[0032] Effective signal peptide coding regions in bacterial host cells include those from Bacillus NCIB 11837 maltodextrinase, Bacillus stearothermophilus α-amylase, Bacillus subtilis protease, Bacillus stearothermophilus β-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and the Bacillus subtilis prsA gene. Descriptions of other signal peptides can be found in Simonen and Palva's paper "Protein Secretion in Bacillus Genus" (1993, Microbiol Rev 57: 109-137).

[0033] The effective signal peptide coding regions of filamentous fungal host cells can be derived from the signal peptide coding regions of Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Rhizopus oryzae aspartic protease, Saprophytic mold cellulase, and Saprophytic villi lipase genes.

[0034] Signal peptides useful to yeast host cells may originate from the genes of Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Signal peptides from other host cell systems have also been extensively studied.

[0035] The control sequence may also be a propeptide coding region, which encodes an amino acid sequence located at the N-terminus of the polypeptide. The resulting polypeptide is called a proenzyme or propeptide (in some cases also called a zymogen). Propeptides are usually inactive and can be converted into mature, active polypeptides by catalytic or autocatalytic cleavage of the propeptide. Propeptide coding regions can be derived from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizopus oryzae aspartic protease, and Thermophilus lactase (WO 95 / 33836, incorporated herein by reference).

[0036] When both a signal peptide and a propeptide region are present at the N-terminus of a polypeptide, the propeptide region is located near the N-terminus of the polypeptide, and the signal peptide region is located near the N-terminus of the propeptide region.

[0037] Furthermore, the addition of regulatory sequences is essential; these sequences can regulate the expression of labeled restriction endonucleases according to the growth status of the host cell. Examples of regulatory systems include those that can turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. In prokaryotic host cells, suitable regulatory sequences include the lactose operon system (lac), the tac operon system, and the tryptophan operon system (trp). In yeast host cells, suitable regulatory systems include the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include the TAKA α-amylase promoter, the Aspergillus niger glucosylase promoter, and the Aspergillus oryzae glucosylase promoter. Other host cell regulatory systems have also been extensively studied.

[0038] Other examples of regulatory sequences include those capable of amplifying genes. In eukaryotic systems, these sequences include the dihydrofolate reductase gene (amplified in the presence of methotrexate) and the metallothionein gene (amplified in the presence of heavy metals). In these cases, the nucleic acid sequence can be operatively linked to the regulatory sequence.

[0039] Another embodiment includes a recombinant expression vector containing a polynucleotide encoding an engineered label restriction endonuclease or a variant thereof, and one or more expression regulatory regions (e.g., promoters and terminators) and a replication origin, depending on the host type to which it is introduced. The various nucleic acid sequences and control sequences described above can be linked together to produce a recombinant expression vector that may include one or more convenient restriction sites for inserting or replacing nucleic acid sequences encoding label restriction endonucleases. Alternatively, the nucleic acid sequences encoding restriction endonucleases can be expressed by inserting the nucleic acid sequences or nucleic acid constructs containing these sequences into a suitable expression vector. When constructing the expression vector, the coding sequence is located within the vector such that it can be operatively linked to suitable control sequences for expression.

[0040] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) as long as they facilitate recombinant DNA manipulation and express labeled restriction endonuclease polynucleotide sequences. The choice of vector typically depends on its compatibility with the target host cell. Vectors can be linear or closed circular plasmids.

[0041] Expression vectors can be self-replicating vectors, meaning they exist as extrachromosomal entities whose replication is independent of chromosome replication, such as plasmids, extrachromosomal elements, mini-chromosomes, or artificial chromosomes. Vectors can contain any mechanism that ensures their own replication. Alternatively, vectors can be vectors that integrate into the genome after being introduced into the host cell and replicate along with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids (which together contain the entire DNA to be introduced into the host cell's genome), or transposons can be used.

[0042] The expression vectors described herein preferably contain one or more selection markers to facilitate the screening of transformed cells. Selection markers are genes whose products confer characteristics such as resistance to biocides or viruses, heavy metal resistance, or auxotrophic traits. Examples of bacterial selection markers include the dal gene of Bacillus subtilis or Bacillus licheniformis, or markers conferring antibiotic resistance, such as resistance to ampicillin, kanamycin, chloramphenicol (Example 1), or tetracycline. Markers suitable for yeast host cells include ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for filamentous fungal host cells include, but are not limited to, amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (phosphinicotinic acid acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotate-5'-phosphate decarboxylase), sC (adenosyl sulfate transferase), and trpC (o-aminobenzoic acid synthase) and their equivalent genes. Examples of markers used in Aspergillus cells include the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus. Selectable markers for insect, plant, and mammalian cells are also well known.

[0043] The expression vector of the present invention preferably comprises one or more elements that allow the vector to integrate into the host cell genome or allow the vector to replicate autonomously within the cell independently of the genome. For integration into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector to integrate into the genome via homologous or non-homologous recombination.

[0044] Alternatively, the expression vector may contain additional nucleic acid sequences to guide its integration into the host cell genome via homologous recombination. These additional nucleic acid sequences enable the vector to integrate into one or more precise locations on the host cell's chromosomal genome. The integrative element can be any sequence homologous to the target sequence in the host cell genome. Furthermore, the integrative element can be a nucleic acid-coding sequence or a non-nucleic acid-coding sequence. On the other hand, the vector can also integrate into the host cell genome via non-homologous recombination.

[0045] To achieve autonomous replication, vectors may also contain origins of replication (OCRs), enabling them to replicate autonomously within the target host cell. Examples of bacterial OCRs include the P15A OCR, as well as OCRs for plasmids pBR322, pUC19, pACYC177 (of which pACYC177 contains the P15A OCR), or pACYC184, which allow the vector to replicate in *E. coli*; and OCRs for pUB110, pE194, pTA1060, or pAM31, which allow the vector to replicate in *Bacillus*. Examples of OCRs used for yeast host cells include the 2-micron OCR, ARS1, ARS4, combinations of ARS1 and CEN3, and combinations of ARS4 and CEN6. The origin of replication may be mutated, making its function in the host cell temperature-sensitive (e.g., see Ehrlich, "Replication of Staphylococcus aureus plasmids in *E. coli*" (1978), *Proceedings of the National Academy of Sciences* 75:1433).

[0046] To increase the yield of gene products, multiple copies of nucleic acid sequences carrying labeled restriction endonucleases can be inserted into host cells. There are two methods to increase the number of nucleic acid sequence copies: one is to integrate at least one additional sequence copy into the host cell genome; the other is to insert an amplifiable selectable marker gene along with the nucleic acid sequence, and then culture the cells in a medium containing the corresponding selectant reagent to screen for cells containing amplified selectable marker gene copies (thus containing additional nucleic acid sequence copies).

[0047] Expression vectors are commercially available. Suitable commercial expression vectors include the p3xFLAG™ expression vector from Sigma-Aldrich, St. Louis, Missouri, USA, which contains a CMV promoter and hGH polyadenylation site for expression in mammalian host cells; and a pBR322 origin of replication and ampicillin resistance marker for E. coli amplification. Other suitable expression vectors include pBluescriptII SK(-) and pBK-CMV, available from Stratagene, La Jolla, California, and plasmids derived from pBR322 (Gibco BRL), pUC (GibcoBRL), pREP4, pCEP4 (Invitrogen), or pPoly (Lathe et al., Plasmids and Phage Vectors for Removing Complete Inserts (1987) Gene 57:193-201).

[0048] Suitable host cells for expressing polynucleotides encoding restriction endonucleases are known in the art, including but not limited to bacterial cells such as *Escherichia coli*, *Lactobacillus kefir*, *Lactobacillus brevis*, *Lactobacillus minor*, *Streptomyces*, and *Salmonella typhimurium*; fungal cells such as yeast cells (e.g., *Saccharomyces cerevisiae* or *Pichia pastoris* (ATCC accession number 201178); insect cells such as *Drosophila melanogaster* S2 cells and *Spodoptera litura* Sf9 cells; animal cells such as CHO cells, COS cells, BHK cells, 293 cells, and Bowes melanoma cells); and plant cells. Suitable culture media and growth conditions for the aforementioned host cells are well known in the art.

[0049] The polynucleotides to be expressed can be introduced into cells by a variety of methods known in the art. These techniques include, but are not limited to, electroporation, gene gun bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells are well known to those skilled in the art.

[0050] Engineered peptides expressed in host cells can be recovered from cells and / or culture media using one or more known protein purification techniques, including but not limited to lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography. Solutions suitable for lysing bacteria (e.g., E. coli) and efficiently extracting proteins are available from Sigma-Aldrich, Inc., St. Louis, Missouri, USA, under the trade name CelLytic B.TM.

[0051] Chromatographic techniques used for the separation of engineered peptides include, but are not limited to, reversed-phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, and affinity chromatography. Purification conditions depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, and molecular shape, which are readily apparent to those skilled in the art.

[0052] In some embodiments, affinity chromatography can be used to isolate engineered peptides. For affinity chromatography purification, any antibody that specifically binds to the engineered peptide can be used. To produce antibodies, various host animals, including but not limited to rabbits, mice, rats, etc., can be immunized by injecting a compound. The compound can be linked to a suitable carrier (e.g., bovine serum albumin) via a side-chain functional group or linker. Depending on the host species, various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvants (complete and incomplete adjuvants), mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysophosphatidylcholine), Pranic polyols, polyanionic peptides, peptides, oil emulsions, keyhole hemocyanin, dinitrophenol, and some adjuvants that may be useful to humans, such as BCG and Corynebacterium microphyllum.

[0053] The following are examples of applying the method of the present invention to the preparation of desired mutants. Example

[0054] Example 1: fuGFP wild-type (WT) and G68S mutants fuGFP is a variant of green fluorescent protein (3), which appears green under long-wave ultraviolet light excitation. Furthermore, fuGFP exhibits significantly higher thermostability at 37°C than GFP, thus colonies are more likely to express fuGFP when cultured overnight at 37°C. The DNA sequence (SEQ ID NO:1) is translated into protein (SEQ ID NO:2). fuGFP and GFP WT (4) (SEQ ID NO:3) have significantly different protein structures, as compared below (identical residues in each sequence are shown in bold below; even-numbered sequences [12, 14, 16, 18] represent fuGFP, and odd-numbered sequences [13, 15, 17, 19] represent wild-type GFP): 1VSSGEDIFSGLVPILIELEGDVNGHRFSVRGEGYGDASNGKLEIKFICTTGRLPVPWPTL 61 (SEQID NO:12) 1MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL 60 (SEQID NO:13) Query62 62VTTLSYGVQCFAKYPEHMRQNDFFKSAMPDGYVQERTISFKEDGTYKTRAEVKFEGEALV 121(SEQ ID NO:14) 61VTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV 120(SEQ ID NO:15) 122NRIDLKGLEFKEDGNILGHKLEYSFNSHYVYITADKNRNGLEAQFRIRHNVDDGSVQLAD 181(SEQ ID NO:16) 121NRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD 180(SEQ ID NO:17) 182HYQQNTPIGEGPVLLPEQHYLTTNSVLSKDPQERRDHMVLVEFVTAAGLSLGMDELYK 239(SEQ ID NO:18) 181HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK 238(SEQ ID NO:19) One of the GFP mutants, G67S, was found to lose green fluorescence (5), and this mutation is conserved in fuGFP, where the amino acid position is shifted one position compared to wild-type GFP. Therefore, it is reasonable to assume that G68S fuGFP will also lose green fluorescence. On the other hand, fluorescence is restored when the 68th amino acid residue in fuGFP is mutated back to glycine (G).

[0055] Examples 2 and 3 (Introduction) When an additional tag for purification is added to the target protein, the amino acid numbering starting from the N-terminus is the same as the untagged numbering. In Examples 2 and 3 below, an additional 6xHis tag (labeled "NHfuGFP") was added to the N-terminus of the fuGFP protein. The DNA sequence of NHfuGFP WT is located in SEQ ID NO:4, and its protein sequence is located in SEQ ID NO:5. The DNA sequence of NHfuGFP G68S is located in SEQ ID NO:6, and its protein sequence is located in SEQ ID NO:7.

[0056] Example 2: Construction of an antibiotic resistance conversion template The conversion of antibiotic resistance from template to mutant is a relatively simple functional change. In this construct (Figure 1), the kanamycin resistance gene (KanR) is located in the middle of the ampicillin resistance gene (AmpR), which is divided into two parts: AmpR1 and AmpR2. When a deletion mutation is performed to remove the kanamycin resistance gene, the ampicillin resistance gene is still retained. The primers for the deletion mutation can overlap completely or partially, or they can not overlap if phosphorylation and ligation are performed after PCR.

[0057] The primer pair used for the deletion mutation is named AmpR1R and AmpR2F. When overlapping primers are used, the ampicillin resistance gene is obtained after cell transformation. All transformed cells (E. coli) that do not carry the mutant plasmid also lack the ampicillin resistance gene and therefore cannot grow on ampicillin-containing agar plates. Transformation can be performed directly or post-reaction, for example using a cloning kit such as the ZY Cloning method kit (ZyCloning, Woburn, MA). If overlapping primers are not used, but the protocol employs PCR followed by phosphorylation and ligation, a plasmid carrying the ampicillin resistance gene can be obtained in vitro. However, after transformation into cells, its resistance to ampicillin still needs to be evaluated and confirmed.

[0058] When performing another targeted mutagenesis on the same plasmid, whether targeting a specific gene or editing the sequence, the mutation primers on the ampicillin resistance gene are identical, i.e., completely or partially overlapping, or if they do not overlap, mutation is performed after phosphorylation and ligation. In this invention, a pair of primers is used for each target site, and a pair of primers is used for the ampicillin resistance restoration site, for two separate PCR reactions. After the two PCR reactions are completed, the PCR products can be transformed into competent cells (such as E. coli) by mixing equal volumes without assembly or by in vitro assembly. Due to the extremely low wild-type content, DpnI digestion is not required.

[0059] The transformed E. coli were spread onto agar plates containing ampicillin (preferably 100 μg / ml). The transformed cells will be ampicillin resistant and will form colonies on the agar plates.

[0060] Example 3: Characteristics of template plasmids pKaLNHfuGFPWT and pKaLNHfuGFPG68S The complete pKaLNHfuGFPWT sequence is located in SEQ ID NO:8. It exhibits heterozygous antibiotic resistance, where the kanamycin resistance gene is located in two parts of the ampicillin resistance gene, and fuGFP is controlled by the constitutive expression promoter plac. Figure 3 Therefore, *E. coli* colonies containing pKaLNHfuGFPWT appear green under long-wave ultraviolet light and show as bright spots in a grayscale image. The mutant product, pALNHfuGFPG68S (SEQ ID NO:9), is derived by deleting the kanamycin resistance gene and introducing the G68S mutation. Colonies on plates containing this plasmid do not fluoresce under ultraviolet light. Figure 4 In the grayscale image, it appears as light gray spots.

[0061] The complete sequence of pKaLNHfuGFPG68S is SEQ ID NO:10. E. coli colonies containing this plasmid do not exhibit ampicillin resistance, do not fluoresce under ultraviolet light, and... Figure 4 In the grayscale image, it appears as light gray spots. The mutant product of kanamycin resistance gene deletion and the introduction of the S68G mutation is pALNHfuGFPWT (SEQ ID NO:11). Colonies containing this product bioparticle emit green fluorescence when exposed to ultraviolet light. Figure 4 In the grayscale image, it appears as bright white spots.

[0062] Example 4: pKaLNHfuGFPWT was mutated to pALNHfuGFPG68S, and the kanamycin resistance gene was replaced with the ampicillin resistance gene, and NHfuGFPWT was replaced with NHfuGFPG68S. Set up two independent PCR reactions using the following primers: PCR reaction 1: G68SR: 5' TGCACAGAATACG ACAAGGTCGTCACCAAGGTCGGC 3' (SEQ ID NO:20) Amp2F: 5' CTGGATCTCAAC AGCGGTAAGATCCTTGAG 3' (SEQ ID NO:21) PCR reaction 2: G68SF: 5' CGTATTCTGTGCA GTGTTTTCGAAGTATCCG 3' (SEQ ID NO:22) Amp1R: 5' GTTGAGATCCAG TTCGATGTAACCCACTCG 3' (SEQ ID NO:23) G68SF and G68SR share 13 bp of homology (underlined), and Amp1R and Amp2F share 12 bp of homology (double underlined).

[0063] Prepare a 0.5 μM mixture of forward and reverse primers.

[0064] The reaction reagents are as follows: 4 μL of 0.5 μM primer mixture; 1 μL 3.7 ng template plasmid pKaLNHfuGFPWT; 5 μL Q5 High Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA); The total volume is 10 μL.

[0065] The PCR reaction procedure is as follows: 98℃ for 30 seconds; Then perform 28 cycles: 98℃ for 5 seconds; 55℃ for 10 seconds; and 72℃ for 1 minute.

[0066] It was then kept at 72°C for 2 minutes, and then cooled.

[0067] Take 2 μl of PCR product and electrophoresis it on a 0.8% agarose gel at 200 V for 20 minutes. Figure 4 Mix 1 μl of product from each PCR reaction, then transform into 50 μl of DH10B medium (ZyCloning, Woburn, MA) containing enhancers, and immediately plate onto agar plates containing ampicillin. Incubate the plates overnight at 37°C. After UV observation, out of a total of 289 colonies, 288 were light gray and 1 was bright white. Figure 4 As indicated by the middle arrow.

[0068] Even without DpnI treatment, the remaining wild-type strains account for only 0.3% of the total, meaning this antibiotic conversion method leaves virtually no wild-type residues. Previous methods, lacking DpnI digestion, resulted in numerous false positives that were difficult to detect without sequencing. However, in this method, false positives are minimized. The only remaining wild-type colony on the plate is likely the result of transformation with both the template plasmid and the PCR fragment; that is, *E. coli* simultaneously carries the template and the mutant plasmid, thus preserving a template wild-type colony on the plate. Furthermore, ampicillin resistance could also be caused by other factors, such as cellular mutations in *E. coli* rather than transformation.

[0069] Not all light gray products in Figure 4 are correct mutant products. Errors may occur during in vitro assembly, such as deletions, insertions, homologous double insertions, and mutations during PCR. Even so, this example still demonstrates that the residual template content is very low.

[0070] Example 5: pKaLNHfuGFPG68S was mutated into pALNHfuGFPWT, and the kanamycin resistance gene was replaced with the ampicillin resistance gene, and NHfuGFPG68S was replaced with NHfuGFPWT. Set up two independent PCR reactions with the following primer formulations: PCR reaction 1 S68GR: 5' TGCACGCCATACG ACAAGGTCGTCACCAAGGTCGGC 3' (SEQ ID NO:24) Amp2F: 5' CTGGATCTCAAC AGCGGTAAGATCCTTGAG 3' (SEQ ID NO:25) For PCR reaction 2 S68GF: 5' CGTATGGCGTGCA GTGTTTTCGAAGTATCCG 3' (SEQ ID NO:26) Amp1R: 5' GTTGAGATCCAG TTCGATGTAACCCACTCG 3' (SEQ ID NO:27) G68SF and G68SR have a 13 bp homologous sequence (underlined), and Amp1R and Amp2F have a 12 bp homologous sequence (double underlined).

[0071] Prepare a 0.5 μM mixture of forward and reverse primers.

[0072] The reaction reagents are as follows: 4 μL of 0.5 μM primer mixture; 1 μL 3.7 ng template plasmid pKaLNHfuGFPS68G; 5 μL Q5 High Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA).

[0073] The total volume is 10 μL.

[0074] The PCR reaction procedure is as follows: 98℃ for 30 seconds Then perform 28 cycles: 98℃ for 5 seconds; 55℃ for 10 seconds; 72℃ for 1 minute.

[0075] Then, pre-treat at 72°C for 2 minutes, followed by cooling.

[0076] Take 2 μl of PCR product and electrophoresis it on a 0.8% agarose gel at 200 V for 20 minutes. Figure 5 Mix 1 μl of product from each PCR reaction, then transform into 50 μl of DH10B medium (ZyCloning, Woburn, MA) containing enhancers, and immediately plate onto agar plates containing ampicillin. Incubate the plates overnight at 37°C. Upon UV observation, out of a total of 335 colonies, 31 were light gray and 304 were bright white.

[0077] In this case, the remaining template was difficult to determine. All remaining template colonies, plus other errors caused by in vitro assembly and PCR mutations, accounted for 9.3% of the total colonies. The apparent correct mutation rate was 90.7%, but the actual value may be higher.

[0078] Since the PCR primers used are crude extracts, purified PCR primers can increase the proportion of correct mutations. Mutations and assembly errors mostly belong to different categories; therefore, if the mutation is used for protein function analysis, a mixture of dozens of colonies can usually represent the desired mutation.

[0079] The specific methods and compositions described herein are representative of preferred embodiments and are exemplary, not intended to limit the scope of the invention. Other objects, aspects, and embodiments will arise in those skilled in the art upon reading this specification, all of which are included within the spirit of the invention as defined in the claims. Those skilled in the art will understand that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention can be suitably practiced even in the absence of any elements or limitations not expressly disclosed herein. For example, in each instance herein, the terms “comprising,” “including,” “containing,” etc., in embodiments or embodiments of the invention should be interpreted broadly and without limitation. The methods and processes described herein may be implemented in different sequences of steps and are not necessarily limited to the sequence specified herein or in the claims. It should also be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” used herein and in the appended claims include the plural forms, and the plural forms also include the singular forms. In no event should this patent be construed as limited to the specific embodiments, examples, or methods specifically disclosed herein. Under no circumstances should this patent be construed as being limited to any statement made by any examiner or any other officer or employee of the Patent and Trademark Office, unless such statement is expressly within the general scope of disclosure and also constitutes part of the invention. The terms and expressions used are for descriptive purposes only and not for limitation. The use of such terms and expressions is not intended to exclude equivalents of the shown and described features or any part thereof, but it should be understood that various modifications may be made within the scope of the invention as defined by the claims. Therefore, it should be understood that although the invention has been specifically disclosed by way of preferred embodiments and optional features, those skilled in the art can make modifications and variations to the concepts disclosed herein, including but not limited to sequences of variations, and such modifications and variations should be considered within the scope of the invention as defined by the appended claims. The applicant has not expressly adopted any limitations or reservations in the response documents. The invention has been described broadly and generally herein. Each more specific species and subgenus is grouped... Additional references (all cited): 1.Yang Z., et al, A simple and economical site-directed mutagenesismethod for large plasmids by direct transformation of two overlapping PCRfragments (2022) BIOTECHNIQUES VOL. 73, NO. 5, https: / / doi.org / 10.2144 / btn-2022-0085 2.Lu L., et al, Optimizing DpnI Digestion Conditions to DetectReplicated DNA (2002) BioTechniques 33:316-318 3.https: / / schaechter.asmblog.org / schaechter / 2019 / 05 / the-story-of-free-use-gfp-fugfp.html 4.Tsien R. Y., The green fluorescent protein. (1998) Annu. Rev.Biochem. 67: 509–544. 5.Fu, G. L., GFP Loss-of-Function Mutations in Arabidopsis thaliana(2015) G3 (Bethesda). 5(9): 1849–1855.

Claims

1. A method for site-directed mutagenesis of plasmids using PCR technology, wherein the method involves at least two independent PCR reactions, using a first pair of forward and reverse primers, a second pair of forward and reverse primers, or additional forward and reverse primers, respectively, to induce functional changes from the template plasmid to the PCR product and add at least one additional mutation; wherein, The first PCR reaction uses the first pair of primers for additional mutation, and the second PCR reaction uses the second pair of primers for functional alteration; the method includes: Perform first and second PCR reactions on the plasmid using the first and second pairs of primers in any order; The products of each PCR reaction are recombined; Cell transformation with recombinant products; and A screening method is used to locate cells with functional alterations, thereby distinguishing transformed cells carrying PCR product plasmids with functional alterations from untransformed cells carrying template plasmids.

2. The method of claim 1, wherein DpnI is not used before or after recombination.

3. The method of claim 1, wherein the second PCR reaction using the second primer pair is performed before the first PCR reaction.

4. The method of claim 1, wherein recombination is achieved by linking or mixing reaction products.

5. The method of claim 1, wherein the functional alteration is antibiotic resistance, cell growth conditions, or cell appearance (including fluorescence or color changes).

6. The method of claim 1, wherein the additional mutation may be located in a region of the plasmid that is different from the region controlling the change in function.

7. The method of claim 1, wherein multiple functional changes are initiated by the same pair of primers.

8. The method of claim 1, wherein multiple functional changes are initiated by different primer pairs.

9. The method of claim 1, wherein the functional alteration removes the kanamycin resistance gene from the plasmid but retains the ampicillin resistance gene.

10. The method of claim 1, wherein the plasmid DNA sequence comprises the sequence of SEQ ID NO:

1.

11. The method of claim 1, wherein the plasmid protein sequence comprises the sequence of SEQ ID NO:

2.

12. The method of claim 11, wherein the plasmid protein has a hexahistidine tag added to its N-terminus.

13. The method of claim 12, wherein the primer pairs are SEQ ID NO:20 and 22; and SEQ ID NO:21 and 23.

14. The method of claim 12, wherein the primer pairs are SEQ ID NO:24 and 26; and SEQ ID NO:25 and 27.

Images