A python script to design site-directed mutagenesis primers.
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ABSTRACT:
SUBMITTER: Bi C
PROVIDER: S-EPMC7096704 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ligating two or more DNA fragments is a regular operation for the subcloning and the engineering of vectors. The overlap extension PCR serves as a straightforward method to solve this issue. However, it takes a relatively long time to design the appropriate overlapping primers and the primers for the full-length sequence, and there has not been a professional offline software for such kind of primer design. Here, we propose a Python script to search, calculate and sort thousands of combinations of primers for users according to the predefined parameters. The results of script running and experimental validation show that this script is capable of generating the optimal pairs of primers based on the proper melting temperatures and lengths of the primers, which facilitates gene modification in research.
| S-EPMC9632469 | biostudies-literature
Project description:BackgroundSite-directed mutagenesis (SDM) is a key method in molecular biology; allowing to modify DNA sequences at single base pair resolution. Although many SDM methods have been developed, methods that increase efficiency and versatility of this process remain highly desired.MethodWe present a versatile and simple method to efficiently introduce a variety of mutation schemes using Gibson-assembly but without the need to design uniquely designated Gibson primers. Instead, we explore the re-use of standard SDM primers (completely overlapping in sequence) in combination with regular primers (~ 25 bps long) for amplification of fragments flanking the site of mutagenesis. We further introduce a rapid amplification step of the Gibson-assembled product for analysis and quality control, as well as for ligation, or re-ligation at instances the process fails (avoiding expenditure of added Gibson reaction mixtures).ResultsWe first demonstrate that standard SDM primers can be used with the Gibson assembly method and, despite the need for extensive digestion of the DNA past the entire primer sequence, the reaction is attainable within as short as 15 min. We also find that the amount of the assembled Gibson product is too low to be visualized on standard agarose gel. Our added amplification step (by use of the same short primers initially employed) remedies this limitation and allows to resolve whether the desired Gibson-assembled product has been obtained on agarose gel or by sequencing of amplicons. It also provides large amounts of amplicons for subsequent ligations, bypassing the need to re-employ Gibson mixtures. Lastly, we find that our method can easily accommodate SDM primers with degenerate sequences.ConclusionWe employ our alternative approach to delete, replace, insert, and degenerate sequences within target DNA sequences, specifically DNA sequences that proved very resistant to mutagenesis by multiple other SDM methods (standard and commercial). Importantly, our approach involves the re-use of SDM primers from our primer-inventory. Our scheme thereby reduces the need (and time and money) to design and order new custom Gibson-primers. Together, we provide a simple and versatile protocol that spans only 4 days (including the added amplification step), requires minimal primer sets and provides very high yields and success rates (> 98%).
| S-EPMC8918331 | biostudies-literature
Project description:To meet the demand for the application of high activity and thermostable cellulases in the production of new-generation bioethanol from nongrain-cellulose sources, a hyperthermostable β-1,4-endoglucase Cel12B from Thermotoga maritima was selected for further modification by gene site-directed mutagenesis method in the present study, based on homology modeling and rational design. As a result, two recombinant enzymes showed significant improvement in enzyme activity by 77% and 87%, respectively, higher than the parental enzyme TmCel12B. Furthermore, the two mutants could retain 80% and 90.5% of their initial activity after incubation at 80°C for 8 h, while only 45% for 5 h to TmCel12B. The Km and Vmax of the two recombinant enzymes were 1.97±0.05 mM, 4.23±0.15 μmol·mg(-1)·min(-1) of TmCel12B-E225H-K207G-D37V, and 2.97±0.12 mM, 3.15±0.21 μmol·mg(-1)·min(-1) of TmCel12B-E225H-K207G, respectively, when using CMC-Na as the substrate. The roles of the mutation sites were also analyzed and evaluated in terms of electron density, hydrophobicity of the modeled protein structures. The recombinant enzymes may be used in the hydrolysis of cellulose at higher temperature in the future. It was concluded that the gene mutagenesis approach of a certain active residues may effectively improve the performance of cellulases for the industrial applications and contribute to the study the thermostable mechanism of thermophilic enzymes.
| S-EPMC4517919 | biostudies-literature
Project description:Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.
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Project description:SITE-DIRECTED MUTAGENESIS OF MYCOBACTERIUM TUBERCULOSIS AND FUNCTIONAL VALIDATION TO INVESTIGATE POTENTIAL BEDAQUILINE RESISTANCE CAUSING MUTATIONS
| PRJEB52049 | ENA
Project description:Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient.
| S-EPMC4707547 | biostudies-literature
Project description:C-reactive protein (CRP) is a cyclic pentameric protein whose major binding specificity, at physiological pH, is for substances bearing exposed phosphocholine moieties. Another pentameric form of CRP, which exists at acidic pH, displays binding activity for oxidized LDL (ox-LDL). The ox-LDL-binding site in CRP, which is hidden at physiological pH, is exposed by acidic pH-induced structural changes in pentameric CRP. The aim of this study was to expose the hidden ox-LDL-binding site of CRP by site-directed mutagenesis and to generate a CRP mutant that can bind to ox-LDL without the requirement of acidic pH. Mutation of Glu(42), an amino acid that participates in intersubunit interactions in the CRP pentamer and is buried, to Gln resulted in a CRP mutant (E42Q) that showed significant binding activity for ox-LDL at physiological pH. For maximal binding to ox-LDL, E42Q CRP required a pH much less acidic than that required by wild-type CRP. At any given pH, E42Q CRP was more efficient than wild-type CRP in binding to ox-LDL. Like wild-type CRP, E42Q CRP remained pentameric at acidic pH. Also, E42Q CRP was more efficient than wild-type CRP in binding to several other deposited, conformationally altered proteins. The E42Q CRP mutant provides a tool to investigate the functions of CRP in defined animal models of inflammatory diseases including atherosclerosis because wild-type CRP requires acidic pH to bind to deposited, conformationally altered proteins, including ox-LDL, and available animal models may not have sufficient acidosis or other possible modifiers of the pentameric structure of CRP at the sites of inflammation.
| S-EPMC3271008 | biostudies-literature
Project description:The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.
| S-EPMC5138438 | biostudies-literature
Project description:The protein cross-reactive material 197 (CRM197) is known to catalyze the hydrolytic cleavage of DNA (DNase activity). A suspected metal-binding site (S109, T111, and E112) and suspected DNA-binding motif (T89, K90, and V91) were predicted within the CRM197 protein X-ray crystal structure (4AE0) using METSITE and DNABindProt, respectively. Between these two predicted sites is a groove (K103, E116, T120, E122, F123, and R126) that may assist in DNase activity. Alanine scanning was performed at these sites to determine which amino acids might be important for DNase activity. These mutations individually or in combination either maintained or increased the overall DNase activity compared to the unmodified CRM197. Mutation at the suspected metal-binding site showed similar fluctuations to the overall DNase activity whether the DNase assays were run with Mg2+ and Ca2+ or Mn2+. However, many of the mutations within the suspected DNA-binding motif saw significant differences depending on which metal was used. Only some of the improvements in DNase activity could be attributed to improved folding of the mutants compared to the unmodified CRM197. This study should provide a basis for further mutagenesis studies to remove the DNase activity of CRM197.
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Project description:Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in Escherichia coli as a DsbA fusion protein including a Dsb signal sequence for translocation to the periplasm and a His tag for purification. The missing N-glycosylation results in reduced catalytic activity and thermal stability, therefore enzyme engineering was used to improve these characteristics. The amino acids at four N-glycosylation sites, namely N13, N57, N255 and N268, were mutated by site-directed mutagenesis and combined to double, triple and quadruple enzyme variants. Subsequently, the rHRP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) and biochemically characterized. We found that the quadruple mutant rHRP N13D/N57S/N255D/N268D showed 2-fold higher thermostability and 8-fold increased catalytic activity with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as reducing substrate when compared to the non-mutated rHRP benchmark enzyme.
| S-EPMC6412888 | biostudies-literature