Mutagenesis from Customer-Provided Templates

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Creative Enzymes provides Mutagenesis from Customer-Provided Templates, a core offering within our Site-Directed Mutagenesis (SDM) Services. This service is designed for clients who already possess verified plasmid DNA or expression constructs and seek to introduce targeted mutations with precision and reliability. Whether you aim to investigate structure–function relationships, enhance catalytic performance, or fine-tune enzyme stability, we deliver accurate mutagenesis outcomes through high-fidelity methodologies and rigorous quality control. Our platform integrates scientific consultation, optimized reaction design, and sequence verification, ensuring each mutation is exactly as specified—ready for downstream expression, characterization, or screening.

Introduction to Mutagenesis from Customer-Provided Templates

In enzyme engineering and functional genomics, site-directed mutagenesis remains one of the most powerful tools for probing and enhancing protein properties. However, even when clients supply their own expression vectors, achieving reliable and reproducible mutations requires technical precision and thorough validation.

Traditional in-lab mutagenesis often encounters pitfalls—such as incomplete primer design, low efficiency in PCR amplification, plasmid instability, or off-target mutations. At Creative Enzymes, we leverage optimized protocols, automated reaction systems, and stringent QC standards to overcome these challenges.

By combining client-supplied templates with our mutagenesis expertise, we enable rapid generation of single-site or multiple-site variants with exceptional accuracy. Our end-to-end workflow—from template verification through final sequencing confirmation—ensures high confidence in the integrity of every construct delivered.

Mutagenesis service using customer templates

What We Offer

We provide high-accuracy mutagenesis services using customer-provided templates, offering the following capabilities:

Mutagenesis Using Client-Supplied Templates

We perform mutagenesis directly on customer-provided plasmids or DNA constructs, preserving your vector backbone and cloning context.

Single and Multiple Site Mutagenesis

Introduction of one or more targeted mutations (substitution, insertion, deletion) as per client specification.

Template Verification Prior to Mutagenesis

Optional pre-screening and sequencing verification of the provided plasmid to ensure accuracy before modification.

Flexible Method Selection

Use of PCR-based, recombination-driven, or oligonucleotide-directed strategies depending on construct complexity.

Full Mutation Validation

Complete open reading frame sequencing and restriction digestion verification after mutagenesis.

Library Construction Option

Generation of combinatorial or saturation-mutagenesis libraries for enzyme optimization projects.

Vector Maintenance

Your original plasmid backbone, promoter, and selection markers are fully preserved.

Fast Delivery

Verified mutant constructs are supplied with sequencing reports, vector maps, and mutation confirmation certificates.

Service Workflow

Service workflow for site-directed mutagenesis using customer-provided templates

Service Details

Parameter	Specification
Mutation Types	Point mutation, insertion, deletion, or combinatorial mutations
Template Requirements	Purified plasmid DNA or verified vector construct (≥50 ng/µL)
Vector Compatibility	All standard cloning and expression vectors (bacterial, yeast, insect, mammalian)
Verification Method	Full-length Sanger sequencing
Turnaround Time	7–15 business days depending on complexity
Deliverables	Verified plasmid (≥2 µg), sequencing report, vector map, QC documentation
Optional Add-Ons	Expression testing, protein purification, or mutational library expansion

Connecting Services

This service complements our sibling offering:

Mutagenesis from Creative Enzymes-Synthesized Templates

Together, these two services form the foundation of our core site-directed mutagenesis services, offering complete flexibility for research and industrial needs. Whether you supply your own vector or request a fully integrated synthesis-to-mutation workflow, we ensure accurate, reproducible, and high-quality results for every project.

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Why Choose Us

Precision on Your Constructs

We work directly on your supplied plasmids, ensuring full control over vector configuration and experimental context.

Flexible Mutation Options

Support for single, multiple, and combinatorial mutations tailored to research goals.

Advanced Methodology

Utilization of high-fidelity PCR and recombination-based approaches minimizes undesired mutations.

Rapid Turnaround

Optimized workflows reduce project time without compromising quality.

Comprehensive Validation

Every modified construct is fully sequenced, ensuring exact correspondence to design.

Confidentiality and IP Protection

All client sequences and data are handled under strict confidentiality agreements.

Representative Case Studies

Case 1: Single-Point Mutation for Enhanced Cofactor Preference

Client Need:

A metabolic engineering research group sought to alter the cofactor specificity of a key oxidoreductase enzyme from NADH to NADPH to improve pathway efficiency in a biosynthetic production system. Despite having an existing E. coli expression vector, their previous mutagenesis trials suffered from low reproducibility and background plasmid contamination.

Our Approach:

The client supplied a verified pET-28a(+)-based construct, which underwent preliminary plasmid integrity assessment at Creative Enzymes. Using a high-fidelity PCR-based site-directed mutagenesis method, we introduced a single amino acid substitution (D199S) at a residue known to influence cofactor binding. The mutant plasmid was then sequenced in full to confirm the presence of the correct mutation and the absence of secondary errors before delivery for expression testing.

Outcome:

The engineered oxidoreductase exhibited a sixfold improvement in catalytic efficiency toward NADPH while maintaining substantial NADH activity. The entire project—from mutagenesis design to confirmed plasmid delivery—was completed within two weeks, enabling the client to rapidly proceed with kinetic and structural analyses.

Case 2: Multi-Site Mutagenesis to Enhance Enzyme Stability

Client Need:

An industrial enzyme manufacturer required a thermostable lipase variant suitable for high-temperature biocatalysis in continuous production systems. The goal was to introduce four stabilizing substitutions simultaneously, avoiding the time-consuming, stepwise mutagenesis approach that had previously delayed product development.

Our Approach:

Upon receiving the client's proprietary lipase plasmid, we designed four targeted primers for parallel oligonucleotide-directed mutagenesis. Using our optimized multi-site mutagenesis workflow, all substitutions were introduced in a single round of amplification and ligation. The complete construct underwent full-length sequencing to ensure accurate mutation incorporation and plasmid integrity.

Outcome:

The resulting lipase variant displayed a 9°C increase in melting temperature (Tm) compared to the wild type while maintaining its original catalytic turnover and substrate specificity. The one-step, verified workflow eliminated multiple cloning and verification rounds, shortening the project timeline by approximately 40% and providing a robust template for further industrial enzyme development.

Frequently Ased Questions

Q: What are the requirements for submitting a template?

A: Please provide purified plasmid DNA (>50 ng/µL) and, if available, the full sequence and vector map. This ensures optimal primer design and accurate mutation targeting.
Q: Do you verify the provided template before performing mutagenesis?

A: Yes, we can optionally sequence the provided construct before proceeding. This step ensures that no pre-existing mutations interfere with the desired modification.
Q: What if my plasmid is large or contains repetitive regions?

A: We have experience with complex plasmids up to 20 kb and can adjust our protocols for high-GC or repetitive sequences.
Q: Can you perform multiple-site mutations in one round?

A: Yes. Our proprietary multiplex SDM method allows introduction of several mutations simultaneously, minimizing cloning steps.
Q: How do you ensure confidentiality of client-provided DNA?

A: All client materials are processed under strict confidentiality and non-disclosure agreements, with secure data management and disposal protocols.
Q: Can you assist with downstream protein expression after mutagenesis?

A: Yes. Creative Enzymes offers optional downstream services including protein expression, purification, and functional assays to accelerate your enzyme engineering workflow.

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Project Description:

For research and industrial use only. Not intended for personal medicinal use. Certain food-grade products are suitable for formulation development in food and related applications.

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