DNA Recombination

DNA recombination, also called DNA shuffling, is a cornerstone technology in directed evolution, enabling the creation of large and diverse mutant libraries for protein engineering. By mimicking natural recombination processes, multiple gene sequences are fragmented, recombined, and expressed to generate offspring with novel variations. Unlike random mutagenesis, DNA recombination allows controlled exploration of sequence space, improving functional diversity while minimizing unwanted mutations or truncated sequences. Creative BioMart provides a complete, customized library construction service, including homologous and non-homologous recombination, sequence validation, and transformation into host systems, delivering high-quality mutant libraries tailored to specific protein engineering objectives.

Background: Library Construction by DNA Recombination

Figure 1. The strategy adapted for de novo designing of proteins. (Gulati and Poluri, 2016)

Creating a high-quality mutant library is essential for directed evolution experiments and protein engineering. DNA recombination accelerates this process by mimicking natural sexual reproduction, shuffling gene fragments from homologous or non-homologous sources to generate new variants. The general workflow includes:

• Fragmentation: Gene sequences are digested using DNase, creating fragments that may carry different mutations.
• Reassembly: Full-length genes are reconstituted through PCR, combining fragments into novel sequences.
• Cloning: Amplified sequences are inserted into plasmid vectors for expression.
• Library Generation: DNA variants are expressed in the host system to yield a protein library for downstream screening.

DNA recombination strategies can be homologous, using sequence similarity to guide recombination (e.g., QuikChange Shuffling), or non-homologous, allowing hybrid proteins from sequences with little or no homology (e.g., ITCHY). Both approaches expand sequence diversity while preserving functional integrity, which is critical for downstream protein optimization.

Our Customized Library Construction by DNA Recombination Service

Service Workflow

Comprehensive Services

Creative BioMart provides comprehensive DNA recombination and library construction services, including:

Homologous Recombination

Shuffling of related genes to generate functional variants.

Non-homologous Recombination

Construction of hybrid proteins from unrelated sequences.

Sequence Validation

Ensuring library integrity with minimal wild-type contamination, truncation, or redundancy.

Transformation Into Target Host

Delivery of recombinant libraries into bacterial, yeast, or mammalian systems.

Custom Library Design

Tailored to specific protein engineering projects, maximizing diversity and functional coverage.

Service Features

Our DNA recombination service is optimized for high-quality library construction:

• Minimized wild-type and truncated sequences: Increasing the proportion of functional mutants.
• Broad sequence diversity: Both homologous and non-homologous strategies for maximum exploration of sequence space.
• Customizable library size and composition: Designed to meet specific protein engineering goals.
• Validated protocols: Incorporating best practices in PCR, cloning, and transformation to ensure reproducibility.
• High-throughput sequencing: Confirms variant distribution and integrity of the generated library.
• Compatibility with multiple hosts: Libraries can be expressed in bacterial, yeast, or mammalian systems depending on the project.

Why Choose Creative BioMart

Expertise in Directed Evolution: Years of experience in DNA recombination and library construction.

Customized Library Design: Tailored solutions to match specific protein engineering goals.

High-Quality Libraries: Minimal wild-type contamination and truncated sequences.

Flexible Recombination Strategies: Homologous or non-homologous approaches to maximize diversity.

Integrated Validation: Sequencing and quality control for confident downstream screening.

Comprehensive Service: From consultation to library delivery, a complete one-stop solution.

Case Studies and Real-World Applications

Case 1: Directional cDNA library construction assisted by the in vitro recombination reaction

Ohara, 2001. doi:10.1093/nar/29.4.e22

A novel directional cDNA library construction method based on in vitro site-specific recombination using the bacteriophage λ integrase–excisionase system demonstrated significant advantages over traditional ligation-assisted cloning. This recombinational cloning (RC) approach eliminated the need for restriction digestion, minimized chimeric clone formation, reduced size bias, and achieved much higher cloning efficiency. In tests with long poly(A)+ RNA (7.8 kb), RC produced around tenfold more transformants and a greater proportion of full-length cDNA clones. Moreover, it successfully captured longer (>2 kb) rat brain cDNAs that conventional methods failed to clone, enabling a more comprehensive and representative cDNA library construction.

Figure 2. Crystal structure of C14linkmid in complex with the hydrophobic core of gp41, PDB code: 1GZL. The diaminoalkane crosslinker and the hydrophobic core of gp41, consisting of residues Leu29, Leu30, Leu32, Thr33, Val34, Trp35, Gly36, Ile37, Lys38, Leu40, and Gln41are colored blue (the residues are numbered according to their position in 1GZL). (Bellows et al., 2010)

Case 2. Construction of a plant transformation-ready expression cDNA library for Thellungiella halophila using recombination cloning

Ni et al., 2007. doi:10.1111/j.1744-7909.2007.00483.x

A high-quality entry cDNA library was constructed from salt-stressed Thellungiella halophila (salt cress), an extremophile closely related to Arabidopsis thaliana, to identify salt-tolerance genes. Using a modified cDNA synthesis and an improved recombination-assisted cloning method—eliminating the need for restriction enzymes and ligases—the process was greatly simplified while enhancing library quality. The resulting cDNA library was transferred into the binary vector pCB406 for plant transformation. This transformation-ready library enabled large-scale Arabidopsis transformation, generating extensive transgenic lines for functional gene mining and advancing the discovery of genetic mechanisms underlying salt tolerance in plants.

Figure 3. Agarose gel assays for cDNA quality and cDNA inserts. (A) Comparison of standard (S) and modified (M) SMART cDNA synthesis using salt cress RNA. The modified method added the template-switching oligo after 1 h incubation, yielding longer products after PCR. (B) Agarose gel of 14 representative clones from the cDNA library showing inserts >500 bp. Sequencing confirmed all were true cDNAs with high homology to Arabidopsis genes. (Ni et al., 2007)

Client Feedback on Our DNA Recombination and Library Construction Services

"We collaborated with Creative BioMart to construct a large-scale mutant library for an industrial hydrolase. Using homologous DNA recombination, they generated over 20,000 unique variants with minimal wild-type contamination. High-throughput screening identified several mutants with enhanced substrate specificity and a 3-fold increase in catalytic efficiency. Their expertise in library design and sequence validation significantly accelerated our enzyme optimization program."

— Senior Scientist | Industrial Biotechnology Company

"Our team required a hybrid enzyme library combining two unrelated oxidases for biocatalysis. Creative BioMart applied non-homologous recombination techniques (ITCHY-based) to produce a diverse pool of chimeric proteins. Among the 5,000 variants, we identified a hybrid with superior thermal stability and improved activity, which became the lead candidate for scale-up. Their integrated workflow and attention to detail were outstanding."

— R&D Director | Specialty Enzyme Manufacturer

"We partnered with Creative BioMart to design a combinatorial antibody library for affinity maturation. Their homologous DNA recombination approach produced a highly diverse set of variants while maintaining full-length sequences. The library enabled us to isolate clones with significantly enhanced binding affinities, and the team provided detailed sequencing validation reports, which were critical for our downstream selection."

— Director of Protein Engineering | Biopharmaceutical Company

"For a synthetic biology project, we needed a robust library of transcription factors with combinatorial mutations. Creative BioMart constructed a 50,000-variant library via DNA shuffling, ensuring minimal truncation and high sequence integrity. Transformation into our bacterial host was seamless, and the resulting library facilitated rapid screening and identification of functional variants. Their expertise in library quality control was invaluable."

— Principal Investigator | Academic Synthetic Biology Laboratory

FAQs About DNA Recombination and Library Construction for Protein Engineering

• Q: What is DNA recombination, and how is it different from random mutagenesis?

  A: DNA recombination, or DNA shuffling, mimics natural sexual recombination to generate diverse gene libraries. Unlike random mutagenesis, which introduces single-point mutations, DNA recombination can combine fragments from multiple parental genes, producing offspring with multiple, combinatorial mutations for greater functional diversity.

• Q: What types of DNA recombination do you offer?

  A: We provide both homologous recombination, which relies on sequence similarity between parental genes (e.g., QuikChange Shuffling), and non-homologous recombination, which allows hybrid proteins from unrelated sequences (e.g., ITCHY). Each method is tailored to maximize diversity and functional variation based on your project goals.

• Q: How do you ensure high-quality libraries with minimal wild-type or truncated sequences?

  A: Our workflow incorporates optimized fragment generation, PCR assembly, and sequencing validation. High-throughput sequence analysis ensures a high proportion of full-length, unique variants while minimizing wild-type contamination and truncated or redundant sequences.

• Q: Can the recombined libraries be used in different host systems?

  A: Yes. We can transform libraries into bacterial, yeast, or mammalian hosts, depending on your protein expression requirements and downstream screening needs. Our team advises on the most suitable host system for your project.

• Q: What information do I need to provide to start a DNA recombination project?

  A: Clients should provide parental gene sequences, desired library size, preferred recombination method (homologous or non-homologous), and target host system. Additional specifications, such as desired functional traits or mutational constraints, help us design the optimal library.

• Q: How long does it take to generate a DNA recombination library?

  A: Typical timelines depend on library size and complexity but usually range from 4 to 8 weeks, including fragment generation, recombination, cloning, sequencing validation, and transformation. Detailed project plans and progress updates are provided throughout the process.

References:

1. Ni W, Lei Z, Chen X, Oliver DJ, Xiang C. Construction of a plant transformation‐ready expression cDNA library for Thellungiella halophila using recombination cloning. JIPB. 2007;49(9):1313-1319. doi:10.1111/j.1744-7909.2007.00483.x
2. Ohara O. Directional cDNA library construction assisted by the in vitro recombination reaction. Nucleic Acids Research. 2001;29(4):22e-222. doi:10.1093/nar/29.4.e22