In Vivo phage Recombineering

In vivo recombineering, approaches use phages as engineering tools for other less-studied E. coli phages. E. coli cells carrying the defective prophage and pL operon were infected with the engineered phage at a multiplicity of infection (MOI) of 1 to 3 and allowed to adsorb for 15 minutes. The pL operon involved in general and site-specific recombination is controlled by a temperature-sensitive repressor. Following phage infection, λ Red recombination function was induced by heating mid-log phase bacterial cultures to 42°C. At this point, cells are electroporated with dsDNA or single-stranded DNA (ssDNA) substrates. Phage lysates were then recovered and examined for incorporation of the desired DNA. The yield of recombinant phage obtained using this technique is approximately 0.5-2%. By introducing λ Red system into host bacteria via plasmids (without the rest of the phage) or other recombination mechanisms, this technique can potentially be adapted to other phages and other bacterial species that can be targeted by the phage to be engineered.

Fig 1. In vivo recombineering. (Pires D P, et al. 2016)

Our Services

Creative Biolabs provides phage genetic recombination services to improve phage development and meet customers' needs to obtain the ideal phage. With our extensive experience and advanced technology in genetic recombination, we are able to increase the production of industrially important metabolites. We combine genetic recombination technology with other molecular biology engineering to maximize the accuracy of phage genetic recombination.

Recombineering with dsDNA

dsDNA recombineering is useful for performing replacement knockouts and deletions, which are important for many genetic analyses. The frequency of these recombinants was greater than 10⁴ per 10⁸ viable cells

Recombineering with ssDNA

Using commercially available 40-70 base long oligonucleotides, precise mutations can be made at high frequencies, typically 10⁵/10⁸ viable cells, but some have ratios as high as several percent.

Application of Recombination

In vivo reconstitution is widely used in biotechnology, medicine and research.
The most common application of in vivo recombination is basic research, where the technique is important to most current work in biological and biomedical sciences.
In vivo recombination is used to identify, locate and sequence genes, and to determine their function.

Our Advantages

Expertise: We focus on improving the quality of our service, which is our only focus. We specialize in this area, which is why it is important to support yourself in improving any business improvement.
Flexibility: We are never tied to a particular approach, but instead focus on achieving the results our client's desire. We meet needs, adapt and provide the most appropriate approach and solution based on business objectives.
Depth and breadth of knowledge: We have many years of depth of knowledge and we are involved in business across multiple industries.

Creative Biolabs can meet the needs of customers by providing in vivo bacteriophage recombineering services on time and on budget. We have in-depth knowledge and experience of the tools and processes involved in the phage projects. Our skilled and dedicated scientific researchers ensure that the most suitable methods and techniques are selected for your project. If necessary, please feel free to contact us.

Reference

Pires D P, Cleto S, Sillankorva S, et al. Genetically engineered phages: a review of advances over the last decade[J]. Microbiology and Molecular Biology Reviews, 2016, 80(3): 523-543.

Please kindly note that our services can only be used to support research purposes (Not for clinical use).