What is recombineering? Generally, how do you do it?
Recombineering (recombination-mediated genetic engineering) is a recently developed in vivo technique for making recombinant DNA. As a fast and efficient alternative to classically used in vitro techniques, recombineering takes advantage of lambda phage's homologous recombination proteins collectively known as Red. Previous genetically engineered systems could not successfully insert linear DNA into E. coli due to degradation by nucleases. However, homologous recombination of ssDNA succeeded in the presence of the Red proteins, which inhibited the degrading nuclease in E. coli. Therefore, a defective lambda prophage was engineered with lysis and replication functions inhibited and Red functions retained. After creating cell strains containing this prophage, single-stranded oligos with the desired mutationscould successively be used for recombineering the phage.
We will use recombineering to create phages which will serve as suitable background strains for this project.
Our project requires phage strains with two main characteristics. First, our phage strains must be defective in expression of the N, Q, or cro developmental genes, while still being easy to propagate. Second, the strains must allow easy cloning of heterologous constructs -- our riboregulated N, Q, and cro expression constructs -- into them.
Recombineering allows us to satisfy the first constraint in an elegant way. Specifically, we will use recombineering to insert in-frame amber stop mutations into the coding sequences of N, Q, or cro. The amber stop mutation prevents successful translation of the genes in most E. coli strains, crippling the phages. By stopping translation with only a single point mutation, we minimize the mutation's impact on other regulatory and coding sequences. Finally, phages with amber stop mutations can be easily propagated in special amber suppressor E. coli strains, a technique which has been used for decades in lambda genetics.
Choosing a phage strain which facilitates insertion of heterologous sequences and tolerates their presence requires some care. The process is somewhat different than, for example, cloning heterologous DNA into a bacterial plasmid. Two features make this task slightly more involved. First, wild type lambda phage does not contain many unique restriction sites, making standard cloning techniques difficult. Second, since evolution has optimized lambda's small genome to have a high density of regulation, meaning that many stretches of DNA serve multiple functions.
Recombineering allows us to accomplish these goals in a single step, by modifying commonly used lambda cloning vector strains to be defective in the genes N, Q, or cro. For convenience, we initially chose to work with the Lambda Zap cloning vector (insert paper reference, JM Short et al, NAR 1988), available from Stratagene. This vector is a standard lambda phage strain engineered to contain multiple, unique, restriction sites in an unessential central portion of the lambda genome. (insert figure from NAR paper detailing MCS) Although we initially used this commercial strain for convenience, our method can easily be applied to freely available lambda cloning vectors.
Status and Future Plans
Where are we now? What's happening next?
The double-layer titering assay was used to screen for plaques corresponding to successfully recombineered phage (i.e. amber mutants). Unfortunately, the "cloudy" vs "clear" difference in amber mutant and wild type plaques proved to be more subtle than expected. Therefore, a new approach was taken consisting of carrying out the double-layer assay with the same amber suppressor layer and now a non-suppressor layer that expresses RFP. This modified experiment would simply require identifying plaques that pierce the bacterial lawn under visible light but appear confluent with the surrounding bacteria under fluorescence. After screening extracted plaques via single-layer titering in amber suppressing and non-suppressing cell strains revealed no amber mutants, we decided to go back to the recombineering process and re-design the single-stranded oligos.