IGEM:Caltech/2007/Project/Recombineering

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iGEM 2007

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Recombineering Background Recombineering (recombination-mediated genetic engineering) is a recently developed in vivo technique for making recombinant DNA [1]. 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. Integration 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 these 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 functions in the densely coded phage genome (insert endy ref). Finally, phages with amber stop mutations can be easily propagated in special amber suppressor E. coli strains, a standard decades-old technique in classical lambda genetics. To satisfy the second constraint, we choose a phage strain commonly used for cloning cDNA libraries. This phage strain, named Lambda Zap (by Stratagene) (insert NAR ref) is engineered to contain unique restriction sites in nonessential areas of the phage genome. This makes the strain much easier to work with than standard lambda strains, which contain few uniques restriction sites, often in the middle of critical genes. Note that we chose to work with a commercial strain for convenience, and that our method can be easily ported to freely available lambda phage cloning strains. 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. Relevant Protocols Titering Recombineering Two Layer Plaque Assay References Oppenheim AB, Rattray AJ, Bubunenko M, Thomason LC, and Court DL. In vivo recombineering of bacteriophage lambda by PCR fragments and single-strand oligonucleotides. Virology. 2004 Feb 20;319(2):185-9. DOI:10.1016/j.virol.2003.11.007 | Short JM, Fernandez JM, Sorge JA, and Huse WD. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583-600. DOI:10.1093/nar/16.15.7583 | pmid = 16729053