IGEM:Caltech/2007/Project/Recombineering: Difference between revisions
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Recombineering (recombination-mediated genetic engineering) is a recently developed ''in vivo'' technique for making recombinant DNA <cite>oppenheim</cite>. 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. Cells containing this prophage facilitate homologous-recombination based engineering of novel genetic sequences, hence the term <i>recombineering.</i> | Recombineering (recombination-mediated genetic engineering) is a recently developed ''in vivo'' technique for making recombinant DNA <cite>oppenheim</cite>. 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. Cells containing this prophage facilitate homologous-recombination based engineering of novel genetic sequences, hence the term <i>recombineering.</i> | ||
One can adapt recombineering to introduce short mutations into lambda phage genomes <cite>oppenheim</cite>. Briefly, E. coli cells expressing the proper prophage gene suite are infected with the lambda phage strain to be engineered. The cells are then made electrocompetent and single stranded oligos (about 70 nt) containing the desired mutation, plus flanking homology regions, are electroporated in. The new phages, a mixed population of wild type and engineered, are then allowed to lyse the host cells. The resulting lysate is screened in an appropriate manner to detect the mutation of interest. | One can adapt recombineering to introduce short mutations into lambda phage genomes <cite>oppenheim</cite>. Briefly, ''E. coli'' cells expressing the proper prophage gene suite are infected with the lambda phage strain to be engineered. The cells are then made electrocompetent and single stranded oligos (about 70 nt) containing the desired mutation, plus flanking homology regions, are electroporated in. The new phages, a mixed population of wild type and engineered, are then allowed to lyse the host cells. The resulting lysate is screened in an appropriate manner to detect the mutation of interest. | ||
In our particular application, we are introducing amber stop codons in frame to <i>N</i> and <i>Q</i>. To screen for these mutations, phages are plated for plaques in an amber suppressor strain. A further layer of non-amber suppressing cells, without phages, is immediately plated on top of the amber suppressing layer. Phages without the amber mutation will make plaques that 'punch through' both layers, while amber mutant phages will be unable to penetrate the upper layer. The two types of plaques can be distinguished by eye, allowing us to screen for amber mutants. | In our particular application, we are introducing amber stop codons in frame to <i>N</i> and <i>Q</i>. To screen for these mutations, phages are plated for plaques in an amber suppressor strain. A further layer of non-amber suppressing cells, without phages, is immediately plated on top of the amber suppressing layer. Phages without the amber mutation will make plaques that 'punch through' both layers, while amber mutant phages will be unable to penetrate the upper layer. The two types of plaques can be distinguished by eye, allowing us to screen for amber mutants. |
Revision as of 08:19, 26 October 2007
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