IGEM"Caltech/2007/Project/Recombineering: Difference between revisions
Line 13: | Line 13: | ||
==Background== | ==Background== | ||
'''What is recombineering? Generally, how do you do it? | '''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. | |||
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. | 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. |
Revision as of 14:58, 25 October 2007
|