Team:Harvard/Lambda Red

From 2011.igem.org

bar

To see how we used lambda red to build our selection system, go to our Test page.

For a step-by-step procedure, see our Lambda Red Protocols.

Lambda Red

Summary (adapted from Mosberg and Lajoie et al)[1]

To make targeted genetic changes in E.Coli with larger constructs of tens to thousands of base pairs, unlike the smaller changes made in MAGE, we can use lambda red recombineering to create insertions, deletions and replacements in chromosomal DNA.

How lambda red works (adapted from Yu et al)[2]

Even though homologous recombination can occur naturally, its efficiency can be greatly enhanced through the presence of the phage λ-based recombination system, Red. While it is very efficient, the exact mechanism is not known. For efficient genome editing using lambda red, you can use the ECNR2 strain with your own insertion construct and overhangs (i.e. 30-50bp homology to the locus in which the gene is being inserted). The ECNR2 strain is especially suited for lambda red recombineering, because in addition to having the lambda-phage based recombination system, it also has the mutS gene knocked out to reduce DNA mismatch repair activity, so that the insert (which will not match the original genomic code) is less likely to be excised. To obtain this strain, you can make a request at the Registry of Standard Biological Parts.

Figure 1. How to perform lambda red. First, we run a PCR to get the required insertion product i.e. construct A with an antibiotic resistance marker, and then use lambda red recombination to insert the desired product into the genome.

Lambda Red, Tech generic.gif

References:

1. Mosberg JA, Lajoie MJ, Church GM. Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 2010;186:791-799.[1]

2. Yu, D., H. M. Ellis, et al. (2000). "An efficient recombination system for chromosome engineering in Escherichia coli." Proceedings of the National Academy of Sciences of the United States of America 97(11): 5978-5983.[2]

3. (Supporting material for) Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science, 333(6040):348-53.[3]