Team:Harvard/Lambda Red

Lambda Red

Lambda red recombineering makes use of homologous recombination systems to allow the insertion of constructs into the genome. It is accomplished in two steps, as shown in the diagram below and in our Protocols section. See Lambda Red results section for how we used lambda red to build our selection system.

Lambda red in ECNR2

We used a phage lambda-derived Red recombination system because of its efficiency

How to use ECNR2

For efficient genome editing using lambda red, you can use this strain with your own insertion construct and overhangs. To obtain this strain, you can make a request at [http://partsregistry.org/Part:BBa_K615002 the Registry of Standard Biological Parts.]

Figure 1. Lambda Red, PCR to get the required insertion product (zeocin in this example) 915px

References:

1.[http://www.nature.com/nature/journal/v460/n7257/full/nature08187.html Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest, George M. Church. Programming cells by multiplex genome engineering and accelerated evolution. (2009). Nature, 460(7257):894-8.]

2.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165854/ 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.]

3.[http://www.genetics.org/content/186/3/791 Mosberg JA, Lajoie MJ, Church GM. Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 2010;186:791-799.]

4.[http://www.sciencemag.org/content/suppl/2011/07/13/333.6040.348.DC1/Isaacs.SOM.pdf (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.]