Team:Harvard/Lambda Red

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__NOTOC__
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== Lambda Red ==
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To see how we used lambda red to build our selection system, go to our [https://2011.igem.org/Team:Harvard/Project/Test#Building_the_selection_strain:_Lambda_Red_Recombineering Test] page.  
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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 [https://2011.igem.org/Team:Harvard/Protocols#Lambda_Red Protocols] section. See [https://2011.igem.org/Team:Harvard/Results/One-Hybrid_Selection#Building_the_selection_strain:_Lambda_Red_Recombineering Lambda Red results] section for how we used lambda red to build our selection system.
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===Lambda red in ECNR2===
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We used a phage lambda-derived Red recombination system because of its efficiency
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===How to use ECNR2===
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For a step-by-step procedure, see our [https://2011.igem.org/Team:Harvard/Protocols#Lambda_Red Lambda Red Protocols].
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For efficient genome editing using lambda red, you can use [https://2011.igem.org/Team:Harvard/Results/Biobricks#EcNR2_strain_.28BBa_K615002.29 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.]
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Figure 1. Lambda Red, PCR to get the required insertion product (zeocin in this example)
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= Lambda Red=
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[[File:Lambda_Red,_Tech.gif |frameless|915px]]
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==Summary (adapted from Mosberg and Lajoie et al)[[#References|[1]]]==
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To make targeted genetic changes in E.Coli with larger constructs of tens to thousands of base pairs, unlike the smaller changes made in [https://2011.igem.org/Team:Harvard/Technology/MAGE MAGE], we can use lambda red recombineering to create insertions, deletions and replacements in chromosomal DNA.
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==How lambda red works (adapted from Yu et al)[[#References|[2]]]==
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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.
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For efficient genome editing using lambda red, you can use [https://2011.igem.org/Team:Harvard/Results/Biobricks#EcNR2_strain_.28BBa_K615002.29 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 [http://partsregistry.org/Part:BBa_K615002 the Registry of Standard Biological Parts.]
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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.
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[[File:Lambda_Red,_Tech_generic.gif |frameless|center|915px]]
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==References:==
==References:==
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[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.]
 
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[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.]
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'''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.[http://www.genetics.org/content/186/3/791]
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'''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.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165854/]
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'''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.[http://www.sciencemag.org/content/suppl/2011/07/13/333.6040.348.DC1/Isaacs.SOM.pdf]  
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[http://www.sciencemag.org/content/suppl/2011/07/13/333.6040.348.DC1/Isaacs.SOM.pdf ECNR2 strain details]
 
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Latest revision as of 03:54, 29 October 2011

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Overview | MAGE | Chip-Based Synthesis | Lambda Red | Protocols

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 [http://partsregistry.org/Part:BBa_K615002 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.

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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.[http://www.genetics.org/content/186/3/791]

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

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

Retrieved from "http://2011.igem.org/Team:Harvard/Lambda_Red"