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 and the procedure below.
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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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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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=Applications of Lambda Red recombineering=
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==References:==
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==Kan-ZFB-wp==
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In this case, for the his3 ura3 system, we inserted, in order, a Kanamycin cassette, the zif268 binding site (ZFB-Zinc Finger Binding site), and the weak promoter that has low levels of transcription on its own, but high levels of transcription when bound to the omega subunit that is attached to the ZFP (Zinc Finger Protein). After recombineering, the bacteria were plated on kanamycin agar plates to select for the insert.
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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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==Tet-ZFB-wp==
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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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After we got the selection system working with the zif268 protein and binding site, we swapped out the ZFB for the other sequences, and switched out the Kanamycin cassette for a Tetracycline cassette. This allowed us to change the binding site and select for cells that had the changed binding site, and the new ZFB
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==zeocin substituting rpoZ==
 
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In this case, the rpoZ gene is the bacterial homolog of the omega subunit on the expression plasmid for the ZFP (Zinc Finger Protein). In order to bind the level of transcription of his3-ura3 to the expression of the ZFP, it would need to be knocked out, so that there would be a reduced level of constitutive expression of the his3-ura3. Since rpoZ is a RNA polymerase subunit, knocking it out would reduce the viability of the bacteria, so we could not simply knock it out using MAGE. As a result, we used a zeocin cassette to confer an antibiotic resistance to the bacteria, which we then selected for through zeocin agar plates.
 
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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.

915px

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"