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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__toc__
 
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=Procedure=
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==References:==
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===Part A) PCR to get the required product.===
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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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*We designed primers flanking the region of interest (the zeocin cassette), and added 30 to 50 base pairs of homology matching the ECNR2 genome flanking the rpoZ gene. The lengths of the annealing regions of the primers are chosen so that they have a similar (within 1°C ) melting temperature for the PCR reaction.
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*The PCR reaction is done with the above primers, and a template of a small amount of liquid culture (diluted 30 times) of the omega knockout bacteria hybrid selection strain.
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*The product is run on a gel, and then either gel extracted (if there are side products) or PCR purified (if the reaction ran clear) and eluted in molecular grade water, not EB, since the ions and salts would otherwise cause electrical arcing during electroporation
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== Part B) Lambda-red recombination ==
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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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*1) Activate lambda red through temperature sensitive activation. For the ECNR2 strain, this involved taking the culture that’s grown in LB, at 30°C at around midlog, and placing it in a water bath at 42°C for 15 min to activate the lambda red machinery. This is because it has a temperature inducible promoter.
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*2) Add 1-1.5 mL of mid-log cells in culture to a 1.5 mL microcentrifuge tube. Spin in the centrifuge for 1 minute at 15000rpm (max speed)
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*3) (All steps need to be done on ice and the pipette tips, the cuvettes, and the molecular grade water must be kept cold at all times.)
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*4) Decant the liquid at the top, and use a pipette to remove as much of the liquid as possible without disturbing the bacterial pellet. Change tips. Wash cells with 1 mL of cold water, pipette up and down to mix them well, and spin in centrifuge for 1 minute at max speed.
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*5) Repeat the wash step.
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*6) Tip: When you are removing the liquid after spinning down, it helps to hold the tube so that the bacterial pellet is at the top and then use the pipette to draw out the liquid right below it. This is done so that the salts from the culture media can be thoroughly washed away, as they could interfere with the use of the electroporator from Step 10 (and onwards).
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*7) Resuspend cells with 50 μL of cold water.
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*8) Add 50-200 ng of insertion construct to the microcentrifuge tube with the bacterial pellet. This could also could be accomplished by taking a separate microcentrifuge tube, and adding enough of the insertion construct to almost 50 μL of cold water in an earlier step, so that these steps can be accomplished more quickly. Since the PCR product(insertion construct) from part A was at a concentration of about 70ng/μL to about 200ng/μL for the different times we used this method, we usually added 1-2μL of the construct to about 48-49μL of water in a microcentrifuge tube and placed it on ice before we began part B from step 2. It helps to reduce the amount of time the bacteria are suspended in water since it can lyse the cells because the surrounding solution is hypotonic to the bacterial cytoplasm.
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*9) Add the 50 μL solution to the cells, pipette up and down to mix them well, and then place in a chilled cuvette.
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*10) Set the electroporation machine to Eco1, 1.8 volts. A time constant of 4.8ms is a good number. You can also run a 'blank' to figure out the optimum time constant. A blank is run by filling a cuvette with 50 μL of water, which is put in the electroporator and pulsed, and its time constant is taken as as the standard. We want a time constant as close to that for water as possible, because pure water has no salts.
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*11) Before placing the cuvette in the electroporator, make sure that all water is wiped off of cuvette, so that it is dry, or it can interfere with electrical contact.
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*12) After electroporation immediately place 1 mL of LB in cuvette, pipette up and down to mix the cells in the LB and resuspend them.
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*13) Then place that 1mL of bacteria in LB into  2 mL of LB in a culture tube and allow them to grow for 2 to 3 hours in correct temperature (30°C for ECNR2).
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*14) After three hours, plate out culture in different concentrations of bacteria on specific plates (in this case, of zeocin, since that was in the construct, and it allows us to select for the lambda red insertion).
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*15) Grow up the cultures  on the plates overnight, and use a colony to do PCR or sequencing to check for the insert via its sequence, or the band size on the gel.
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=Applications of Lambda Red recombineering=
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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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==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"