Topoisomerase I mediated TA cloning
See also TOPO TA cloning.
Topoisomerase site
Topoisomerase
Topoisomerase recognition site
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Topoisomerase nick site
|
V
5' C C C T T N N N N N N
3' G G G A A N N N N N N
^
|
Restriction enzyme must nick here
Offset nicking enzyme
- Nt.BstNB I: offset nicking enzyme from NEB.
Nt.BstNB I recognition site
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Nt.BstNB I nick site
|
V
5' G A G T C N N N N N 3'
3' C T C A G N N N N N 5'
Site for topoisomerase-mediated cloning
Upstream of the BioBricks prefix
Topoisomerase recognition site
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|
V
5' C C C T T N N N G A C T C 3'
3' G G G A A N N N C T G A G 5'
^
|
---------
Nt.BstNB I recognition site
Downstream of BioBricks suffix
Nt.BstNB I recognition site
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|
V
5' G A G T C N N N A A G G G 3'
3' C T C A G N N N T T C C C 5'
^
|
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Topoisomerase recognition site
Possible enzymes to generate 3' T overhang
None of these enzymes will work because it appears as if the topoisomerase enzyme needs some duplex DNA in order to function.
Each can accommodate the topoisomerase site CCCTT
- BmrI
- offset cutter that leaves 3' single base overhang
- two sites in sopC (incD) repeat region of BBa_I50000
- no sites in BBa_I50020
- no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
- pretty high activity in all 4 NEB buffers
- BciVI
- offset cutter that leaves 3' single base overhang
- no sites in BBa_I50000
- one site in BBa_I50020
- no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
- Austin says this is a very bad enzyme.
- XcmI
- long recognition sequence with internal N9 that leaves 3' single base overhang
- no sites in BBa_I50000
- no sites in BBa_I50020
- no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
- 100% activity only in NEBBuffer 2
Topoisomerase sites internal to vector components are not important because topoisomerase only operates near double stranded breaks.
Based on the preexistence of sites in the designed parts, XcmI looks like a good bet but it is not as robust to buffer conditions as BmrI.
Topoisomerase I
Most of the papers reference Topoisomerase I from vaccinia. It seems to be available commercially at Epicentre. html protocol
- Anselm Levskaya - I've gotten Vaccinia WR strain isolate and am presently cloning the topoisomerase IB gene out. Classically this is purified
by using a phosphocellulose column but I'm going to just try a His-Tagged approach since it's a small protein.
Vector preparation
Reference
J. A. Heyman, J. Cornthwaite, L. Foncerrada, J. R. Gilmore, E. Gontang, K. J. Hartman, C. L. Hernandez, R. Hood, H. M. Hull, W. Y. Lee, R. Marcil, E. J. Marsh, K. M. Mudd, M. J. Patino, T. J. Purcell, J. J. Rowland, M. L. Sindici, and J. P. Hoeffler. Genome-scale cloning and expression of individual open reading frames using topoisomerase i-mediated ligation. Genome Res, 9(1088-9051 (Print)):383–92, 1999. pubmed
- Vectors pcDNA3.1/GS and pYES2/GS
v
HindIII site
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5' N N N N N A A G C T T N N N N N 3'
3' N N N N N T T C G A A N N N N N 5'
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HindIII site
^
- Cut with HindIII
5' C C C T T A
3' G G G A A T T C G A
A G C T T A A G G G 3'
A T T C C C 5'
- Ligate on oligos (TOPO-H and TOPO-4)
- TOPO-H destroys the HindIII site
- TOPO-4 provides recessed end
- In the drawings below, " | " refers to a break in the backbone on that strand.
TOPO-H oligo
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5' N N N N N A A G C T C G C C C T T A T T C C G A T A G T G
3' N N N N N T T C G A G C G G G A
------- -----------
HindIII overhang TOPO-4 oligo
TOPO-4 oligo HindIII overhang
------------ -------
A G G G C G A G C T T N N N N N 3'
G T G A T A C C T T A T T C C C G C T C G A A N N N N N 5'
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TOPO-H oligo
- Purify and cut again with HindIII to remove circular vector.
- Add TOPO-5 oligo and topoisomerase.
- Vaccinia topoisomerase I cleaves after and remains covalently attached to second T in CCCTT sequence.
Topoisomerase nick
v
TOPO-H oligo
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5' N N N N N A A G C T C G C C C T T A T T C C G A T A G T G 3'
3' N N N N N T T C G A G C G G G A | A T A A G G C T A T C A C A A C 5'
------- ----------- -------------------------------
HindIII overhang TOPO-4 oligo TOPO-5 oligo
TOPO-5 oligo TOPO-4 oligo HindIII overhang
------------------------------- ------------ -------
C A A C A C T A T C G G A A T A | A G G G C G A G C T T N N N N N 3'
G T G A T A C C T T A T T C C C G C T C G A A N N N N N 5'
------------------------------------------------
TOPO-H oligo
^
Topoisomerase nick
- Add TOPO-10X stop buffer
- Purify away free oligonucleotides and unbound topoisomerase I
Questions
- The authors add a primer TOPO-5 into the mix when they add the isomerase. It seems like this primer should not be necessary. Does the topoisomerase I "need" some duplex DNA extending out from where it will nick in order to cleave the backbone? Austin thinks most enzymes do need duplex DNA.
- How much of a performance hit will we take if we don't do all the purification steps they do ... it kind of seems like a lot of work (even though you only have to prep the vector once for multiple reactions).
- Can a restriction enzyme and topoisomerase I cleave simultaneously or will they occlude one another?
- Does TOPO TA cloning have a better success rate than TA cloning or is it just simpler?
