Topoisomerase I mediated TA cloning

See also TOPO TA cloning.

Topoisomerase
Topoisomerase recognition site -      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 -             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'

Upstream of the BioBricks prefix
Topoisomerase recognition site -            |             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 -                    |                     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' ^                  |                    -                    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
 * 1) Heyman-GenomeRes-1999 pmid=10207160


 * Vectors pcDNA3.1/GS and pYES2/GS

v              HindIII site --- 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' ---              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 --- 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' 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 - 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

 * 1) 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.
 * 2) 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).
 * 3) Can a restriction enzyme and topoisomerase I cleave simultaneously or will they occlude one another?
 * 4) Does TOPO TA cloning have a better success rate than TA cloning or is it just simpler?

Topoisomerase I

 * 1) Shuman-PNAS-1987 pmid=2823264
 * 2) Shuman-JBC-1990 pmid=2170398
 * 3) Shuman-JBC-1991 pmid=1314832
 * 4) Shuman-PNAS-1991 pmid=1658796
 * 5) Shuman-JBC-1992 pmid=1324909
 * 6) Shuman-JBC-1992b pmid=1314832
 * 7) Shuman-JBC-1994 pmid=7798275