DNA ligation by selection

Figures & data

Figure 1.  LBS cloning vectors and approach.

Modified pUC18 sister cloning vectors pKos293-172-2 (3.6 kb; insert acceptor) and pKos293-172-A76 (3.9 kb; insert donor), showing restriction sites at the insert edges (A, B, and C). Both vectors contain a common XhoI site used for assembly of inserts. Vector pKos293-172-2 contains a unique Cm^R marker and EcoRV restriction site, while vector pKos293-172-A76 contains a unique Kn^R marker and StuI restriction site. For LBS, both acceptor and donor vectors are digested at the XhoI site and B site, the common site between the two adjacent fragments. The donor vector, pKos293-172-A76 in this example, is treated with StuI. The desired two fragments are ligated together, selected on Ap and Kn, and analyzed by gel electrophoresis after restriction with NotI/EcoRI. LBS, ligation by selection; Cm^R, chloramphenicol resistance gene; Kn^R, kayamycin resistance gene; Ap^R, ampicillin resistance gene.

Table 1.  Oligonucleotide Primers and Linkers

Primer	Sequence	Restriction Enzyme Site
Cm-For	5′-AATCCTGGTGTCCCTGTTGA-3′
Cm-Rev	5′-CTCGAGTTATTCAGGCGTAGCACCAG-3′	XhoI
Kan-For	5′-GCAGAGCGAGGTATGTAGGC-3′
Kan-Rev	5′-CTCGAGTGCGCGGAACCCCTATTTGT-3′	XhoI
UDG-L 1	5′-AGCTAGCGGCCGCCCTCAGCTATATCGCTATCGATGAGCTCAATGCATCGATCACTAGCTGAGGG-3′	N. BbvCIA, SacI,
		N. Bbv CIA,
UDG-L 2	5′-AATTCCCTCAGCTAGTGATCGATGCATTGAGCTCATCGATAGCGATATAGCTGAGGGCGGCCGCT-3′	N. Bbv CIA, SacI,
		N. Bbv CIA,
Eco RV L-1	5′-TACTGAGCTGTAGTGATATCATCTGCTGGACTTT-3′	Eco RV
EcoRV L-2	5′-GCGCAAAGTCCAGCAGATGATATCACTACAGCTCAG-3′	Eco RV
StuI L-1	5′-TACTGAGCTGTAGTAGGCCTATCTGCTGGACTTT-3′	StuI
StuI L-2	5′-GCGCAAAGTCCAGCAGATAGGCCTACTACAGCTCAG-3′	StuI

Restriction endonuclease recognition sites are underlined. Single-stranded overhangs used to insert linkers into vector shown in bold italics. For, forward; Rev, reverse; UDG-L, uracil-DNA glycosylase-based ligation.

Figure 2.  Dendrogram of a plan for a ten fragment, four-cycle recursive LBS synthesis of a 4.3-kb polyketide synthase module gene.

In parentheses are shown the unique resistance marker used for selection and the insert size for each LBS cycle. LBS, ligation by selection.