Thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR

Abstract

Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.

Keywords: amplification of circular plasmids; directed evolution; error-prone PCR; random mutagenesis libraries; thermostable DNA ligase.

Figure 1.

Schematic of swapping selection marker of a plasmid by thermostable DNA ligase-mediated PPCP. The white boxed-region of the template plasmid (Vector 1) represents the original selection marker, which after the whole-plasmid PCR is replaced by a second selection marker built in the double-stranded PPCP primers (the black boxed-region in the primers and Vector 2). The procedure features the incorporation of a thermostable ligation step into the standard PCR procedure to ligate the nick remaining in the extended DNA strand, resulting in nick-free, circular plasmid DNA products. At the end of the PCR cycles, the template plasmid is eliminated by plating the plasmid-transformed host cells under the second selection pressure.

Figure 2.

Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh-amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.

Figure 3.

Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh-kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh-xynA1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.

Figure 4.

Schematic of directed evolution by in situ error-prone PCR using the thermostable DNA ligase-mediated PPCP method to create random mutagenesis libraries. (a) In situ error-prone PCR using PPCP. Primers (amplified from the vector with a swapped selection marker, e.g. from amp^r to kan^r by PPCP as described in the legend to Fig. 1) anneal to and cover the entire template plasmid sequence minus that of the target sequence (slashed boxed region); PPCP is performed as described in the legend to Fig. 1, except that the target DNA sequence is synthesized under error-prone conditions by using mutagenic DNA polymerase. (b) Transforming the circular PCR products into host cells and selecting for the second marker while screening mutants for desired mutation. (c and d) The process of (a) and (b) can be repeated for multiple rounds of PPCP mutagenesis, marker selection and functional screening.

Figure 5.

Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh-xynA1 as template and the PPCP primer pair from pHsh-kan. Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh-xynA1. The size of xynA1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh-celB.