DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides.The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs.
Timeline & development
Since the first reports of hybrid gene synthesis, PCR-based gene cross-overs and PCR-based gene synthesis were published, the idea of directing evolution initialized the development of various methods for the shuffling of gene libraries, which permit homologous recombination in vitro. To date, however, all of these methods have not been without disadvantage or difficulty. In the well-established protocol of Stemmer, DNase is used to fragment DNA requiring careful optimization of the digest conditions, e.g. time, temperature, amount of nuclease and DNA.
Other methods such as the staggered extension process and random-priming is limited by the DNA composition, and matters are complicated further by the lack of controllability of the range of fragment sizes generated. Methods such as RACHITT also require DNase digests and are even more labor intensive. The race for the best method is still on. Simple comparisons can be helpful but need to be taken with caution since the gene length, the homology of the shuffled gene libraries and the intended cross-over rate would have to be taken into account. Besides the homology-dependent methods, which are related to the presented data, homology-independent methods have also been developed based on DNA fragment fusion [e.g. thio-ITCHY and SHIPREC].
Nucleotide Exchange and Excision Technology (NExT) DNA shuffling is based on the random incorporation of ‘exchange nucleotides’. The occurrence and position of these exchange nucleotides in the DNA will dictate the subsequent fragmentation pattern without the need for further adjustment. The highly homologous libraries with a few members to be able to analyze our fragmentation and shuffling results was used inthis method.
The key advantages of our method are
- calculable experimental setup aided by a computer program,
- reproducible end-point reactions without adjustments,
- no gel purification required,
- efficient reassembly with a proofreading polymerase,
- gene recombination including very short fragments of only a few bases,
- low error rate and
- practically no contamination with unshuffled clones.
- Genes used in the NExT DNA shuffling procedures were the 657 bp chloramphenicol acetyl transferase I (CAT) wild-type gene and variants coding for an N-terminally 10 amino acid truncated C-terminally 9 amino acid truncated or double-truncated CAT or C-terminally 26 amino acid truncated CAT.
- For shuffling and error-prone PCR genes were amplified using the primers Pr-N-shuffle (5′- ATTTCTAGATAACGAGGGCAA-3′) and Pr-C-shuffle (5′-ACTTCACAGGTCAAGCTTTC-3′) for the wild-type and N-terminally truncated genes, Pr-N-shuffle and Pr-Cdxshuffle (5′-CTTCACAGGTCAAGCTTATCA-3′) for the C-terminally truncated and for the double-truncated genes.
- Priming sites were located shortly before and after the gene adding in total 45 nt to the genes and contained the restriction sites XbaI and HindIII for cloning into the vector pLisc-SAFH11 thus replacing part of the original plasmid.
- Plasmids were transformed by electroporation in Escherichia coli strain RV308 using standard methods.
- Mutated variants of the various clones were obtained by error-prone PCR using 2.5 U Taq polymerase in a 50 ml reaction supplemented with vendor-supplied buffer and with 7 mM MgCl2, 0.5 mM MnCl2, 0.4 mM each dNTP and 50 ng template.
- The PCR protocol was as follows: 1 cycle of 94oC, 3 min; 30 cycles of 92oC, 1 min; 60oC, 1 min; 72oC, 2 min; 1 cycle of 72oC, 7 min.
Uridine exchange PCR
The uridine versus thymidine exchange PCR mixture contained 50 ng template (0.017 pmol of a 4340 bp plasmid), 25 pmol of each primer, 0.2 mM of dATP, dGTP and dCTP each, a 0.2 mM mixture of dUTP:dTTP in various ratios, 5 U Taq DNA polymerase , and 5 ml of 10* PCR buffer containing 160 mM (NH4)2SO4, 670 mM Tris–HCl, pH 8.8 (at 25oC), 15 mM MgCl2, 0.1% Tween-20 for the reactions shown in the ethidium bromide stained gels and 5 ml 10*PCR buffer containing 100 mM Tris–HCl, pH 9.0 (at 25 oC), 500 mM NaCl, 15mMMgCl2, 1% Triton X-100 for the reactions shown in the autoradiographed gel. The volume was adjusted to 50 ml with H2O. Before adding to the reaction, the 100 mM nucleotide stock solutions were diluted in water to 10 mM for dATP, dGTP and dCTP, and to 1 mM for dUTP and dTTP. For radioactive experiments, 0.5 ml of a 3.3 mM [32P]dCTP solution or 0.5 mCi, respectively, were added. The cycler program was as follows: 1 cycle of 94oC, 1 min;25 cycles of 92oC, 30 s; 62oC, 20 s; 72 oC, 2 min; final incubation 72 oC, 4 min. To obtain sufficient product, four 50 ml reactions were combined, separated on a 1% agarose gel, purified using one column of a PCR clean-up kit and eluted with 50 ml of 10 mM Tris, pH 8.0. For radioactive experiments, the clean-up kit was used without the gel step. The concentration of the PCR product was determined by taking the baseline corrected 260 nm value of an absorption spectrum from 220 to 350 nm of a 1:30 diluted 5 ml aliquot in a 140 ml microcuvette. Product yield for 200 ml of PCR was 10–17 mg.
Enzymatic digest and chemical cleavage
About 15 mg in 45 ml (minimal 7 mg) of the purified PCR product was supplemented with 6 ml supplied uracil-DNA glycosylase (UDG) 10 X buffer and 2 U E.coli UDG, adjusted to 60 ml with water and digested for 1 h at 37oC. The DNA was cleaved by adding piperidine to a final concentration of 10% (v/v) and heated for 30 min at 90oC in a thermocycler with a heated lid. Piperidine is toxic and should be handled in a hood. Alternatively, piperidine was replaced by a 5 M NaOH stock solution added at 10% (v/v) to the cleavage reaction.
Fragments were purified directly from the piperidine or NaOH cleavage using the gel extraction kit according to the manufacturer manual. The capture buffer included was added and neutralized (20 ml of 3 M Na-acetate, pH 5.3). After two washing steps, fragments were extracted two times with 25 ml of 10 mM Tris pH 8.0 and pooled. Two centrifugation steps with transfer to a fresh tube ensured that oligonucleotides were not contaminated with the matrix. For the extraction of fragments from polyacrylamide-urea gels, the excised slices were crushed and incubated either with 1 ml water or diffusion buffer containing 0.5 M ammonium acetate, 10 mM magnesium acetate, 1mMEDTA, 0.1% SDS, pH 8.0 in a thermomixer at 37oC, 1000 r.p.m. overnight. The water extracted oligonucleotides were precipitated by adding sodium acetate, MgCl2, and 2-propanol. The diffusion-buffer-extracted fragments were purified with the purification kit as described above. Initially, fragments were quantified by mixing with SYBR Green II (Molecular Probes) and measuring fluorescence emission intensity relative to a 60 bp oligonucleotide calibration curve.
Denaturing polyacrylamide urea gel
Gels were composed of 6.7 M urea, 11.3% polyacrylamide/ bisacrylamide (37.5:1), 1·X TBE, ammonium peroxodisulfate and TEMED. Gels (10 cm * 8 cm ·* 1 mm) were prepared freshly, as older gels did not run properly, and electrophoresed in a small basic unit heated to 56oC with an attached temperature-controlled water bath. Before loading, the cleaved DNA was concentrated to 7 ml in a speed-vac in order to evaporate the piperidine, supplemented with 25 ml of deionized formamide and heated to 80oC for 3 min in a thermocycler; 9 ml of the sample were loaded on the gel. For the radioactive experiments, 7 ml of the DNA-formamide sample was additionally supplemented with 3 ml of 60% sucrose solution, which improved loading, and 7 ml H2O and then 15 ml were loaded. Oligonucleotides of 20, 38, 48, 58, 65 or 68 nt, as well as a 100 bp ladder with added bromophenol blue dye, served as a visible length standard. For radioactive experiments, the oligonucleotides were kinased with [g-32P]ATP and purified by size exclusion. After heating and a 10 min pre-run at 100 V, the gel was loaded and run at 170 V until the dye was 1– 2 cm from the bottom of the gel. The gel was stained with 30 ml, 1.2 mg/ml ethidium bromide for 5 min. Note that with longer incubation times the smaller fragments start to elude from the gel and exposure to UV light bleaches the gels.
Gene reassembly and amplification
For the reassembly 2 mg of the purified DNA fragments (typically 20 ml) were mixed with 4 ml of a mix of 10 mM of each dATP, dTTP, dCTP and dGTP (800 mM final), 4 U Vent DNA Polymerase (NEB) with 1–4 ml of 25 mM MgSO4 and 5 ml supplied 10 X buffer. For the experiments with Taq, 1 or 4 ml of the dNTP mix were tested (without noticeable differences in yield) and 5 U of the enzyme were used. The volume was adjusted to 50 ml. In case of the Vent polymerase, even <1 mg of DNA was sufficient. Cycles for the reassembly were as follows: 1 cycle of 94oC, 3 min; 36 cycles of 92oC, 30 s; 30oC, 60 s + 1oC per cycle (cooling ramp 1oC/s); 72oC, 1 min + 4 s per cycle; final incubation at 72oC, 3 min. Ten microliters of the reassembly product (this volume was chosen to ensure diversity) were amplified using a standard PCR reaction (25 pmol primers, 0.2 mM dNTPs, 25 cycles, 40 s elongation time) with the appropriate primers listed in cloning steps. Amplified genes were cloned via the XbaI and HindIII restriction sites, and plasmids prepared from E.coli grown on plates without selection pressure were named, e.g. pNd10_Cd9_control# and equivalents. Clones were sequenced using the cycle sequencing kit and analyzed in an Genectic analyzer- sequencer.
RESULTS AND DISCUSSION
The NExT procedure was developed and tested by increasing the functionality of truncated mutants of chloramphenicol acetyltransferase I (CAT), which mediates resistance against the antibiotic chloramphenicol. Directed evolution was independently applied to four sets of variants truncated at the genetic level. The first library of CAT mutants was shortened by ten amino acids at the N-terminus (CAT_Nd10) while maintaining the start methionine, the second library by 9 amino acids at the C-terminus (CAT_Cd9), the third library by 26 amino acids at the C-terminus (CAT_Cd26), and the fourth library was truncated at both ends by 10 and 9 amino acids (CAT_Nd10_Cd9), respectively. Besides testing the NExT method, these experiments were set up to elucidate the structure–function relations of this thermostable enzyme. We also wanted to test the applicability of our structure perturbation strategy to improve the thermostability of already thermostable enzymes. Detailed data for the NExT shuffling were obtained using test ‘libraries’ with three to six members selected from error-prone PCR diversification steps containing 14–49 mutations within the genes of 627 (CAT_Nd10, CAT_Cd9) to 579 (CAT_Cd26) bp in length. The small number of library members with a manageable set of mutations ensured that almost all mutations found in recombined clones could be unambiguously traced to parental segments. The melting temperature of the truncated CAT_Nd10 variant was increased by 24oC as detected by circular dichroism measurements.
Evaluation of NExT shuffling
The NExT DNA shuffling procedure described so far has been applied to the directed evolution of a 600 bp long CAT gene truncated at both ends (CAT_Nd10_Cd9). In the course of these experiments, a defined library of six clones with different mutation patterns between nucleotides 12 and 383 was shuffled based on a 33.3% uridine exchange PCR. Eight shuffled clones taken from control plates without selection pressure were sequenced. The unique mutation pattern of these clones showed that all clones tested were derived from at least two (e.g. clone 1) to four (e.g. clone 4) parental clones. Within the 372 bp stretch amenable to analysis, this resulted in one cross-over per 93 to 186 bp with a mean fragment length of 114 bp. Sequencing also determined the error level of this procedure. Within 4425 bases sequenced, four alterations were found (one A to G and one T to C transition, a 1 bp insertion and a 1 bp deletion) giving a mutation rate of 0.09%. This is remarkably lower than an error rate of 0.7% reported previously for DNase shuffling.
As our fragment distribution and cross-over rate were comparable to previous experiments, we are inclined to attribute the previously reported error rates more to the DNase digest and to the UV damage due to gel visualization rather than the fragment size and the polymerase. A low mutation rate is particularly important when shuffling of longer DNAs is envisioned as this will avoid dilution of the gene pool with dysfunctional or undesired molecules. Using a proofreading polymerase for the amplification of the gene assembly could further lower the error rate. In another experiment, four parental truncated CAT genes (CAT_Cd26) containing a total of 49 mutations spread from bases at position 9–575 were shuffled and five clones sequenced. A detectable mean fragment length of 86 bases was found, including fragments down to only 8 bases. The mean fragment length in this experiment is smaller than in the previous one, as more mutations result in a better detection of the fragment length. The short fragments used was purified to significant amountsor, more likely, by efficient priming with frequent strand switching for each PCR cycle. In general, gene assembly is a complex process mainly but not only determined by fragment length.
A complete directed evolution series based on error-prone PCR and NExT DNA shuffling was applied to improve the enzymatic activity of truncated CAT_Nd10, which grew on plates up to only 25 mg/ml chloramphenicol in the presence of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG), and CAT_Cd9, which failed to grow at all. After optimization, several clones of both libraries grew even at 400 mg/ml chloramphenicol / IPTG, demonstrating the efficacy of this technique. In addition, the preferred method was applied to TEM-1 b-lactamase using a dUTP fraction of 30%. The fragmentation and assembly worked in the first experiment only ensuring sufficient material to start with but without any prior or intermediate tests or analysis steps.