In vitro generation of genetic diversity for directed evolution by error-prone artificial DNA synthesis

Abstract

Generating genetic diversity lies at the heart of directed evolution which has been widely used to engineer genetic parts and gene circuits in synthetic biology. With the ever-expanding application of directed evolution, different approaches of generating genetic diversity are required to enrich the traditional toolbox. Here we show in vitro generation of genetic diversity for directed evolution by error-prone artificial DNA synthesis (epADS). This approach comprises a three-step process which incorporates base errors randomly generated during chemical synthesis of oligonucleotides under specific conditions into the target DNA. Through this method, 200 ~ 4000 folds of diversification in fluorescent strength have been achieved in genes encoding fluorescent proteins. EpADS has also been successfully used to diversify regulatory genetic parts, synthetic gene circuits and even increase microbial tolerance to carbenicillin in a short time period. EpADS would be an alternative tool for directed evolution which may have useful applications in synthetic biology.

Fig. 1. Working principle of in vitro generation of genetic diversity by error-prone artificial DNA synthesis (epADS).

At the beginning of the in vitro generation of genetic diversity by epADS, the target DNA of interest, either it is a protein-coding gene, regulatory genetic parts like promoter and riboswitch, or synthetic circuit, was in silicon designed into overlapped oligonucleotides sequences covering the DNA of interest. And then, chemical synthesis of the designed oligonucleotides with a high error rate was carried out under specific conditions (like high water content of DNA synthesis reagents, mixed dNTPs monomers, and specific reaction programs tested in this work). During this process, base errors like indels, and base substitutions may take place in portions of the synthesized oligonucleotides. After that, the synthesized oligonucleotides were assembled into double-stranded DNA of interest by different methods like annealing (<200 bp) and PCR ( > 200 bp). For an even longer DNA sequence (>2 Kb), the target DNA sequence was broken into several blocks and each block was obtained via the above methods, then these blocks were integrated via fusion PCR or by the following cloning step. Thereafter, the obtained double-stranded DNA with possible errors incorporated was cloned into suitable vectors or expression platforms. Finally, the constructed library was ready for mutant selection, screening or characterization via different approaches like solid plate screening, continuous selection in liquid culture, droplet microfluidics screening or fluorescence-activated cell sorting (FACS).

Fig. 2. Characterization of mutations generated by epADS under different suboptimal conditions and phenotypic characterization of three genetic parts generated by epADS.

a Types and frequency of mutations in four genes encoding fluorescent proteins (EmGFP, mCherry, BFP, and mBanana) generated by epADS with long-term used DNA synthesis solvents in the preliminary test. b Types and frequency of mutations in the gene encoding the fluorescent protein (EmGFP) generated by epADS in M1, M3, M5, and M6 test. A comparative test of in vitro generation of genetic diversity by ep-PCR was also carried out as described in “Methods” section. c Types and distributions of mutations in the gene encoding the fluorescent protein (EmGFP) generated by epADS of M6 test. Types of mutations were represented by numeric values on the Y-axis, 1: Base insertion (not observed); 2: Base deletion; 3: Base transition; 4: Base transversion. d Diversification in fluorescent strength of three genetic parts encoding fluorescent proteins EmGFP, Cherry, and mBanana modulated by epADS.

Fig. 3. Construction of serials of synthetic gene circuits encoding a red-green-blue (RGB) bio-palette system.

a Working principle of the Red-Green-Blue (RGB) bio-palette system. b Schematic illustration of the fluorescent phenotype of the RGB bio-palette system. c Color of cultures and cell pellets of serials of synthetic gene circuits encoding the RGB bio-palette system under illumination with room light or ultraviolet light. d Structure design of serials of synthetic gene circuits (P1~P6) encoding the RGB bio-palette system, otherwise indicated, T7 promoter, P_trc promoter, and T7 terminator was used for regulation of the expression of specific genetic parts.

Fig. 4. Phenotype characterization of regulatory genetic parts (P_trc promoter) modulated by epADS.

Fluorescent profiles of 12 colonies (a–i) from the P6-P_trcM mutant library generated by epADS of P_trc promoter were monitored to reflect its regulation strength on the downstream gene (mBanana). Fluorescent spectrum analysis was carried out with an excitation wavelength of 488 nm. The peak at 553 nm represented the emission signal of mBanana (red arrow), and the peak at 610 nm represented the emission signal of mCherry (black arrow).

Fig. 5. Phenotype characterization of regulatory genetic parts (tryptophan riboswitches) modulated by epADS.

a Structure illustration of two tryptophan riboswitches Blc3 and Blc4. b Structure design of two synthetic gene circuits P1-Blc3M and P1-Blc4M used to evaluate modulation of tryptophan riboswitches by epADS. c, d Fold of change in fluorescent strength of mCherry regulated by tryptophan riboswitch with/without tryptophan addition was determined to reflect its regulation activity on the downstream gene. 12 colonies from the P1-Blc3M mutant library and 13 colonies from the P1-Blc4M mutant library cultured in M9 medium with/without tryptophan (1 mM) were assayed for their fluorescent strength.

Fig. 6. Phenotypic characterizations of two synthetic gene circuits encoding an RGB bio-palette system modulated by epADS.

a Synthetic gene circuit P5 was constructed based on the pET28a(+) plasmid with the incorporation of genes encoding EmGFP, Cherry, and SBFP2 regulated by the T7 promoter and P_trc promoter respectively. The fluorescent strength of EmGFP, Cherry, and SBFP2 from colonies of the P5M6 mutant library generated by epADS was determined. b Synthetic gene circuit P6 was also constructed based on the pET28a(+) plasmid with the incorporation of genes encoding EmGFP, Cherry, and mBanana regulated by T7 promoter and P_trc promoter respectively. The fluorescent strength of EmGFP, Cherry, and SBFP2 from colonies of the P6M6 mutant library generated by epADS were determined.

Fig. 7. Combination of epADS with ALE for increasing microbial tolerance to antibiotics.

The bla gene generated by epADS was cloned and transformed into E. coli DH5a cells to obtain the parent culture for ALE with a stepwise increase of carbenicillin concentration. a Distribution ratio of different types of mutations in the starting library of ALE (AmpM6-R0). b Distribution ratio of different types of mutations in the evolved library of ALE (AmpM6-R15). Growth of cultures with the evolved bla gene after 24 h (c) and 48 h (d) of cultivation under various concentrations of carbenicillin. Results presented were obtained from tests with three biological replicates of the ALE experiment and four replicates of the growth inhibition assay.