Organisms that permit hypermutation of target genes without off-target mutagenesis of the host genome enable the accelerated, continuous evolution of genes for new or enhanced functions. We develop and optimize an orthogonal DNA replication system in Escherichia coli that uses components from bacteriophage Φ29.
Organisms that permit hypermutation of target genes without off-target mutagenesis of the host genome enable the accelerated, continuous evolution of genes for new or enhanced functions. We develop and optimize an orthogonal DNA replication system inthat uses components from bacteriophage Φ29.
The minimal system requires just two Φ29 genes to maintain the replicon and replicons can be efficiently engineered in vivo. We generate a highly mutagenic Φ29 DNA polymerase that introduces mutations at a frequency approaching 10per base per generation . Our system is stable for hundreds of generations and enables the continuous, accelerated evolution of new gene functions. We demonstrate the rapid evolution of a tetracycline resistance gene to confer resistance to tigecycline at higher levels than achieved with previously reported systems. We further evolve a 1,000-fold increase in β-lactamase activity for a third-generation cephalosporin in just 3 days.The high-fidelity replication of an organism’s genome is essential for maintaining genetic integrity and limits mutations that could be detrimental to survival or reproduction; therefore, the natural evolution of new gene function, resulting from the accumulation of mutations and selection within a population, is a slow process.. Classical directed evolution experiments rely on cycles of in vitro genetic diversification followed by transformation into an appropriate host for selection. These types of experiments are laborious and limited in terms of evolutionary depth and scale. Continuous evolution strategies, enabled by in vivo mutagenesis and selection of target genes, may overcome these limitations and allow for the accelerated exploration of fitness landscapes at scale. However, the sequence that can be explored may be a function of the mutational spectrum that is accessed, the copy number of the genes of interest and whether selective pressure is continuous or intermittent.. This restriction limits both the number of generations for which the selection can be performed and the phenotypes that can be evolved.sidesteps some of the limitations of the initial approaches. However, these approaches are limited to evolving small genes that can be packaged into viruses and are often run noncontinuously for practical reasons. Other strategies rely on the localization of a mutagenic moiety, such as an error-prone polymerase or a deaminase, to a target gene. These approaches are generally restricted by their mutagenic window and limited orthogonality; these limitations may lead to off-target mutations that can culminate in selection escape. By contrast, orthogonal replication systems, where a dedicated error-prone DNA polymerase specifically maintains an episome harboring the target genes without interfering with genome replication, can enable straightforward and robust continuous evolution experiments. The first such system, OrthoRep, exploits natural linear plasmids from yeast and has enabled a range of continuous evolution experiments at high mutation ratesas a host were realized by a recent orthogonal replication system, EcORep; this system uses components from the PRD1 bacteriophage, which naturally infects a range of Gram-negative bacteria, including. However, the initial mutation rates achieved by this system were only modestly higher than the genomic mutation rates encodes 27 protein-coding genes and a prohead RNA that is essential for genome packaging at each end. The genome is capped at its 5′ ends by covalently bound terminal proteins ; these proteins prime replication by the Φ29 DNAP, A synthetic replication operon was designed on the basis of genes 3, 2, 6 and 5, encoding the TP, DNAP, DSB and SSB, respectively. The synthetic replicon was designed to encode an antibiotic resistance gene and a gene of interest and was flanked by the left and right origins of replication derived from the Φ29 genome., Φ29 synthetic replicons could be established by electroporating a PCR-derived product into cells harboring the Φ29 synthetic replication operon on a single-copy plasmid.and the genes encoding the Φ29 SSB and DSB. The Φ29 synthetic replicons extracted from cells could be transformed with a higher efficiency ; error bars indicate the s.d. We note that ‘Plasmid extract’ includes the replicon and copurified plasmid encoding the replication operon; thus, the quantity of replicon transformed is lower than 300 ng., Stability of the TcR–GFP Φ29 synthetic replicon over 550 generations with or without tetracycline, as assessed by maintenance of GFP fluorescence using flow cytometry , DNAP , double-stranded DNA-binding protein and single-stranded DNA-binding protein ; a combination of these proteins has been used to amplify portions of the Φ29 genome or heterologous genes in vitro. We arranged these genes into an operon under the control of an IPTG-inducible promoter, PtacIPTG, on a single-copy plasmid and GFP, flanked by the left and right Φ29 origins of replication to inhibit host nucleases , as well as the Φ29 DSB and SSB to further protect the electroporated replicon DNA. Upon electroporation of the Φ29 synthetic replicon into cells bearing the helper plasmids and the synthetic replication operon, we observed a small number of tetracycline-resistant colonies that exhibited green fluorescence. These experiments suggested that the Φ29 synthetic replicon was established in these cells and by experiments where we could reduce the replicon copy number by downregulating expression from the replication operon by targeting the promoter driving the operon with dCas9 . Extracting the established replicons from cells bearing the helper plasmids and the synthetic replication operon and electroporating them into fresh cells enabled substantially improved transformation efficiencies, even when the helper plasmids providing genes encoding Gam and Φ29 DSB and SSB were not present in the recipient cells . This revealed the replication operon itself to lead to a mild reduction in cell fitness , whereas cells harboring both the replication operon and the Φ29 synthetic replicon exhibited substantially slower growth and a lower maximum cell density . Without tetracycline, the replicon was lost in less than 50 generations. In the presence of tetracycline, the replicon was stably maintained for the entire 550 generations tested. Therefore, the Φ29 synthetic replicon can be stably maintained for many generations, as required for continuous directed evolution experiments.. We generated a set of single-copy plasmids harboring variants of the Φ29 synthetic replication operon; in each member of the set, an individual gene was disrupted. We then electroporated the Φ29 synthetic replicon into cells containing each of these plasmids and assessed the formation of colonies that exhibited tetracycline resistance and green fluorescence. This experiment revealed that only two genes, encoding TP and DNAP, are essential for replicative maintenance of the Φ29 synthetic replicon . We passaged cells harboring the Φ29 synthetic replication operon, with or without the genes encoding the SSB and DSB proteins, and a TcR–GFP Φ29 synthetic replicon for 50 generations and assessed whether the Φ29 synthetic replicons remained intact by colony PCR . We only observed full-length replicons, suggesting that the absence of the SSB and DSB did not notably impair replication fidelity. Our results are in agreement with in vitro efforts to replicate the Φ29 genome using only TP and DNAP and the self-replication of TP-encoding and DNAP-encoding replicons in synthetic protocellsAs the establishment of new Φ29 synthetic replicons from PCR amplicons was a low-efficiency process, we sought to develop a strategy for engineering replicons that were established in cells to introduce new DNA sequences of interest. The lambda Red recombination system, from the lambda phage, has been extensively used for engineering bacterial genomes and plasmids through the introduction of linear DNA flanked by short stretches of homology to the target site. To assess whether this recombination system could be used to engineer Φ29 synthetic replicons, we tested replacing the tetracycline resistance gene on the replicon with a kanamycin resistance gene. We electroporated 0.5 μg of a PCR product consisting of a kanamycin resistance gene flanked by 60-bp homologies into cells harboring a Φ29 synthetic replication operon plasmid, a tetracycline-resistance-conferring Φ29 synthetic replicon and a plasmid encoding the lambda Red recombination system components under arabinose-inducible control , we randomized the first codon following the start codon and we randomized codons 2–7 to synonyms . Error bars indicate the s.d. CFU, colony-forming unit.We passaged cells transformed with the Φ29 synthetic replication operon library and a Φ29 synthetic replicon to enrich operons with favorable properties. To select for operons that supported increased replicon copy numbers, we picked colonies exhibiting stronger GFP fluorescence. We identified an operon, Φ29-opt and faster growth while maintaining a Φ29 synthetic replicon to higher cell densities . We hypothesize that the improved growth is because of the increased capacity to maintain the Φ29 synthetic replicon, ensuring stable transfer of the replicon to daughter cells at every division. We next tested whether Φ29-opt could support longer Φ29 synthetic replicons. We used lambda Red recombination to insert variable lengths of thegene onto a KanR–GFP replicons to yield replicons with lengths of 6.6 and 10.5 kb. The longer replicons were established and maintained, as assessed by genotyping and sequencing of the Φ29 DNAP; in in vitro experiments, these individual DNAP mutants are reported to mutate the DNA that they replicate while retaining strand-displacement activity, An orthogonal replication system enables hypermutation of a target DNA sequence without interfering with the high-fidelity replication of genomic DNA., Error-prone mutants of the Φ29 DNAP do not increase the genomic mutation rate but do increase the replicon mutation rate. The mutagenic T7 DNAP mutant has previously been reported. We used fluctuation analysis to calculate the mutation rate from the frequency of a TAG stop codon reversion in a genomically or replicon-integrated chloramphenicol resistance gene . After ten generations of growth, with each DNAP variant and a wild-type control, we measured the fraction of chloramphenicol-resistant cells arising from a point mutation in the TAG stop codon to generate sense codons. These experiments allowed us to determine the apparent mutation rates of the replicon in the presence of each DNAP using fluctuation analysis). To determine the mutational spectra of these error-prone DNAP mutants, we grew cells harboring a Φ29 synthetic replicon for up to 50 generations and measured the accumulation of mutations using next-generation sequencing . We sequenced replicons from cells growing on high tigecycline concentrations and found that certain mutations had convergently emerged in our three independent replicates . Of note are W233, which was substituted to C or S in every clone, A393, which was substituted to S or D in every clone, P193, which was substituted to S, T or N in 79% of clones, G388, which was substituted to V in some clones from all replicates but in all clones from the third replicate, and V355, which was substituted to F in two of the three replicates . We cloned select sequences from each replicate into a standard circular plasmid to further validate the performance of these mutants with the residues that were substituted in at least two independent evolution replicates shown in stick representation. All observed substitutions are summarized in Supplementary Data) with the residues that were substituted in at least two independent ceftazidime resistance evolution replicates shown in stick representation. All observed substitutions are summarized in Supplementary DataNext, we sought to rapidly evolve TEM-1 β-lactamase for improved activity against the third-generation cephalosporin ceftazidime. Third-generation cephalosporin antibiotics differ from earlier generations because of their improved resistance to β-lactamase activity. We passaged cells harboring a replicon encoding the TEM-1 β-lactamase in increasing concentrations of ceftazidime, ranging from 2 to 500 µg ml. We sequenced replicons from cells growing on high ceftazidime concentrations and found that certain mutations had convergently emerged in our four independent replicates . Of note are E102, which was substituted to K in every sequenced clone, R162, which was substituted to S or N in every sequenced clone, E237, which was substituted to K in some clones in two independent evolution replicates, and E166, which was substituted to D in some clones from three of the four independent evolution replicates . We cloned select sequences from each replicate into a standard circular plasmid to further validate the performance of these mutants . By contrast, the ceftazidime-resistant pools, which were the input to this evolution, grew poorly or not at all at greater than 10 µg mlof cefotaxime. We sequenced replicons from cells growing on high cefotaxime concentrations and found that certain mutations had convergently emerged between replicates 1 and 3 and between replicates 2 and 4 . Replicates 2 and 4 had diverged most from the ceftazidime-resistant clones, with P165T, A170D and M180T substitutions in every replicon sequenced from these replicates, in addition to other substitutions. We cloned select sequences from each replicate into a standard circular plasmid to further validate the performance of these mutants . Given that these evolutions are run with multiple replicons per cell, it would in principle be possible for multiresistance to either emerge in a single genotype or for distinct resistances to be conferred by separate genotypes. In this case, all obtained clones fall into the former category. We hypothesize that the improved heritability associated with a single genotype conferring multiresistance may provide a selective advantage.using a synthetically designed operon encoding components from bacteriophage Φ29, which naturally infects only Gram-positive bacteria. This phage has served as a long-standing model system to study protein-primed replication. Here, we report sustained in vivo replication using Φ29 components. We engineered the operon for improved performance and found that only two of the genes are needed for orthogonal replication. Moreover, we used the lambda-phage-derived recombination system to precisely and efficiently engineer the replicons and showed that extracted replicons could be efficiently transformed into fresh cells harboring the optimized synthetic replication operon. We developed an error-prone DNAP with a mutation rate approaching 10. We expect that this system will readily integrate with selection regimes where a desired activity is coupled to bacterial growth. Future work will focus on using the Φ29-based orthogonal replication system to rapidly evolve protein function, predict mutations that will confer resistance to antibiotics and other drugs and rapidly generate synthetic phylogenies for proteins with new and useful functions.DH10B. We genomically integrated the Φ29 synthetic replication operons, under the control of a PtacIPTG promoter, using lambda Red recombination by the plasmid pEcCas, as described previouslyGenes 2, 3, 5 and 6 from bacteriophage Φ29 were codon-optimized and arranged into a synthetic operon with synthetic ribosome-binding sites using the operon calculator from de novo DNA. The resultant design was ordered as gBlocks from Integrated DNA Technologies, assembled using overlap extension PCR and cloned into a single-copy plasmid backbone using HiFi Gibson assembly or genomically integrated.Linear replicons used for electroporation were obtained by PCR using PrimeSTAR Max DNAP . The initial Φ29 synthetic replicon was designed to encode tetracycline resistance and GFP, flanked by the full-length left and right origins of replication from the Φ29 genome. The primers used for amplifying the replicon annealed at the terminal ends of both origins of replication. The PCR products were purified using a QIAquick PCR Purification Kit .and the Φ29 SSB and DSB). Both plasmids had a CloDF13 origin of replication and a gentamicin resistance marker. To test the essentiality of the four genes in our Φ29 synthetic replication operon, we generated plasmids where the genes encoding SSB and/or DSB were disrupted or deleted. Like the intact replication operon, these plasmids had a bacterial F plasmid replication origin and a chloramphenicol resistance gene. All circular plasmids were constructed using HiFi Gibson assembly from multiple fragments.Colonies of strains harboring the synthetic replication operon, on a single-copy plasmid or the genome, with or without a helper plasmid were inoculated into 10 ml of 2×YT and grown overnight at 37 °C with shaking . Then, 5 ml of this culture was transferred into 250 ml of 2×YT in a 2-L flask and grown at 37 °C with shaking until an optical density at 600 nm of 0.3–0.4 was reached. When helper plasmids were used, we supplemented the medium with 10 mM arabinose at this point and grew the cells for a further 30 min. Subsequently, the cultures were chilled on ice before being pelleted by centrifugation , washed twice with cold 10% glycerol and resuspended in a final volume of 500 µl. Then, 100 µl of the resultant electrocompetent cells were added to an electroporation cuvette , the synthetic replicon was added to the cells and electroporation was performed using an Eppendorf Eporator at 2,500 V. Immediately after electroporation, 1 ml of SOB medium was added to the cuvette and the cells were recovered in a 2-ml tube at 37 °C with shaking for 2 h before being plated on an agar plate . For the initial Φ29 synthetic replication operon, colonies could be observed after 1–2 days of incubation at 37 °C. For Φ29-opt, colonies appeared after 16–24 h of incubation. We note that how fast colonies appear might be influenced by what is encoded by the replicon.Extracted replicons could be transformed with a much higher efficiency than PCR products and without needing to provide a helper plasmid. To extract these replicons, a 10-ml overnight culture of a strain harboring a Φ29 synthetic replicon was pelleted by centrifugation . Next, we used the QIAprep spin miniprep kit to extract the replicon, following the manufacturer’s guidelines. We used Millipore-filtered water for the elution and heated the columns, to which the water was added at 55 °C for 10 min before the final centrifugation. All extracted replicons were stored at −20 °C. For the initial Φ29 synthetic replication operon, colonies could be observed after 1–2 days of incubation at 37 °C. For Φ29-opt, colonies appeared after 16–24 h of incubation. We note that how fast colonies appear might be influenced by what is encoded by the replicon.To assess the stability of the synthetic replicons, we passaged cells in 2 ml of 2×YT with or without tetracycline to select for the replicon. Replicates were passaged in 24-well deep-well plates at 37 °C with shaking. For each passage, we transferred 2 µl into 2 ml of fresh medium and allowed the cells to grow for at least 16 h to reach the stationary phase. Each 1,000-fold dilution and growth to stationary phase was considered to be ten generations of growth . All samples were analyzed by flow cytometry using a BD LSRFortessa cell analyzer to determine the fluorescence of single cells. Samples were manually gated for single bacterial cells. Gating of green fluorescent cells was set relative to the negative control not expressing GFP. The orthogonal replicon stability was calculated on the basis of the proportion of single GFP-fluorescing cells.Cultures to be measured were first pelleted by centrifugation before resuspension in PBS. These samples were then transferred into 96-well flat-bottom clear plates to measure GFP fluorescence . We used qPCR to determine the DNA copy numbers for all samples with a Vii 7 real-time PCR system with 384-well block .Cells harboring a synthetic replication operon and a replicon to be modified were transformed with plasmid pLF118 encoding the lambda Red components under the control of an arabinose-inducible promoter. Transformant colonies were then inoculated into 10 ml of 2×YT and grown overnight at 37 °C with shaking . Then, 5 ml of this culture was transferred into 250 ml of 2×YT in a 2-L flask and grown at 37 °C with shaking until an ODof 0.3 was reached. We then added arabinose to a final concentration of 10 mM to the culture and grew the cells for a further 30 min. Subsequently, the cultures were chilled on ice before being pelleted by centrifugation , washed twice with cold 10% glycerol and resuspended in a final volume of 500 µl. Then, 100 µl of the resultant electrocompetent cells were added to an electroporation cuvette , the purified PCR product consisting of the gene of interest flanked by at least 30-bp homologies to the existing replicon was added to the cells and electroporation was performed using an Eppendorf Eporator at 2,500 V. Immediately after electroporation, 1 ml of SOB medium was added to the cuvette and the cells were recovered in 10 ml of SOB in a 50-ml tube at 37 °C with shaking for 1 h, transferred to 50 ml of 2×YT supplemented with gentamicin to select for pLF118 and grown for 1 h at 37 °C with shaking, before being plated on an agar plate . When determining the efficiency of the recombination, we used 0.5 µg of purified PCR product; however, for other experiments, lower quantities were also sufficient.To construct a library of the Φ29 synthetic replication operon, we generated a PCR product for each gene in the operon with overlaps to the adjacent gene. We used primers that randomized the RBS spacer region, saturated the first codon following the ATG start codon and synonymized the following six codons of each gene. We then assembled these genes by overlap extension PCR using primers that amplified the entire operon and appended overlaps to the plasmid backbone and cloned them into a plasmid backbone with a bacterial F plasmid replication origin and a spectinomycin resistance gene using HiFi Gibson assembly . The resultant plasmid library was then electroporated into cells harboring the initial Φ29 synthetic replication operon on a chloramphenicol-resistance-conferring plasmid with the same F plasmid origin of replication and a TetA–GFP Φ29 synthetic replicon. We selected for maintenance of the replicon and the plasmid library members by supplementing tetracycline and spectinomycin. To select for operon variants with improved growth, we cultured the cells in 250 ml in 2-L flasks at 37 °C with shaking for three passages , before plating the cells for single colonies. We then picked large colonies with bright green fluorescence to ultimately identify the Φ29-opt operon. The Φ29-opt operon was used for all experiments after Fig.The Φ29-opt operon and the hygromycin resistance marker were PCR-amplified from plasmid pFR329 with primers Gen-integration_opt_F and Gen-integration_opt_R . The resultant PCR amplicon was then genomically integrated using lambda Red. This was achieved using the same protocol as in the above section detailing Φ29 replicon engineering, except that the strain used was DH10B transformed with pLF118 without a replicon.Bacterial colonies were grown overnight at 37 °C in 2×YT with the relevant antibiotics in a 96-well plate. Overnight cultures were diluted 1:100 and monitored for growth in a 200-µl volume in a clear flat-bottom 96-well plate. Measurements of ODwere taken every 5 min on a Tecan Infinite M1000 microplate reader set at 37 °C with shaking. Doubling times were calculated by fitting the data with the logistic growth equation in GraphPad Prism and taking the ln2/DH10B. Point mutations that convert the TAG stop codon to sense codons can confer chloramphenicol resistance. The insertion site was adjacent to thegene, distal from the origin of replication, where the copy number is expected to be approximately one. We transformed this strain with the plasmids encoding each of the DNAPs to be tested. Untransformed cells were used as a negative control. For the Φ29 DNAPs, the plasmids encoded the entire operon; identical plasmids were used to test the replicon mutation rates. The error-prone T7 DNAP was cloned under the control of a salicylic acid-inducible promoter but leaky expression was sufficient to observe substantial mutagenesis of the genome. We inoculated transformant colonies into 2 ml of 2×YT supplemented with the necessary antibiotics in 24-well plates and grew the cells overnight at 37 °C with shaking. A total of 12 biological replicates were performed for each strain. All samples were then diluted and plated on plates with or without 20 μg ml) as a function of the cell numbers on the selective and non-selective plates for all 12 biological replicates. The mutation rate per generation per base pairTo measure the replicon mutation rates, we generated a KanR–CmR replicon by lambda Red recombination. The CmR gene used was identical to the one that was genomically integrated. Cells harboring this replicon and a single-copy plasmid harboring the WT Φ29-opt operon were transformed with a single-copy, spectinomycin resistance-conferring plasmid to replace the initial plasmid. The resultant transformants were inoculated into 2 ml of 2×YT supplemented with the necessary antibiotics in 24-well plates and grown overnight at 37 °C with shaking. A total of 12 biological replicates were performed for each strain. All samples were then diluted and plated on plates with or without 100 μg mlMeasuring mutational spectra using Illumina unique molecular identifier consensus sequencing Four Φ29 DNAPs maintaining a KanR–CmR replicon were subjected to passaging in at least three biological replicates. At 0 , 20 and 50 generations , the linear replicon was purified by minimal PCR amplification and prepared for Illumina sequencing with UMIs using the NEB Ultra II FS library prep kit and NEB unique dual index UMI adaptors according to manufacturer’s instructions. Libraries were quantified, pooled and sequenced on an Illumina NextSeq2000 with P3, 200-cycle XLEAP reagent kit, reading a 12-mer UMI. A 0.33% PhiX spike-in gave a measured error rate of 0.25%. The NextSeq2000-generated fastq files were demultiplexed with DRAGEN BCL-Convert , separating UMIs with override cycle settings ‘Y104;I8U12;I8;Y104’. Reads were trimmed and filtered for quality gene and 100 bp of terminal ends under selection). To generate UMI consensus sequences, UMIs were first summarized using the umi-toolsgroup function and UMIs with three or more copies were identified. For each >3-member UMI family, SAMtools consensus was used to generate a consensus sequence. Consensus sequences were then realigned and files containing bases or indels at each position were generated with igvtools count. In brief, for every DNAP and generation count, files containing bases or indels at each position were processed to generate a mutation frequency for every base mutation . Each specific mutation was processed as follows: the mutation rate per base per generation was the slope of linear regression of mutation frequency across 0, 20 and 50 or 0 and 20 generations. This yielded a set of specific mutation rates per generation for every position, for every mutation, for every DNAP. As this method has no strand information, base-pair substitutions were aggregated . Mutational spectra show the median of positive mutation rates for each substitution mutation and the overall DNAP mutation rate was taken as the mean of all the summed median mutation substitution rates of A, C, G and T.Cells harboring a genomic Φ29-opt synthetic replication operon and a Φ29 replicon encoding TetA or TEM-1 β-lactamase were transformed with plasmid pFR364 encoding Φ29-opt with the N62D;F65S error-prone DNAP. Transformant colonies were inoculated into 2×YT supplemented with the necessary antibiotics . After overnight growth, the resultant cells were tenfold diluted into 5 ml of 2×YT supplemented with spectinomycin and ceftazidime or tigecycline in 24-well plates and incubated at 37 °C with shaking. Tetracycline was supplemented at the lowest concentration of ceftazidime in the ceftazidime evolution but not at subsequent steps. The evolution for cefotaxime resistance began from the final ceftazidime-resistant pools. Ceftazidime and carbenicillin were supplemented during the evolution for cefotaxime resistance. For each passage, we transferred 500 µl of culture into 5 ml of fresh medium. After completing the passages, we plated the cells on agar plates supplemented with tigecycline, cefotaxime or ceftazidime and sequenced individual colonies using Nanopore sequencing of colony PCR products. We also cloned select sequences into a ColE1 plasmid backbone for validation.Google ScholarMoore, C. L., Papa, L. J. 3rd & Shoulders, M. D. A processive protein chimera introduces mutations across defined DNA regions in vivo.Cravens, A., Jamil, O. K., Kong, D., Sockolosky, J. T. & Smolke, C. D. Polymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering.Ravikumar, A., Arzumanyan, G. A., Obadi, M. K. A., Javanpour, A. A. & Liu, C. C. Scalable, continuous evolution of genes at mutation rates above genomic error thresholds.Google ScholarBlanco, L. & Salas, M. Characterization and purification of a phage Φ29-encoded DNA polymerase required for the initiation of replication.Hermoso, J. M., Mendez, E., Soriano, F. & Salas, M. Location of the serine residue involved in the linkage between the terminal protein and the DNA of phage Φ29.Mendez, J., Blanco, L., Esteban, J. A., Bernad, A. & Salas, M. Initiation of Φ29 DNA replication occurs at the second 3′ nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication.Mendez, J., Blanco, L. & Salas, M. Protein-primed DNA replication: a transition between two modes of priming by a unique DNA polymerase.Gutierrez, C., Sogo, J. M. & Salas, M. Analysis of replicative intermediates produced during bacteriophage Φ29 DNA replication in vitro.Soengas, M. S., Gutierrez, C. & Salas, M. Helix-destabilizing activity of Φ29 single-stranded DNA binding protein: effect on the elongation rate during strand displacement DNA replication.Camacho, A. & Salas, M. Mechanism for the switch of Φ29 DNA early to late transcription by regulatory protein p4 and histone-like protein p6.Mencia, M., Gella, P., Camacho, A., de Vega, M. & Salas, M. Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage Φ29.Cetnar, D. P. & Salis, H. M. Systematic quantification of sequence and structural determinants controlling mRNA stability in bacterial operons.Salis, H. M., Mirsky, E. A. & Voigt, C. A. Automated design of synthetic ribosome binding sites to control protein expression.Blanco, L. & Salas, M. Replication of phage Φ29 DNA with purified terminal protein and DNA polymerase: synthesis of full-length Φ29 DNA.Google Scholar Cambray, G., Guimaraes, J. C. & Arkin, A. P. Evaluation of 244,000 synthetic sequences reveals design principles to optimize translation inde Vega, M., Lazaro, J. M., Salas, M. & Blanco, L. Mutational analysis of Φ29 DNA polymerase residues acting as ssDNA ligands for 3′–5′ exonucleolysis.de Vega, M., Lazaro, J. M., Salas, M. & Blanco, L. Primer-terminus stabilization at the 3′–5′ exonuclease active site of Φ29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases.Li, X. T., Thomason, L. C., Sawitzke, J. A., Costantino, N. & Court, D. L. Positive and negative selection using the tetA–sacB cassette: recombineering and P1 transduction inRains, C. P., Bryson, H. M. & Peters, D. H. Ceftazidime. An update of its antibacterial activity, pharmacokinetic properties and therapeutic efficacy.Halper, S. M., Hossain, A. & Salis, H. M. Synthesis success calculator: predicting the rapid synthesis of DNA fragments with machine learning.Ng, C. Y., Farasat, I., Maranas, C. D. & Salis, H. M. Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration.Reis, A. C. & Salis, H. M. An automated model test system for systematic development and improvement of gene expression models.Hall, B. M., Ma, C. X., Liang, P. & Singh, K. K. Fluctuation analysis CalculatOR: a web tool for the determination of mutation rate using Luria–Delbruck fluctuation analysis.We thank C. Piedrafita and S. Grazioli for helpful discussions in the development of mutation rate analyses. This work was supported by the Medical Research Council UK . F.B.H.R. was supported by a UK Research and Innovation Marie Skłodowska-Curie Actions guarantee fellowship and an Investigator Grant from the National Health and Medical Research Council Australia.F.B.H.R., R.T. and K.C.L. performed the experiments. K.C.L. generated and analyzed the next-generation sequencing data. F.B.H.R. conceptualized and established the Φ29-based orthogonal replication system. J.W.C. supervised the project. F.B.H.R. and J.W.C. wrote the paper with input from all other authors.The Medical Research Council has filed a provisional patent application related to this work, on which F.B.H.R., R.T. and J.W.C. are listed as inventors. J.W.C. is the founder of and a shareholder in Constructive Bio. K.C.L. declares no competing interests.thanks Heinz Neumann, Vitor Pinheiro and the other, anonymous, reviewer for their contribution to the peer review of this work.This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
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