General methods<\/p>\n
All E. coli and phage strains created in this study are listed in Supplementary Table 1<\/a>, plasmids in Supplementary Table 2<\/a> and all oligonucleotides in Supplementary Table 6<\/a>. Phage T7\u2206DNAP is available upon request and relevant plasmids are available from Addgene. Phage T7 was obtained from ATCC (BAA-1025-B2). Cloning of all plasmids was carried out using MDS42 cells (E-6265-05K; Scarab Genomics). Plasmids were constructed with NEBuilder HiFi DNA Assembly (New England Biolabs) unless otherwise stated. Native E. coli and T7 genes were amplified by PCR directly from genomic DNA. Other genes were synthesized as gBlocks Gene Fragments (Integrated DNA Technologies), unless otherwise stated. PCR reactions were performed using PrimeSTAR GXL DNA Polymerase (Takara) for cloning and using Rapid Taq DNA Polymerase (Vazyme) for genotyping. All oligonucleotides were synthesized by Integrated DNA Technologies. Sanger sequencing was performed by 1st BASE. Nanopore sequencing was performed by Plasmidsaurus. Sample preparation for sequencing was done according to each company\u2019s protocol.<\/p>\n Culture media<\/p>\n E. coli was grown in LB medium or on LB agar (Bio Basic Asia Pacific) with added kanamycin (50\u2009\u03bcg\u2009ml\u22121), ampicillin (100\u2009\u03bcg\u2009ml\u22121), streptomycin (100\u2009\u03bcg\u2009ml\u22121), chloramphenicol (20\u2009\u03bcg\u2009ml\u22121), tetracycline (10\u2009\u03bcg\u2009ml\u22121) or hygromycin (200\u2009\u03bcg\u2009ml\u22121) where appropriate. EG selection and growth experiments were carried out without antibiotics in standard M9 minimal media (50\u2009mM Na2HPO4, 20\u2009mM KH2PO4, 1\u2009mM NaCl, 20\u2009mM NH4Cl, 2\u2009mM MgSO4, 100\u2009\u03bcM CaCl2, 134\u2009\u03bcM EDTA, 13\u2009\u03bcM FeCl3\u00b76H2O, 6.2\u2009\u03bcM ZnCl2, 0.76\u2009\u03bcM CuCl2\u00b72H2O, 0.42\u2009\u03bcM CoCl2\u00b72H2O, 1.62\u2009\u03bcM H3BO3 and 0.081\u2009\u03bcM MnCl2\u00b74H2O). Carbon sources were used as indicated in the text.<\/p>\n Electrocompetent cell preparation<\/p>\n E. coli cells were inoculated in 10\u2009ml LB and grown overnight at 37\u2009\u00b0C with shaking at 225\u2009rpm, with appropriate antibiotics if applicable. The next morning, cells were diluted 1:50-fold in the same media to a final volume of 500\u2009ml, grown to an optical density at 600\u2009nm (OD600) of 0.5 and incubated on ice for 20\u2009minutes. The cells were spun down at 4,000g for 10\u2009minutes at 4\u2009\u00b0C. The supernatant was discarded and the cells were resuspended in 50\u2009ml of chilled sterile dH2O. The cells were washed once again with sterile dH2O followed by 16% (w\/v) chilled glycerol. The cells were then resuspended in 1\u2009ml of chilled 16% glycerol and used immediately for transformation by electroporation or they were flash frozen and stored at \u221280\u2009\u00b0C.<\/p>\n Scarless gene deletion by homologous recombination<\/p>\n Gene deletions were performed as previously described50<\/a>. We created plasmid pPBG01 (number 253997; Addgene) (Supplementary Data 13<\/a>) for arabinose-inducible \u03bb-Red recombination by cloning genes gam, beta and exo from E. coli C321.\u2206A.opt (87359; Addgene) into a CloDF13 vector (69669; Addgene).<\/p>\n E. coli MG1655rpsLK43R was used for all gene deletions. It was created by PCR amplification of rpsLK43R from E. coli DH10b, which we electroporated into competent and induced MG1655 cells harbouring pPBG01. After recovery, the cells were plated on LB agar with streptomycin to select for successful recombination. We further created a double-selection cassette of negative selection gene rpsL (from MG1655) and positive selection gene hygR (104405; Addgene) combined by overhang extension PCR. For gene deletions, we amplified the rpsL-hygR cassette by PCR using primers containing homology to the regions flanking the target gene to be deleted. We electroporated the PCR product into competent, induced MG1655rpsLK43R cells harbouring pPBG01 and selected for genomic integration with hygromycin. Successful gene deletions were verified by genotyping. To achieve scarless gene deletion, the rpsL-hygR cassette was subsequently removed by a second round of recombineering by electroporating 10\u2009\u03bcg of a 90-base oligonucleotide covering the genomic homology regions targeting the lagging strand. Loss of the rpsL-hygR cassette was selected for with streptomycin and verified by genotyping and Sanger sequencing.<\/p>\n Phage plaque assay<\/p>\n Phage from clarified phage lysate (or from rebooting in cell-free TXTL extract, see later) were serially diluted tenfold in LB medium. Overnight cultures of E. coli host strains were prepared, diluted 4-fold in LB and incubated with shaking for 1\u2009hour at 37\u2009\u00b0C. For each 100-mm petri dish, 100\u2009\u00b5l cells were mixed with 1\u2009\u00b5l of serially diluted phage and incubated for 5\u2009minutes at 37\u2009\u00b0C. The phage-cell mixtures were then mixed with 4\u2009ml pre-warmed molten 0.35% top LB agar and immediately poured uniformly onto 100-mm petri dishes containing 15\u2009ml of solidified 1.5% bottom LB agar. The top agar was left to solidify for 1\u2009hour and then incubated for 4\u2009hours at 37\u2009\u00b0C for plaque formation.<\/p>\n Phage titre determination<\/p>\n Overnight cultures of E. coli were diluted and embedded in top agar as mentioned earlier, but without phage. Plates were dried for 1\u2009hour. Each phage was serially diluted, and 5\u2009\u00b5l was spotted onto the top agar and left to dry for 15\u2009minutes. The plates were incubated at 37\u2009\u00b0C, facing down, for 4\u2009hours. The number of plaques was counted at each dilution to determine plaque-forming units (PFUs). The MOI was determined by calculating the ratio of PFU of phage added to the colony-forming units (CFUs) of cells used for phage infection. We empirically determined that phage lysates are, on average, approximately 1\u2009\u00d7\u20091013 PFU\u2009ml\u22121, and diluted overnight cell cultures are approximately 1\u2009\u00d7\u20091011 CFU\u2009ml\u22121.<\/p>\n T7\u0394DNAP phage assembly and rebooting in TXTL<\/p>\n T7 genomic DNA excluding the T7 DNAP gene, gp5, was PCR amplified in 5 fragments of 10.0\u2009kb (F1), 4.4\u2009kb (F2), 3.5\u2009kb (F4), 10.0\u2009kb (F5) and 10.0\u2009kb (F6), with 25\u201330\u2009bp overlapping sequences. Fragment F3 containing the trxA gene was amplified from the E. coli genome, with overlapping sequences with F2 and F4 to replace T7 DNAP. Purified PCR fragments were mixed, including 300\u2009ng of F3 and 100\u2009ng of each of the other fragments. They were then assembled by Gibson Assembly using NEBuilder HiFi DNA Assembly (New England Biolabs). The assembly mix, together with 50\u2009ng of T7 genomic DNA, was mixed using the myTXTL Linear DNA Expression Kit (Arbor Biosciences), according to the manufacturer\u2019s protocol, and incubated overnight at 37\u2009\u00b0C to generate a mixture of rebooted wild-type T7 and T7\u0394DNAP phage. To select for the mutant phage, a plaque assay was performed by infecting MG1655\u0394trxA containing pSJ55 (253998; Addgene), which expresses wild-type T7 DNAP. Wild-type T7 not expressing trxA would not propagate in this strain. Only T7\u0394DNAP trxA would be able to propagate in this strain and form plaques. We confirmed successful generation of T7\u0394DNAP trxA by genotyping and Sanger sequencing the genomic region from which T7 DNAP was deleted (GenBank:PZ151113) (Supplementary Data 1<\/a> and Supplementary Table 1<\/a>).<\/p>\n Phage infection kinetic assay<\/p>\n Infection kinetics were carried out in 96-well plates using an BioTek Synergy HTX Microplate Reader (Agilent). Overnight cultures of E. coli host strains were prepared, diluted 1:1 in LB medium and 100\u2009\u03bcl was added to each well. In each well, 20\u2009\u00b5l of different serial phage dilutions were added to tune MOI. Each condition was replicated in three different wells. A lid was added to the 96-well plates to reduce evaporation during acquisition. The microplate reader was set to 37\u2009\u00b0C with continuous orbital shaking at 300\u2009rpm. OD600 was measured every 3\u2009minutes and monitored for at least 2\u2009hours.<\/p>\n Yeast assembly<\/p>\n Large phagemids were assembled by transformation-associated recombination in yeast65<\/a> as previously described66<\/a>. S. cerevisiae BY4741 (MAT\u03b1 his3\u03941 leu2\u03940 met15\u03940 ura3\u03940) was obtained from ATCC (201388). We modified the cell wall digestion step slightly: we only used 1\u2009\u00b5l zymolyase and we measured the OD660 every 10\u2009minutes after 30\u2009minutes of zymolyase digestion. Digested cells were resuspended by swirling and inverting the tubes.<\/p>\n Phagemid assembly was assessed by genotyping the resulting yeast colonies, amplifying 350\u20131,000\u2009bp across the intersections of assembled fragments. Positive yeast colonies were cultured, phagemids were isolated with the Zymoprep Yeast Plasmid Miniprep Kit (Zymo Research) and electroporated into E. coli MDS42 under selection. Individual colonies were genotyped again with the same primers, and phagemids were isolated from positive clones using the Monarch Plasmid Miniprep Kit and verified by nanopore sequencing.<\/p>\n General LySE strain preparation<\/p>\n All LySE experiments were conducted with E. coli MG1655. Cells were first transformed with APs containing different T7 DNAP variants by electroporation as described earlier and selected with ampicillin. The protein sequences of the T7 DNAP variants are given in Supplementary Table 3<\/a>. Cells containing the AP were transformed with the phagemid by electroporation and selected on media containing both ampicillin and kanamycin. Genotyping was performed at each step to confirm successful transformation.<\/p>\n LySE cycle<\/p>\n To propagate the T7\u0394DNAP phage, MG1655 with pSJ55 was grown overnight in LB medium with ampicillin, then diluted with equal volumes of LB the next day. T7\u0394DNAP phage lysates were mixed with the diluted cells at a volume ratio of 1:1,000 (approximate MOI\u2009=\u20090.1) and incubated at 37\u2009\u00b0C with shaking for 2\u2009hours until there was no further reduction in OD600. The phage lysates were then washed once with equal volumes of chloroform to remove residual cells and debris.<\/p>\n The LySE cycle begins with an overnight culture of MG1655 cells containing AP and phagemid. Overnight culture cells were diluted with equal volumes of LB the next day. To 100\u2009\u00b5l of diluted cells, 20\u2009\u00b5l of phage was added (approximate MOI\u2009=\u200910; high MOI) and incubated at 37\u2009\u00b0C with shaking for 2\u2009hours until there was no further reduction in OD600. The phage lysates were washed once with equal volumes of chloroform. Next, to 1\u2009ml overnight culture of MG1655 cells containing only AP diluted with equal volumes of LB, we mixed 10\u2009\u00b5l phage lysates containing phagemids and incubated at 37\u2009\u00b0C with shaking for 1\u2009hour for complete transduction of phagemids (approximate MOI\u2009=\u20091; low MOI). Phagemid packaging efficiency was determined by serial dilution of the transduced cells, followed by spot plating on LB agar with kanamycin to determine the CFU of transduced phagemid. To continue the LySE cycle, transduced cells were diluted in selection media and grown to confluency. The exact protocol for selection and culture recovery is specific to each evolution experiment but should include addition of ampicillin to maintain the AP. The next LySE cycle is continued by adding phage at high MOI to lyse cultured cells.<\/p>\n Fluctuation assay<\/p>\n We performed Luria\u2013Delbr\u00fcck fluctuation analysis to quantify the mutation rate per generation of LySE for each T7 DNAP variant. We cloned pSJ51, a phagemid constitutively expressing a chloramphenicol resistance gene (CmR) containing a premature stop codon (Q38TAG). MG1655 containing pSJ51 and an AP encoding the T7 DNAP variant to be tested was grown overnight, diluted and lysed as per standard LySE protocols. After one generation of phagemid replication and transduction, transduced cells were serially diluted tenfold and spotted on LB agar with kanamycin to quantify total transduction CFU and on LB agar with chloramphenicol to quantify stop-codon reversion CFU, respectively. After overnight incubation at 37\u2009\u00b0C, CFUs from 3 independent replicates were counted. The apparent mutation rate, \u00b5 (substitutions per base pair per generation, s.p.b.) was calculated as \u00b5\u2009=\u2009m\u2009\/\u2009(R\u2009\u00d7\u2009C). Mutation frequency (m) was calculated based on the ratio of cells grown on chloramphenicol to that grown on kanamycin. R is the number of distinct mutation sites that make the resistance gene effective, that is, yielding a sense codon. For TAG, 8\/9 possible single base substitutions yield a sense codon. Averaged over the three positions in the codon, R\u2009=\u2009(8\/9)\u2009\u00d7\u20093\u2009=\u20098\/3. C is the gene copy number. As T7 packages concatemeric phagemid DNA via a head-full mechanism, multiple CmR copies are expected per phage, while only a single reverted TAG is sufficient to confer chloramphenicol resistance. We therefore determined C by taking the fraction of phagemid to T7 genome size, so C\u2009=\u200939,937\/3,469\u2009=\u200911.5.<\/p>\n Molecular dynamics simulation<\/p>\n We used the crystal structure published by ref. 29<\/a> (PDB:1T7P<\/a>) as the basis for our molecular dynamics simulations of the wild-type and T523R mutant T7 DNAP. 1T7P contains a growing DNA strand terminated with a dideoxy cytosine nucleotide, and the incoming nucleotide is dideoxyguanosine triphosphate (ddGTP). To represent the real biomolecules as closely as possible, we manually added 3\u2032-hydroxyl groups to the chain-terminating nucleotide of the growing DNA strand and ddGTP. Wild-type and mutant T523R T7 DNAP variants were created in silico, and subsets of these containing DNA substitutions of the leading cytosine nucleotide on the template strand were implemented to study the effect of T523R on base mispairing. The structure is dissolved in water under physiological conditions using the solution builder on CHARMM-GUI. We placed each protein structure in a 130\u2009\u00c5\u2009\u00d7\u2009130\u2009\u00c5\u2009\u00d7\u2009130\u2009\u00c5 simulation box with water containing 215\/216 sodium ions and 183 chloride ions (150\u2009mM) to balance protein charges at a pH of 7.0. For each condition, we ran a 5,000-step steepest descent energy minimization. This was followed by a number of particles, volume and temperature (NVT) equilibration step with a simulation time of 125\u2009ps (125,000 steps) at 303.15\u2009K. The subsequent number of particles, pressure and temperature (NPT) production simulation is 10\u2009ns (5,000,000 steps) at 303.15\u2009K. All simulations used the CHARMM36m force field and were run using CUDA-supported GROMACS (v.2023.3) on high-performance computing facilities (National University of Singapore HPC).<\/p>\n Genomic fluctuation assay<\/p>\n E. coli MG1655 was grown from glycerol stocks overnight in LB. To determine total CFU, the cells were serially diluted tenfold and spotted on LB agar. To determine frequency of rifampicin resistance, 2\u2009ml of cells were spun down and the cell pellet was plated onto selective LB agar with 50\u2009\u00b5g\u2009ml\u22121 rifampicin. After overnight incubation at 37\u2009\u00b0C, CFUs from 3 independent replicates were counted. Mutation frequency was calculated based on the ratio of cells grown on rifampicin to that grown on LB. To calculate substitutions per base pair (s.p.b.), the mutation rate was normalized by the number of mutations in the rpoB gene that impart rifampicin resistance (77 known point mutations, divide observed mutation rate by 77\/3)31<\/a>. We determined E. coli genomic mutation rates to be 2.39\u2009\u00b1\u20091.10\u2009\u00d7\u200910\u221210 s.p.b., comparable to those previously reported16<\/a>,18<\/a>.<\/p>\n Illumina NGS and data analysis<\/p>\n We cloned pSJ77 (GenBank:PZ151114), a 39-kb BAC phagemid by yeast assembly of fragments from pBeloBAC11 (60342; Addgene), pRS316 (110533; Addgene) and yeast genomic DNA. E. coli MG1655 containing pSJ77 and an AP encoding a T7 DNAP variant was grown overnight, diluted and lysed as per standard LySE protocols. After one generation of phagemid replication and transduction into MG1655 with no plasmids, transduced cells were diluted tenfold in LB with kanamycin and recovered overnight. The recovered mutated BAC-phagemid library was purified using the ZymoPURE Plasmid Miniprep Kit (Zymo Research). Illumina NGS of the BAC-phagemid library was performed by Bio Basic Asia Pacific. NGS library preparations were constructed following the manufacturer\u2019s protocol. For each sample, 200\u2009\u03bcg DNA was randomly fragmented by Covaris to an average size of 300\u2013350\u2009bp. The fragments were treated with End Prep Enzyme Mix for end repairing, 5\u2032 phosphorylation and 3\u2032 adenylation to add adaptors to both ends. Size selection of adaptor-ligated DNA was then performed using DNA Cleanup beads. Each sample was then amplified by PCR for eight cycles using P5 and P7 primers, with both primers carrying sequences that can anneal to the flow cell to perform bridge PCR, and the P7 primer carrying a six-base index allowing multiplexing. The PCR products were cleaned up and validated using an Agilent 2100 Bioanalyzer. The qualified libraries were sequenced paired-end (PE150) on the Illumina NovaSeq system. Fastp (v.0.23.0) was used for quality control and preprocessing, including removal of adaptor sequences, PCR primers, reads with more than 14\u2009N bases and reads with less than 40% bases above a Phred quality score of 20 (Q20). The cleaned data were then mapped to the reference genome using the Sentieon pipeline (v.202112.02). A custom python script using the pysam (v.0.23.0) module was used to align the NGS reads with Q score \u226530 to the reference sequence and count the nucleotide positions from which the experimental sample deviates from the reference sequence. To correct for sequencing errors, the observed fraction of mismatches at each nucleotide position mutated by the wild-type T7 DNAP was subtracted from the fraction of mutations for engineered T7 DNAP variants. Corrected mismatch rates and A:T\u2009\u2192\u2009G:C and C:G\u2009\u2192\u2009T:A mutation rates were binned into 20 equal-width bins and then calculated and plotted. For mutational rates spread across 39\u2009kb, corrected mutation rates were binned every 100\u2009bp and then plotted. Overall mutational spectra, and for every 10,000\u2009bp, were calculated by taking the fraction of each of 12 mutation types relative to the overall mutation rate for each T7DNAP variant. We yielded an average of >14,000 reads per position for each of the sequenced samples.<\/p>\n LySE evolution of tetA<\/p>\n We cloned pSJ78 (254000; Addgene), a phagemid constitutively expressing the tetA tetracycline efflux gene. E. coli MG1655 containing pSJ78 and pSJ139 (T7 DNAP v9; 254001; Addgene) was grown overnight, diluted and lysed as per standard LySE protocols. After one generation of phagemid replication and transduction into MG1655 with pSJ139, transduced cells were diluted 10-fold with LB with ampicillin, kanamycin and 0.1\u2009\u03bcg\u2009ml\u22121 tigecycline. The cells were incubated overnight at 37\u2009\u00b0C with shaking at 225\u2009rpm. The next day, the cells were diluted with equal volumes of LB and T7\u0394DNAP phages were added to lyse the culture, starting another round of LySE. Evolution cycles were repeated another four times for a total of five evolution cycles, using the best growing cultures from the previous cycle as the starting material for the next cycle. Tigecycline concentrations were incrementally increased from LySE E1 to E5, with concentrations 0.1\u2009\u03bcg\u2009ml\u22121, 0.25\u2009\u03bcg\u2009ml\u22121, 0.5\u2009\u03bcg\u2009ml\u22121, 0.75\u2009\u03bcg\u2009ml\u22121 and 1\u2009\u03bcg\u2009ml\u22121, respectively. After the 5th LySE cycle, the best growing culture was streaked on LB agar with kanamycin and 1\u2009\u03bcg\u2009ml\u22121 tigecycline. Individual colonies were picked and inoculated separately in 1\u2009ml LB with kanamycin in 24-well plates and grown overnight. A total of 32 evolved clones were spotted on LB agar with increasing concentrations of tigecycline to test resistance. The 32 clones were PCR amplified for the tetA cassette using PrimeSTAR GXL DNA Polymerase and the amplicon was sequenced by Sanger sequencing.<\/p>\n For ALE, E. coli MG1655 containing pSJ78 and pSJ55 (wild-type T7 DNAP) was grown overnight in LB with kanamycin and ampicillin and then diluted 10-fold with LB with kanamycin, ampicillin and 0.1\u2009\u03bcg\u2009ml\u22121 tigecycline. The cells were incubated overnight at 37\u2009\u00b0C with shaking at 225\u2009rpm. The next day, the cells were diluted another tenfold to continue ALE. A total of five passages were performed, each time with increasing concentrations of tigecycline identical to the LySE schedule. Cells at the fifth passage were spotted on LB agar with increasing concentrations of tigecycline to test resistance. The cells were also lysed by addition of T7\u0394DNAP, the lysate washed with chloroform and phagemids transduced to fresh MG1655 with no plasmids. The transduced cells were diluted tenfold with LB with kanamycin and then grown overnight. The recovered E5T ALE cells were then spotted on LB agar with tigecycline to test resistance after transduction.<\/p>\n Quantitative real-time PCR<\/p>\n Total RNA was first isolated from the E. coli cells. Cells were grown overnight and diluted 1:50 with LB and appropriate antibiotics and cultured until OD600\u2009=\u20090.5. Then, 500\u2009\u00b5l of cells were transferred into an Eppendorf tube, spun down and the pellet dried by dabbing on a paper towel. The pellet was resuspended in 100\u2009\u00b5l of Tris-EDTA buffer pH 8.5 with 15\u2009mg\u2009ml\u22121 lysozyme, vortexed for 10\u2009seconds and incubated at room temperature for 5\u2009minutes with shaking. To the mixture, 400\u2009\u00b5l of TRK Lysis Buffer (Omega Biotek) with 4\u2009\u00b5l 2-mercaptoethanol was added and total RNA was extracted immediately using the E.Z.N.A RNA Isolation Kit (Omega Biotek). Purified RNA was quantified with NanoDrop (Thermo Fisher Scientific). One microgram of RNA was converted to cDNA using the GoScript Reverse Transcriptase (Promega). Quantitative real-time PCR (qPCR) was performed with GoTaq qPCR (Promega) using the CFX Opus 96 Real-Time PCR System (Bio-Rad). Fold changes were normalized to 16S rRNA and are based on relative expression values calculated using the \\({2}^{-\\varDelta \\varDelta {C}_{{\\rm{T}}}}\\) method.<\/p>\n LySE evolution of EG assimilation pathway<\/p>\n The gox0313 gene (UniProt: Q5FU50<\/a>) was synthesized by GentleGen. We cloned pAN29 (253996; Addgene), a phagemid containing metabolic pathway genes for complete assimilation of EG. E. coli MG1655 containing pAN29 and pSJ139 (T7 DNAP v9) was grown overnight, diluted and lysed as per standard LySE protocols. After 1 generation of phagemid replication and transduction into MG1655 with pSJ139, transduced cells were diluted 10-fold with LB with ampicillin and kanamycin and incubated overnight at 37\u2009\u00b0C with shaking at 225\u2009rpm. The next day, 5\u2009ml of recovered cells were pelleted by centrifugation at 3,900g for 3\u2009minutes, washed 3\u00d7 with M9 medium and then resuspended in M9 medium with EG, with or without glucose at concentrations as indicated in the text until the OD600 was approximately 0.2. No antibiotics were used for selection. To each well in a 24-well plate, 1\u2009ml of the cell suspension was added and cultured overnight at 37\u2009\u00b0C with shaking at 300\u2009rpm in a Thermo-Shaker PST-60HL-4 Microplate Reader (BioSan). Every hour, the OD600 was measured to monitor cell growth. Evolution cycles were repeated another four times for a total of five evolution cycles, using the best growing cultures from the previous cycle as the starting material for the next cycle. After the 5th LySE cycle, the best growing culture was streaked on M9 minimal agar plates supplemented with 2\u2009g\u2009l\u22121 of glucose and 10\u2009g\u2009l\u22121 of EG. Individual colonies were picked and inoculated separately in 1\u2009ml LB with kanamycin in 24-well plates and grown overnight. The following day, the cultures were washed with M9 media at 3,900g for 1\u2009minute. Subsequently, the cells were inoculated in M9 medium supplemented with 10\u2009g\u2009l\u22121 EG in 12\u2009ml culture tubes for 6\u2009hours to reach OD600\u2009=\u20090.1. For each culture, 200\u2009\u00b5l was transferred into a sterile 96-well microplate as triplicates and incubated overnight with continuous orbital shaking at 600\u2009rpm. Growth rates were determined by measuring the OD600 at 12 hours, 18 hours, 24 hours, 36 hours, 42 hours and 48\u2009hours post-inoculation using the microplate reader. Eight clones were PCR amplified for the phagemid using PrimeSTAR GXL DNA Polymerase and the amplicon was sequenced by nanopore sequencing.<\/p>\n For ALE, 5\u2009ml E. coli MG1655 containing pAN29 was grown overnight in LB with kanamycin, washed 3\u00d7 with M9 medium, and then resuspended in M9 medium with EG, with or without glucose at concentrations as indicated in the text until the OD600 was approximately 0.2. To each well in a 24-well plate, 1\u2009ml of the cell suspension was added and cultured overnight. The next day, 1\u2009\u03bcl of cells was added to 10\u2009ml of LB with kanamycin and grown overnight, before 5\u2009ml was taken and washed following the same washing and resuspension procedure as described previously for the next round of ALE. Evolution cycles were repeated another four times for a total of five evolution cycles, using the best growing cultures from the previous cycle as the starting material for the next cycle.<\/p>\n Statistical analysis<\/p>\n Significance was determined by two-tailed unpaired two-sample t-tests performed with Microsoft Excel Analysis ToolPak v.16.97.2.<\/p>\n Software<\/p>\n SnapGene 8.2.2 was used for plasmid design. GraphPad Prism 11.0.0 was used to generate all graphs. Illustrations were created in Adobe Illustrator 30.2.1 and BioRender.com<\/a>.<\/p>\n Reporting summary<\/p>\n