P recision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads

Abstract

Genetic circuits have many applications, from guiding living therapeutics to ordering process in a bioreactor, but to be useful they have to be genetically stable and not hinder the host. Encoding circuits in the genome reduces burden, but this decreases performance and can interfere with native transcription. We have designed genomic landing pads in Escherichia coli at high‐expression sites, flanked by ultrastrong double terminators. DNA payloads >8 kb are targeted to the landing pads using phage integrases. One landing pad is dedicated to carrying a sensor array, and two are used to carry genetic circuits. NOT/NOR gates based on repressors are optimized for the genome and characterized in the landing pads. These data are used, in conjunction with design automation software (Cello 2.0), to design circuits that perform quantitatively as predicted. These circuits require fourfold less RNA polymerase than when carried on a plasmid and are stable for weeks in a recA+ strain without selection. This approach enables the design of synthetic regulatory networks to guide cells in environments or for applications where plasmid use is infeasible.

Silica Nanostructures Produced Using Diatom Peptides with Designed Post‐Translational Modifications

“…E. coli MG1655 containing 7 repressors on the genome and two available landing pad sites was used for genome integration and protein expression. [113] LB-Miller medium (BD #244620) was used for strain propagation. 2xYT liquid medium (BD #244020) and LB + 1.5% agar (BD #214010) plates were used for transformation recovery and strain maintenance.…”

“…Therefore, SETD7 was integrated into a landing pad site on the genome of E. coli MG1655 cells carrying orthogonal repressors (including LacI, TetR, and CinR) on the genome (Methods). [113] The plasmids for R5 and PKA expression were co-transformed into this strain and normal growth (final OD 600  9.0 in TB media) was recovered.…”

Single-cell measurement of plasmid copy number and promoter activity

“…A problem with using bulk measurements to calculate promoter activity is that each cell has a different number of copies of the promoter because of differences in the copy number of the plasmid or genome on which it is carried. Plasmid copy number is dictated by its replication origin and can change depending on the genetics of the host strain 22 and growth conditions 23 and the copy number of the genome varies depending on the distance to the origin and growth rate 24 , 25 . The average plasmid copy number has been estimated with bulk DNA measurements, but no method has been developed to count the plasmid copy number in single living cells.…”

Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing

“…Such detail is lost with more typical fluorescence-based assays 6,11 , but is essential for developing the low-level biophysical or machine learning based models of genetic parts that are essential for predictive biodesign workflows [44][45][46][47] . While rich, highcontent characterization data can normally only be produced for a small set of samples 8,13,36,48 , the approach presented here removes this limitation, allowing us to more systematically explore the genetic design space of a large pooled library and glean several design principles.…”

“…The design of synthetic terminators has so far focused on increasing their strength to achieve near perfect termination 6 as a way of insulating multiple genetic parts and devices from each other 7 and the host genome 8 . Attempting to engineer terminators that function poorly in terms of efficiency or which can be tuned for manipulating transcript isoform ratios has not yet been explored.…”