Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly

Abstract

In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminator parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.

Figure 1.

Design and implementation of a synthetic-sequence-guided DNA assembly strategy. (A) Computational approach for generating UNSes to facilitate isothermal assembly. In all, 10⁵ random 40-bp sequences were generated in MATLAB, and then eliminated if they contained the indicated sequences (see ‘Materials and Methods’). (B) ‘Part’ and ‘destination’ vectors for UNS-guided assembly. Each part vector contains a multiple cloning site (MCS) flanked by two UNSes (e.g. U₁ + U₂, U₂ + U₃) and a common terminal UNS (U_X). The MCS contains BioBrick and BglBrick cloning sites. Rare unique restriction sites (red arrows) flank the UNSes. Destination vectors contain only U₁ and U_X, and internal restriction sites. (C) Diagram of a five-piece assembly, including four part vectors (P.V.) and a destination vector. All part vectors are digested around U_N and U_N+1 except the last, which is digested around U_N and U_X to permit assembly into U₁U_X of the destination vector. Part vector cloning and assembly into a destination vector takes ∼3 days total. Only ∼1 day is required if the desired part vectors have already been generated. (D) Restriction digest of 16 clones from a representative five-piece assembly in which each part was an identical 380-bp sequence. Red arrow indicates the expected 1.6 kb insert. (E) Effect of different UNSes on mCherry expression in a part vector with or without a P_trc promoter (N = 6, error bars = SEM).

Figure 2.

Systematic variation of gene expression via UNS-guided assembly. (A) Promoter library. Schematic shows U₁U₂ part vector expressing mCherry and indicates the location of the promoter to be varied (red text). The y-axis shows mCherry expression from U₁U₂ part vectors with different BioFAB promoters; x-axis lists the relative strength of each promoter as measured by the BioFAB (N = 6, error bars = SEM). (B) Terminator testing. Schematic shows the product of assembling a promoter-less U₂-EGFP-U₃-U_X part vector downstream of a U₁-P_trc-mCherry-Term-U₂ part vector in pDestET. ‘Term’ (red text) represents one of the terminator arrangements listed on the x-axis, and is located between mCherry and EGFP in the final construct. Read-through frequency is reported as the ratio of EGFP to mCherry fluorescence, normalized to the no-terminator control (N = 6, error bars = SEM). (C) Assembly of a fluorescent protein expression library. U₁U₂ part vectors contained mCherry, the [T_B1006]²-T_T7 terminator (T₃ in figure), and one of four BioFAB promoters. U₂U₃ part vectors were the same but contained EGFP. These eight parts were assembled into pDestET, and 60 resulting clones pooled and tested for insert size via restriction digest. Arrows indicate the backbone (black), correct-size insert (red) and a minor incorrect assembly product (gray). In all, 97.9% of inserts are the correct size by densitometry (∼59/60 clones correct). (D) Fluorescence of 54 sequenced clones, with the color of each circle indicating a unique set of promoter sequences. Dashed lines indicate the mean mCherry or EGFP fluorescence of all clones with a given promoter sequence; the promoter corresponding to each dashed line is indicated at its intercept.

Figure 3.

Optimization of deoxychromoviridans production. (A) UNS-guided assembly strategy for a promoter library of vioB, vioA and vioE. Each part vector contained a vio gene, a [T_B1006]²-T_T7 terminator (T₃ in figure) and one of 6 BioFAB promoters. (B) TOP10 E. coli and TOP10 E. coli transformed with the assembled library, grown for 36 h on LB-agar plates. (C) Distribution of deoxychromoviridans yields from liquid cultures of individual clones. Yields were measured by extracting deoxychromoviridans from each culture and measuring its absorbance (see ‘Materials and Methods’), normalized to the highest absorbance obtained. Inset: restriction digest of 60 pooled library clones. Arrows indicate the backbone (bottom arrow), expected insert size (top arrow) and a minor, incorrect insert (middle arrow). 95.2% of inserts are the correct size by densitometry (∼57/60 clones correct). (D) Plot of individual clones’ deoxychromoviridans production as a function of their promoter strengths. The color of each dot indicates its level of production. The dashed oval highlights a cluster of strains with high production. The highest production strain has medium-to-strong vioB and vioE expression (P199 and P048, respectively) but weak vioA expression (P001). The intersect of the dashed lines indicates this point’s projection onto the vioA–vioB plane, and the solid line connects the point to its projection.

Figure 4.

Facile construction of genetic circuits for integration into mammalian chromosomes. (A) Construction of an AND gate circuit. Four parts were assembled into pDestRmceBAC, a BAC modified to enable site-specific chromosomal integration in mammalian cells. Parts A and B in all constructs are an HS4 insulator alone and an AND-gated reporter construct plus HS4, respectively. Parts C and D are the two inputs to the AND-gated reporter, but different versions were constructed to verify AND gate functionality; these parts also contain HS4 sequences (striped boxes). (B) Analytical restriction digests of assembled AND gate circuit variants with XhoI. Except for ABC₁D₂-1 and ABC₂D₃-3, all clones yielded the expected digest pattern (18/20 correct).