A plasmid system with tunable copy number

Plasmids are one of the most commonly used platforms for genetic engineering and recombinant gene expression in bacteria. The range of available copy numbers for cloning vectors is largely restricted to the handful of Origins of Replication (ORIs) that have been isolated from plasmids found in nature. Here, we introduce two systems that allow for the continuous, finely-tuned control of plasmid copy number between 1 and 800 copies per cell: a plasmid with an anhydrotetracycline-controlled copy number, and a parallelized assay that is used to generate a continuous spectrum of 1194 ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our systems enable the precise control of gene expression, and our results highlight the importance of tuning plasmid copy number as a powerful tool for the optimization of synthetic biological systems.

In the ColE1 Origin, reversible binding of the inhibitory RNA-i to RNA-p exercises control over the copy number by inhibiting replication (left branch). Production of RNA-p transcript may result hybridization with the ORI, which leads to plasmid replication (right branch). C) Predicted plasmid copy number for hyperbolic (upper) and exponential (lower) initiation models [15] plotted as a function of RNA-p transcription initiation rate. Inset shows the predicted dependence of plasmid copy number on host cell division time. Model parameters: = 0.545 / min, ρ = 0.65, r = 0.01 / min, ki = 0.95 / min. D) Plasmid copy number measurements made by digital droplet PCR of the aTcinducible pUC-pTet plasmid at increasing aTc concentrations. E) Growth rates of cells hosting the pUC-pTet plasmid at increasing plasmid copy numbers. We see a marginal effect of plasmid copy number on cellular growth rates, inset shows individual growth curves colored by aTc concentration. F) Plasmid copy number measurements of a plasmid which contains an additional copy of the inhibitory RNA under the control of an IPTG-inducible promoter. G) Growth rate vs plasmid copy number of the IPTG-inducible plasmid shows a strong metabolic burden at high levels of RNA-i expression.
cess is classified as inhibitor-dilution copy number control 80 [18], owing to the fact that the copy number is controlled 81 through a combination of replication inhibition and dilu-82 tion of plasmids and regulatory components due to cell 83 division.

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Specifically, as the ratio of RNA-p transcription rate 85 k p to RNA-i transcription rate k i is increased, plasmid 86 copy number N p will also increase according to where is the RNA degradation rate, ρ is the RNA-p DNA isolates by measuring the ratio of the bla gene on 126 the plasmid to the genomic dxs gene [20]. 127 This plasmid showed robust induction of copy num-128 ber with aTc induction (Fig. 1D). Copy numbers ranged 129 from 1.4 copies per cell in the complete absence of aTc to 130 roughly 50 copies per cell at full induction (100 ng mL −1 131 aTc). We saw a very similar relation in the plasmid yield 132 from overnight cultures grown at varying aTc concentra-133 tions (Sup.). 134 We subsequently measured the growth rate of cells har- 135 boring the pUC-pTet plasmid as a function of the aTc 136 concentration in a microplate reader (Fig. 1E) surements of fluorescence and growth rate could be made. 196 We first observe clear differences in growth rates as 197 a function of both plasmid copy number and aTc con-  To first corroborate protein production measurements 231 with plasmid copy number, we observed the pUC-pTet-   Fig. 4. Massively Parallelized Copy Number Assay. A) Procedure for massively parallelized copy number assay. The promoter upstream of the priming RNA was randomized with site directed mutagenesis. 2. Plasmids were ligated and cells were transformed with this library of plasmids with high efficiency and grown until OD600 of 1.0, 3. at which point a fraction of the culture was diluted and regrown and the remaining fraction was saved for plasmid extraction. 4. Sequencing libraries were generated from isolated plasmids and sequenced using next generation sequencing. Growth rates and Copy numbers were then calculated from the frequencies of sequencing counts present at each time point. Digital PCR was performed to measure the copy number of individual constructs so that absolute copy numbers could be estimated from sequencing measurements. B) Ranked list of plasmids generated through mutations to the promoter driving the priming RNA in this work (n = 830). (lower) The distribution of copy numbers arising from mutations to the priming RNA promoter. C) Ranked list of plasmids generated through mutations to the promoter driving the inhibitory RNA in this work (n = 364). D) Agreement between plasmid copy number as measured by sequencing counts and digital droplet PCR. E) The relationship between plasmid copy number and growth rate as measured across 830 variants of the pUC19 plasmid. The size of each data point is inversely proportional to the error in the measurement, with the top, middle two and lowest quartiles representing an error of less than 5 %, between 5 % and 25 %, and more than 25 %, respectively. Data from Fig. 1E is plotted (red to blue gradient points) A linear metabolic burden seems to account for the observed relation reduction in growth rate with increasing copy number. fluorescence with plasmid copy number (Fig. 3A). We and grown again to an OD 600 of 1.0 (Fig. 4A, step 3).

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Excess culture from each step of dilution was set aside 311 for sequencing (Fig. 4A, step 4).

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For each recovered mutant, the abundance of each con- 318 4B-C) and their growth rates (Fig. 4D). Our method 319 was able to generate 1,194 plasmids, each with a distinct 320 plasmid copy number ranging from less than 1 copy per 321 cell to close to 800 copies per cell (Table S1).

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To make sure our sequencing-based method yields ac-323 curate plasmid copy number measurements, we next 324 sought out to calibrate our sequencing-based numbers to 325 absolute copy numbers made using digital droplet PCR We sought to demonstrate the potential for using plas-369 mid copy number to control complex biosynthetic pro-   (Table S1). We also measured the vi-398 olacein production from cell extracts and found a clearly 399 positive relation between the plasmid copy number and 400 Violacein production (Fig. 5E). Interestingly, we ob-

Sponge sites
Tunable plasmid copy number Nc with decoy binding sites S d .   The pUC-pLac-RNAi plasmid, whose copy number 611 can be reduced through the addition of IPTG, was con-   ter about 600 minutes (about 12 generations). We also 41 observe a stabilization in growth rate around this time.

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Only the data points past this 600 min threshold are used 43 in Fig. 3B and 3C of the main text, as well as supple- Growth rate measurement particularly lack of preci-53 sion for the low aTc conditions. Considering aTc con-54 centration of 1 ng mL −1 and above we observe a constant 55 growth rate with a 10% decrease for the highest aTc con-56 dition of 100 ng mL −1 .

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In supplementary figure S5, we plot fold change his-59 tograms and corresponding gamma distributions: with Γ the gamma function, a = k1 γ2 = x 2 σ 2 the mean