Plasmid Crosstalk in Cell-Free Expression Systems

Although cell-free protein expression has been widely used for the synthesis of single proteins, cell-free synthetic biology has rapidly expanded to new, more complex applications. One such application is the prototyping or implementation of complex genetic networks involving the expression of multiple proteins at precise ratios, often from different plasmids. However, expression of multiple proteins from multiple plasmids may inadvertently result in unexpected, off-target changes to the levels of the proteins being expressed, a phenomenon termed plasmid crosstalk. Here, we show that the effects of plasmid crosstalk—even at the qualitative level of increases vs decreases in protein expression—depend on the concentration of plasmids in the reaction and the type of transcriptional machinery involved in the expression. This crosstalk can have a significant impact on genetic circuitry function and even interpretation of simple experimental results and thus should be taken into consideration during the development of cell-free applications.


Figure S1
. Relative strengths of P T7,strong, P T7,weak , P σ70,strong , and P σ70,weak .Reactions were run with 5 nM of plasmid expressing sfGFP from one of the four promoters.The inset provides a higher resolution view of the P σ70,weak output.Reactions were incubated at 37°C and background fluorescence was subtracted.The shaded region indicates the standard deviation of technical triplicates.
Figure S2.Protein expression curves corresponding to the data in Figure 1.GFP was transcribed from one of four promoters: (A) P T7,strong , (B) P T7,weak , (C) P σ70,strong, and (D) P σ70,weak , each with three different types of empty vector and no additional plasmid added.In all subpanels, data were collected after a three-hour incubation at 37°C and background fluorescence was subtracted.The shaded region indicates the standard deviation of technical triplicates.Figure S3.Protein expression time course data corresponding to the data in Figure 1.sfGFP was transcribed from one of four promoters: (A) P T7,strong , (B) P T7,weak , (C) P σ70,strong, and (D) P σ70,weak , each with three different types of empty vector and no additional plasmid added.In all subpanels, background fluorescence was subtracted.The shaded region indicates the standard deviation of technical triplicates.
Figure S4.Assessment of plasmid crosstalk at the protein level in a lysate with basal T7 RNAP levels.The experimental design corresponds with that of the data in Figure 1.While the quantitative results for this lysate are not identical to those for the lysate used in Figure 1, most trends are consistent with a noteworthy exception of the results at low reporter concentrations for T7 reporters with EmptyT7 added.In all subpanels, data were collected after a three-hour incubation at 37°C and background fluorescence was subtracted.The shaded region indicates the standard deviation of technical triplicates.Figure S5.Protein expression time course data corresponding to the data in Figure S4.GFP was transcribed from one of four promoters: (A) P T7,strong , (B) P T7,weak , (C) P σ70,strong, and (D) P σ70,weak , each with three different types of empty vector and no additional plasmid added.In all subpanels, background fluorescence was subtracted.The shaded region indicates the standard deviation of technical triplicates.
Figure S6.Assessment of plasmid crosstalk at the mRNA level.The same experimental design used for the data in Figure 2 was used, except the lysate was prepared by a different researcher than in Figure 2. All trends are qualitatively consistent, though quantitative values do not match exactly, as one would expect due to batch-to-batch and operator-level variability in cell-free lysate preparation.Data were processed as described in Figure S5.In all subpanels, reactions were run for 3 h at 37°C and background fluorescence was subtracted.The shaded region indicates the standard deviation of technical triplicates.Figure S8.Repression of mRFP expression by expressed and purified LacI-sfGFP.RFP levels generally decrease with increasing concentration of purified LacI-sfGFP.At concentrations of repressor below ~150 µg/mL, the purified repressor enables stronger repression (compared to the fluorescence in the absence of repressor) of RFP levels than the expressed reporter.Above that threshold, however, increasing plasmid levels severely represses RFP expression, mediating much stronger repression than purified LacI.It is worth noting that the plasmid-expressed repressor curve does not seem to represent a function, with multiple fluorescence values corresponding to the same repressor concentration.This behavior arises from the fact that GFP fluorescence slightly decreases at high plasmid concentrations (as seen in Figure 5B), resulting in another (lower) fluorescence value being correlated to the same repressor concentration.

Figure S7 .
Figure S7.Example data analysis for data depicted in Figure 2 and Figure S6.(A) Background fluorescence of dye with no aptamer drops substantially as temperature in the plate reader equilibrates to 37°C after reaction assembly.(B) A representative 3JDB fluorescence curve with aptamer being expressed but without background subtraction also shows a drop early in the time course, though not as substantial.(C) A representative 3JDB fluorescence curve with background subtraction has negative fluorescence values at early time points due to the initial reduction in background fluorescence observed in panel A. (D) The final data as presented in Figure 2 are presented without negative fluorescence values since they are due to these artifacts.In all subpanels, reactions were run for 2 h at 37°C.The shaded region indicates the standard deviation of technical triplicates.
Figure S9.SDS-PAGE gel for LacI-sfGFP.The size of the LacI-sfGFP fusion is approximately 65 kDa.The protein ladder is a Trident Prestained (GTX50875).Elution #4 was used in the experiments described in Figure 5 and Figure S8.

Figure S10 .
Figure S10.Plasmid crosstalk in PUREfrex versus crude lysate.(A) In PUREfrex, minimal crosstalk is observed, as addition of 10 nM EmptySigma70 does not substantially alter sfGFP levels.(B) In a T7 RNAP-enriched crude lysate, EmptySigma70 induces positive crosstalk at low concentrations of the sfGFP plasmid.In all subpanels, data were collected after a three-hour incubation at 37°C.The shaded region indicates the standard deviation of technical triplicates.

Figure S11 .
Figure S11.Comparison of simulated (based on the txtlsim toolbox, dotted curves) and experimental (solid curves) data for crosstalk at the mRNA level.The experimental data are reproduced from Figure 2. Green represents the "no empty" condition, such that a green curve below other colors indicates positive crosstalk.Positive crosstalk is observed across many conditions in the experimental data but could not be recapitulated in the model.

Table S1 .
Description of plasmid parts and DNA sequences in this paper.P T7,strong sfGFP Plasmid encoding sfGFP expression under P T7,strong P T7,strong Stability hairpin RBS sfGFP Terminator Kanamycin resistance cassette ColE1 origin LacO mRFP Plasmid encoding mRFP expression under P σ70,weak and LacO operator P σ70,weak LacO Stability hairpin RBS mRFP Terminator Kanamycin resistance cassette ColE1 origin

Table S3 .
Initial parameter values and search space lower and upper boundaries for the simulation results in Figure S11.Initial guesses were sourced from Singhal et al., Synthetic Biology, 2021.The lower and upper boundaries indicate the boundary for the parameter search space.Some parameters were kept constant in all runs.