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Novel Utilization of Terminators in the Design of Biologically Adjustable Synthetic Filters Mei-Ting Lin, Chun-Ying Wang, Hui-Juan Xie, Chantal Hoi Yin Cheung, Chiao-Hui Hsieh, Hsueh-Fen Juan, Bor-Sen Chen, and Che Lin ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00174 • Publication Date (Web): 25 Feb 2016 Downloaded from http://pubs.acs.org on February 25, 2016
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ACS Synthetic Biology
Novel Utilization of Terminators in the Design of Biologically Adjustable Synthetic Filters Mei-Ting Lin†, Chun-Ying Wang +, Hui-Juan Xie‡, Chantal Hoi Yin Cheung£, Chiao-Hui Hsieh£, Hsueh-Fen Juan£, Bor-Sen Chen+, Che Lin†* †
Institute of Communications Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan Department of Electrical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan ‡ Department of Biomedical Engineering, National Cheng Kung University, Tainan City 701, Taiwan £ Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan +
KEYWORDS: synthetic biology, intrinsic terminator, dynamic modeling, Escherichia coli, synthetic filter ABSTRACT: Terminators, which signal the end of transcription processes, are typically placed behind the last coding sequence of an operon to prevent interference between transcript units in most biologically synthetic systems. Here, we seek to extend the usability of terminators in genetic system design by using terminators as regulatory genetic parts. Terminators with different impacts on their upstream and downstream genes are characterized in detail via dynamic modeling to predict the behavior of the overall genetic system. Some nonlinear effects of terminators observed in our terminator measurements potentially facilitate regulation of gene expression. Through dynamic modeling in silico, we find that such genetic systems may behave like genetic filters. In agreement with the simulations, we successfully implement genetic high-pass and bandpass filters in vivo, demonstrating the potential of using terminators as regulatory parts. The genetic bandpass filter in this work is implemented through the interdependence between genetic parts, in which the termination efficiency of a terminator varies with the strength of the upstream promoter. This design strategy for a bandpass filter requires fewer base pairs than the conventional strategy of concatenating high-pass and low-pass filters. Our results show that this novel utilization of terminators as regulatory parts may provide a new perspective for efficient design of genetic circuits. We believe that further exploration of the complicated dynamics of terminators is important in the development of synthetic biology.
Synthetic biology aims to combine well-characterized biological genetic parts into controllable, predictable devices to perform particular functions in organisms. They may provide novel solutions for applications that improve lives, for example, by enabling the production of inexpensive 1antimalarial drugs and the large-scale production of biofuels. 3 Aiming to build very-large-scale-integration biological devices in the future, researchers have begun to use fundamental electronic-like genetic circuits as building blocks, 4-8 9-12 13-15 for instance, logic gates, oscillators, toggle switches, 16-19 and filters. In order to predict the response of a system built from biological genetic parts, proper characterization of each genetic component is necessary. The majority of research on the characterization of genetic parts concentrates on the initiation of transcription and translation. That is, most works focus on characterizing the promoter and ribosomal binding site (RBS).20, 21 Relatively little research has been done for the characterization of terminators that are responsible for transcription termination. A terminator not only regulates a
downstream gene with various termination efficiencies, but also affects the upstream gene, most likely through by influencing mRNA stability. The functions and dynamics of terminators are much more complicated than are those of simple termination. However, few reports discuss the effect of the terminator, much less consider it as a regulatory part. This led us to investigate further the possibility of using terminators as a regulatory part in genetic circuit design. Transcription terminators of prokaryotes may be categorized into two major types: rho-dependent terminators and rhoindependent terminators. Most researchers in synthetic biology utilize the latter terminators to construct genetic circuits; we also focus on rho-independent terminators in this work. Rhoindependent terminators, also known as intrinsic terminators, utilize a stem-loop structure followed by a U-rich sequence to signal the end of transcription. The mechanism and features of intrinsic terminators have been studied extensively, and many algorithms have been proposed to detect and predict which 22-25 DNA regions can serve as terminators in the genome. On
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the basis of these fundamental studies, many terminators can be generated and used in genetic circuit design. For example, Chen et al. generated and characterized a total of 582 synthetic terminators.26 Major contributors to termination strength were identified with an aim to create terminators with high efficiencies for large-scale bio-engineered genetic circuits. Cambray et al. also characterized 61 terminators and established a linear regression model to relate termination efficiency to sequence features.27 In both studies, it was shown that creating terminators with a wide range of termination efficiency is possible. Nevertheless, only a small fraction of such terminators with high termination efficiency are used to construct engineered genetic systems in the literature. In most synthetic genetic systems, setting one terminator at the end of the last coding sequence of an operon is a routine arrangement for transcription termination, in which it is expected to provide complete termination and guarantee the independence between 28 transcript units. Here, the terminator is simply used as a full stop for the transcription process. There has been a shift of viewpoint on the function of terminators controlled by an external signal. Logic gates have been built by using a terminator that is inverted by 29, 30 recombinase to reduce its efficacy of termination. Different arrangements of terminators and specific recognition sites of recombinase lead to various logical functions. Terminators in the above-mentioned works are controlled in an “all-or-none” fashion, in which complete termination is expected when the terminator sequence aligns with its functional direction. Although terminators have been used judiciously to control logical functions in these works, this manner of control is an idealistic view of terminators, ignoring their complicated and sometimes nonlinear characteristics. In a 31 work by Pfleger et al., hairpins were formed randomly between the coding sequences to regulate the relative expression of the genes in one operon design. They used the hairpins as intrinsic terminators to regulate the level of gene expression via different abilities of the hairpin to stop the transcription. They conducted numerous experiments and selected the best genetic construction from the results. Instead of using a randomly formed hairpin, we seek to utilize a terminator as a regulatory part that can be designed before actual implementation of the genetic system. We concentrated on using the terminators as regulatory parts that can be selected to control gene expression in a biologically synthetic circuit. To this end, we first attempted to characterize the complicated and nonlinear effects of terminators on their downstream and upstream genes. To characterize the terminators, we constructed a genetic device with an operon structure for terminator measurement and considered the impact of the terminator in the design parameters of our dynamic model. There is evidence that the efficiency of a terminator, originally regarded as a constant within one genetic system, may vary with the activity of its 26 upstream promoter. We also observed this nonlinear effect in our measurements, thus prompting us to examine the potential of using terminators as genetic regulatory parts. Through these nonlinear effects, we successfully implemented biological bandpass and high-pass filters that are consistent with the prediction of dynamic modeling. Most previous designs for biological bandpass filters were achieved through the concatenation of low-pass and high-pass filters with various efficiencies of protein repression. In our work, we
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implemented a bandpass filter through the nonlinear effect of the terminator on gene expression, thereby reducing the complexity of our bandpass filter design by nearly half. In addition to the bandpass filter, the other genetic circuits designed with a terminator as a regulatory part in this work behaving like high-pass filters are also consistent with the simulation, thus demonstrating the potential of using terminators as regulatory parts.
RESULTS AND DISCUSSION Characterization of intrinsic terminators with impacts on the downstream and upstream genes. Genetic parts with well-defined characteristics are fundamental building blocks for constructing reliable and predictable genetic systems. Hence, before attempting to use terminators as regulatory parts in the engineered genetic system, we need to properly measure and characterize the terminators. Three terminators of various efficiencies (single terminator BBa_B0011, double terminator BBa_B0014, and double terminator BBa_B0015) are selected from the Registry of Standard Biology Parts (http://parts.igem.org/) as candidates to be measured and characterized by both genetic devices, as shown in Figures 1(a) and (b). Both devices employed an operon structure containing two reporter genes to measure the inserted terminator. The upstream and downstream reporter genes are red fluorescent protein (rfp, BBa_E1010) and green fluorescent protein (gfp, BBa_E0040), respectively; the promoter, PTet (BBa_R0040), drives the transcription initiation. Through observations on RFP and GFP, the inserted terminators are characterized. Without the repressor protein TetR in the genetic device shown in Figure 1(a), PTet constitutively drives the transcription process, and we can obtain basic characteristics of terminators. In addition, some studies indicate that the promoter strength, which is mediated by a protein or external
Figure 1. Genetic devices for measurement of the terminators. The purple T represents the terminator to be measured by the devices. The terminator is characterized through observations on RFP and GFP. (a) The constitutive measurement device. The promoter PTet constitutively drives the transcription process, and the upstream RFP and downstream GFP form at a constant level. (b) The inducible measurement device. The constitutively produced repressor TetR inhibits PTet, and varied induction of aTc results in different strengths of PTet and in varied expressions of the downstream gene.
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signal, influences the termination efficiency of a terminator, even when it is under a common promoter. To further observe this phenomenon, we include a constitutive promoter–RBS pair that generates the repressor protein TetR to inhibit PTet to construct the device shown in Figure 1(b). In the presence of anhydrotetracycline (aTc), aTc binds to TetR and inhibits its operation, consequently switching on PTet again. That is, aTc induces the device shown in Figure 1(b), and varying the induction of aTc leads to varied strengths of the upstream promoter PTet. Data presented in Figures S1 and S2 (Supporting Information) show that terminators affect the expression of both downstream GFP and upstream RFP. We define two parameters, and , to quantify the impact of the terminator on downstream and upstream genes, respectively. First, the termination efficiency, , quantifies the fraction of arriving transcription elongation complexes that are obstructed by the terminator from passing through the terminator. For example, a terminator with 100% TE implies that it can disrupt all the arriving transcription elongation complexes, thus preventing downstream production of mRNA. That is, no GFP would be synthesized in our measurement construction. Second, quantifies the fraction of upstream expression that is influenced by the terminator. A positive represents
enhanced expression of upstream RFP by the terminator, and negative represents attenuation of RFP expression by the terminator. Both parameters are introduced into dynamic models that describe the generation of the proteins of interest. By analyzing the steady states of the dynamic models, and are derived as: 1 and 1 1, 2 respectively, where and are the measured fluorescence intensities of GFP and RFP in the terminator measurement devices, and and denote the fluorescence intensities of GFP and RFP measured from the control devices that lack an inserted terminator between two fluorescent genes. To avoid confusion, and obtained from the genetic device shown in Figure 1(a) are renamed as and , , respectively, and are denoted by the red horizontal lines in Figures 2 and 3. Details of the derivation are available in the Supporting Information. Identified values of and are presented in Figures 2 and 3, respectively.
Figure 2. Termination efficiencies of terminators, TE, in both the constitutive and inducible measurements for (a) single terminator B0011, (b) double terminator B0014 (B0012+B0011), and (c) double terminator B0015 (B0010+B0012). The red horizontal line denotes !"# estimated from the constitutive device shown in Figure 1(a). Blue bars denote TE values estimated from the inducible device shown in Figure 1(b) at various aTc concentrations. Error bars are s.d. calculated from at least three measurements.
Figure 3. Impact of terminators on the expression of the upstream gene, $ , in both the constitutive and inducible measurements for (a) single terminator B0011, (b) double terminator B0014 (B0012+B0011), and (c) double terminator B0015 (B0010+B0012). The red horizontal line denotes $ , !"# estimated from the constitutive system shown in Figure 1(a) and blue bars denote %&' values estimated from the inducible system shown in Figure 1(b) at various aTc concentrations. Error bars are s.d. calculated from at least three measurements.
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Results in Figure 2 show that the termination efficiencies of the terminators differ. Even for the same terminator, in the inducible system also varies with the aTc concentration. The relationships between and the values estimated from the inducible systems of each terminator are not consistent. While the of B0011 is lower than most of the values obtained from the inducible measurement the values of B0014 and B0015 are almost the highest compared with their inducible counterparts. Another interesting observation is the trend of with respect to the aTc concentration. The values of B0011 and B0014 decrease with increasing aTc concentration, and after specific concentrations, the values of B0011 and B0014 gradually increase. However, of B0015 exhibits a monotonic increase with respect to aTc concentration. A similar trend of TE in the B0015 measurement was also reported in the work of Chen et al., in which the transcription process of the measurement device was driven by promoter PBAD activated by arabinose.26 Although the components of our measurement device are different from theirs, the trend of the TE of B0015 is similar; the is low at low promoter activities (low concentrations of arabinose) and high at high promoter activities (high concentrations of arabinose). Note that in Figure 3, most values are positive, indicating that the terminator arrangement downstream of the gene enhances gene expression in most situations. This agrees with previous
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studies that indicate that the terminator protects the upstream transcripts from degradation by RNase and thus enhances expression of the upstream gene.32 However, the magnitude of also does not remain constant. Compared with that of , the variation of , between these three terminators is smaller. The estimated from the inducible measurement also varies with aTc concentration. The change in in the B0014 measurement (Figure 3(b)) is worth noting. When the aTc concentration reaches 50 ng/mL, the of B0014 dramatically increased to 2.5. That is, compared with the control measurement, B0014 increased the RFP by 250% via induction with 50 ng/mL aTc. This indicates a threshold effect at an aTc concentration of 50 ng/mL. When aTc exceeds this threshold, the of B0014 begins to decrease gradually and finally stays at around 0.6. A similar threshold effect may also be observed with B0011 (Figure 3(a)). To further observe how the mRNA stability changes according to the inserted terminator, mRNA expression levels of GFP and RFP in the genetic device shown in Figure 1(a) were determined by qRT-PCR analysis. In the control measurement (no inserted terminator between RFP and GFP), a rapid mRNA half-life of 2.06 and 2.04 min with an identical trend were observed (Figure 4(a) and (b)). For the three selected terminators (BBa_B0011, BBa_B0014, and
Figure 4. The mRNA half-life of GFP and RFP with a terminator used as a regulatory system and a comparison of the mRNA expression levels by qRT-PCR. (a) The mRNA half-life of GFP (green) and RFP (red) of four selected regulatory terminators (WT, BBa_B0011, BBa_B0014, and BBa_B0015). The mRNA half-life of wild-type terminator of GFP (green) and RFP (red) are very stable with an identical trend and a rapid halflife of 2 min (b). The mRNA half-life of GFP and RFP mRNA were 14.71 and 4.52 min in B0011 (c); 11.63 and 8.35 min in B0014 (d); and 6.13 and 4.22 min in B0015 (e). The expressions of RFP within these three systems are similar and stable in a range of 4.22 - 8.35 min and a longer mRNA half-life of GFP of 6.13-14.71 min. The mRNA half-life represents mean results of three replicates and normalized based on gapA expression levels.
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BBa_B0015), we observed a stable mRNA half-life of reporter genes in a minute scale, where the mRNA half-life of RFP was 4.22 - 8.35 min compared to GFP 6.13 -14.71 min (Figure 4(a) and 4 (c)-(e)). Overall, the inserted terminators increase the half-lives of both upstream and downstream genes. From the results shown in Figure 2, 3 and 4, we observed consistent trends between mRNA half-life and the two parameters that characterize the inserted terminator. First, the trend of mRNA half-life of downstream GFP is negatively related to , i.e., of B0011 < of B0014 < of B0015 and the half-life of GFP for B0011 > that for B0014 > that for B0015. This indicates that higher termination efficiency would lead to less stable formation of downstream mRNA. Second, the half-life of upstream RFP follows a similar trend as , for genetic systems with different terminators. The , of B0014 is the largest and the half-life of the upstream RFP is also the longest for the B0014 system. The , for B0011 and B0015 are smaller and the corresponding half-lives are also shorter. Higher , indicates a larger enhancement for the terminator towards the upstream gene, leading to a longer mRNA half-life. Construction of genetic filters as a design example of using terminators as regulatory parts. From earlier results, we can clearly observe some of nonlinear effects of the terminators on the gene expression. These nonlinear effects complicate gene expression, thereby limiting the usefulness of terminators. Nevertheless, through better characterization, these nonlinear effects may also provide more versatility in the use terminators as regulatory parts in a synthetic genetic system. In general, the expression of downstream genes of terminators remains low, especially those with high termination efficiencies. To use terminators as regulatory parts, it is hence necessary to include an amplifying system downstream of the terminators. The Lux system, which is intrinsically endowed with high sensitivity and high expression, is thus used here to amplify the attenuated signal immediately after the terminators. The Lux system is a quorum sensing system correlated with population density. Using signal molecules, it serves as a communication system between bacteria. Genes within different cells are triggered simultaneously to achieve specific functions, such as biofilm formation and virulence. In the Lux system, the complex of the activator protein LuxR (BBa_C0062) and the signal molecule homoserine lactone (HSL) activates the promoter PLux (BBa_R0062). HSL, also known as AHL, is synthesized from the substrates, S-adenosylmethionine and acyl–acyl carrier protein, via catalysis with enzyme LuxI (BBa_C0061). The full genetic system design is illustrated in Figure 5(a). Unlike the previous terminator measurement device, LuxI is arranged directly behind the regulatory terminator depicted as a purple T, and the reporter gene GFP is driven by PLux. TetR and LuxR are the repressor and activator of the promoters PTet and PLux, respectively. Both regulated proteins, TetR and LuxR, are driven by a constitutive promoter–RBS pair and are unregulated by aTc and the regulatory terminator. In this genetic system design, we would like to focus our attention on the expression of RFP and LuxI. Here, both proteins are driven by PTet and are affected by the terminator inserted in between for regulation. The expression of LuxI catalyzes various amounts of AHL, which differently activate PLux and drive expression of GFP. Consequently, regulation of the terminator
on its downstream gene, luxI, can be observed through the reporter protein GFP. The dynamic models used for the overall genetic circuit design depicted in Figure 5(a) are described as follows: () * + , * + .( * + + /0 + () 123* + ,123* + .(123* + + /4 + () *56 + 1 + × *89,: ;
