A DNA Bubble-Mediated Gene Regulation System ... - ACS Publications

Feb 1, 2017 - School of Chemistry and Chemical Engineering, and. § ... of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atl...
0 downloads 0 Views 3MB Size
Download PDF
Letter pubs.acs.org/synthbio

A DNA Bubble-Mediated Gene Regulation System Based on Thrombin-Bound DNA Aptamers Jing Wang,†,‡,§,# Le Yang,†,‡,§,# Xun Cui,† Zhe Zhang,‡ Lichun Dong,*,†,§ and Ningzi Guan*,‡ †

School of Chemistry and Chemical Engineering, and §Key Laboratory of Low-grade Energy Utilization Technologies & Systems of the Ministry of Education, Chongqing University, Chongqing 400044, China ‡ School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, 30332, United States S Supporting Information *

ABSTRACT: We describe here a novel approach to enhance the transcription of a target gene in cell-free systems by symmetrically introducing duplex aptamers upstream to a T7 promoter in both the sense and antisense strands of doublestranded plasmids, which leads to the formation of a DNA bubble due to the none-complementary state of the ssDNA region harboring the aptamer sequences. With the presence of thrombins, the DNA bubble would be enlarged due to the binding of aptamers with thrombins. Consequently, the recognition region of the promoter contained in the DNA bubble can be more easily recognized and bound by RNA polymerases, and the separation efficiency of the unwinding region can also be significantly improved, leading to the enhanced expression of the target gene at the transcriptional level. The effectiveness of the proposed gene regulation system was demonstrated by enhancing the expression of gf p and ecaA genes in cell-free systems. KEYWORDS: gene expression regulation, DNA bubble, aptamer, promoter

A

Accordingly, a gene regulation system with universal applicability is highly expected. It is well recognized that the transcription is initiated with the formation of RNA polymerase (RNAP)-promoter complex via the binding of RNAP to the recognition region of the promoter, which is followed by the DNA strand separation through melting of the unwinding region of the promoter. Then, the promoter-bound RNAP polymerizes the first few nucleotides (up to 10). After the transcription is initiated, the transcript becomes long enough to form a stable hybrid with the template strand, helping to stabilize the transcription complex. Subsequently, RNAP changes to its elongation conformation, loses its σ-factor, and moves away from the promoter. Among the transcription processes, the formation of the RNAP−promoter complex and separation of the unwinding region are the two rate-limiting steps.10 Therefore, the expression of a target gene can be potentially enhanced by strengthening the recognition capability of RNAP to the recognition region and speeding up the separation of the unwinding region. In this sense, nucleic acid aptamers, which include the DNA aptamer (single-stranded DNA molecule) and RNA aptamer (single-stranded DNA molecule) that can be engineered to bind to specific molecules tightly,11 could be utilized to realize the potential strategies. Accordingly, RNA

rtificial control of biofunctions through regulating gene expression is one of the most important and attractive technologies to build novel living systems useful in the areas of chemical synthesis, nanotechnology, pharmacology, cell biology, etc.1−4 The strategies to regulate gene expression through external stimulus, such as heat, electric field, pH, and light, are restricted since a demanding growth environment is required for the growth of organisms.5 It has also been well demonstrated that the regulators by deploying synthetic biology technologies with function-encoding DNA molecules are effective in fundamentally controlling the expression of target genes.6 For optimizing gene expression by using these conventional methods, it is important to control the expression of the individual genes for genetic circuits to operate at the desired level and achieve the ideal dynamic ranges, particularly regulating the toxic genes or those that may interfere with the host machinery.7,8 Accordingly, various molecular tools have been designed to regulate gene expression at both the transcriptional level (e.g., constitutive/inducible promoters with high dynamic ranges)9 and translational level (e.g., RNA-based regulators, libraries of ribosomal binding sites (RBSs)).5 However, a large portion of gene regulation systems, which were theoretically predicted to perform well, executed poorly in practice; furthermore, it is also uncertain whether the same gene regulation system could work well when being transferred to other species or environments since the current molecular tools are strictly specific in hosts and conditions.5 © 2017 American Chemical Society

Received: December 22, 2016 Published: February 1, 2017 758

DOI: 10.1021/acssynbio.6b00391 ACS Synth. Biol. 2017, 6, 758−765

Letter

ACS Synthetic Biology

Figure 1. A DNA bubble can be induced by introducing duplex aptamers upstream to the promoter; with the presence of thrombins, the gene expression at the transcriptional level can be enhanced. (a) The DNA bubble can only be induced by introducing the duplex aptamer upstream to the promoter and with the existence of specific ligands (thrombins in this study). (b) The effect of the DNA bubble-mediated gene regulation is dependent on the distance between the duplex aptamers and the promoter (DBDAP): (i) when the distance is too long, the regulation system would not play its function to enhance gene expression; (ii) the expression of target gene can be enhanced to some extent at a little long DBDAP; (iii) the expression of the target gene can be enhanced to the maximum extent at a suitable DBDAP; (iv) when DBDAP is too short, the recognition region of the promoter is sequestered by the bound thrombins, resulting in a weakened expression of the target gene.

aptamers have been extensively used to regulate the transcription or translation of target genes, often by coupling with the binding event and the ensuing conformational change.12,13 Nucleic aptamers can be chosen against any ligands of interest

from a combinatorial library by using an iterative affinity selection procedure. In addition, the current hybridization rules can facilitate the predictive and rational design of nucleic acid domains,11,14 which endow the aptamer-based gene regulation 759

DOI: 10.1021/acssynbio.6b00391 ACS Synth. Biol. 2017, 6, 758−765

Letter

ACS Synthetic Biology

Figure 2. The effect of the introduction of aptamer(s) 12 bp upstream to the promoter on the expression of the gf p gene. (a) GFP concentration of templates introduced with no, single, or duplex aptamers 12 bp upstream to the promoter after a 6 h reaction of cell-free protein synthesis experiments with/without the existence of 1.2 μM thrombins (DBDAP = 12 bp); (b) Kinetics of GFP concentration of templates introduced with no, single, or duplex aptamers 12 bp upstream to the promoter during a 6 h reaction of cell-free protein synthesis experiments with/without the existence of 1.2 μM thrombins (DBDAP = 12 bp); (c) Michaelis−Menten plots of the transcription rate versus T7 promoter concentration under 0.20 μM T7 RNA polymerase. The ATP incorporation was used to describe the transcription rate.

accelerated, leading to the enhanced expression of the target gene at the transcriptional level. The DNA bubble in this approach is manipulated through a physical method based on the binding of DNA aptamers with its corresponding ligands, the mechanism is generally applicable for other microbes without considering the different specificities. However, the enhancing effect of the DNA bubble-mediated gene regulation is dependent on the distance between the duplex aptamers with the promoter (DBDAP),16 which can be classified into the following scenarios: (i) when the distance is too long, both the sequences of the recognition region and the unwinding region cannot be contained in the induced DNA bubble, whose function to enhance gene expression cannot be fulfilled (Figure 1); (ii) when partial sequences of the recognition region are contained in the induced DNA bubble while no sequence of unwinding region is contained, the recognition capability of RNAP to the promoter can be strengthened while the separation efficiency of the unwinding region is not affected; the expression of the target gene can be enhanced to some extent (Figure 1);17 (iii) when the sequences of the recognition region and partial sequences of the unwinding region are contained in the induced DNA bubble at a suitable range of DBDAP, both the recognition of RNAP to the promoter and

systems with the ability to regulate gene expression for a wide variety of species through a simple method.15 In a previous study,15 an approach by using aptamers to repress gene expression at the transcriptional level has been described by placing a thrombin-bound ssDNA aptamer downstream to the T7 promoter. In the absence of thrombin, T7 RNA polymerases are able to transcribe the templates. Once the DNA aptamer was bound by thrombins, the transcription from the T7-aptamer promoter can be repressed. On the contrary, the transcriptional level of the target gene is enhanced in this study by symmetrically introducing duplex aptamers (two sequences of the same ssDNA aptamers) upstream to the T7 promoter in the sense and antisense strands of a doublestranded plasmid, respectively, which leads to the formation of a DNA bubble due to the none-complementary state of the DNA aptamer.15 With the presence of ligands (thrombins in this study), the DNA bubble would be enlarged around the duplex aptamers in the local structure of the double-stranded plasmid due to the binding of aptamers with thrombins. Consequently, the recognition region of promoter contained in the enlarged DNA bubble can be more easily recognized and bound by RNAPs, and the separation of the unwinding region of promoter contained in the DNA bubble can also be 760

DOI: 10.1021/acssynbio.6b00391 ACS Synth. Biol. 2017, 6, 758−765

Letter

ACS Synthetic Biology

Figure 3. Effect of DBDAP (indicated by the base numbers between the promoter and aptamer) on gene expression. (a) Km of Michaelis−Menten plots (transcription rate versus T7 promoter concentration under 0.20 μM T7 RNA polymerase. The ATP incorporation was used to describe the transcription rate under T7 RNA polymerase (0.20 μM) for the modified T7 promoters with different DBDAPs; (b) Kcal of Michaelis−Menten plots (transcription rate versus T7 promoter concentration under 0.20 μM T7 RNA polymerase. The ATP incorporation was used to describe the transcription rate) under T7 RNA polymerase (0.20 μM) for the modified T7 promoters with different DBDAPs; (c) GFP concentration as a function of DBDAP.

in the plasmid (Figure 2a,b), demonstrating that the proposed gene regulation system cannot affect the expression of the gf p gene without the presence of thrombins. Moreover, in the presence of 1.2 μM of thrombins, GFP concentration of the template introduced with a single aptamer in one strand also exhibited no significant change compared with that of the template with no aptamer being introduced (Figure 2a,b), indicating that it has no effect on gene expression by introducing a single aptamer upstream to promoter in one strand of the double-stranded plasmid even with the presence of thrombins. On the contrary, the GFP concentration of template with duplex aptamers being introduced in the doublestranded plasmid exhibited a 50% increase compared with that of the template with no aptamer being introduced (Figure 2a,b), clarifying that the introduction of duplex aptamers in both the sense and antisense strands of the double-stranded plasmid can significantly enhance the gene expression with the presence of thrombins. Subsequently, kinetic assays were performed on templates introduced with duplex aptamers, a single aptamer, and no aptamer, respectively. According to the Michaelis−Menten plots of transcription rate versus concen-

the separation efficiency of the unwinding region can be improved, leading to the enhanced expression of the target gene to the maximum extent (Figure 1); (iv) when DBDAP is too short, although the sequences of both the recognition region and unwinding region are contained in the DNA bubble, the recognition region of the promoter is presumably sequestered by the bound thrombins, resulting in the weakened recognition of the recognition region by RNAP and a reduced expression of the target gene (Figure 1). For demonstrating the effectiveness of the DNA bubblemediated gene regulation, the expression of the gfp gene was investigated by introducing a single or duplex aptamers 12bp upstream to the T7 promoter (DBDAP = 12 bp) (Figure 2a,b) in a double-stranded plasmid containing gf p gene in a cell-free system. In the situation without thrombins, the green fluorescent protein (GFP, encoded by gf p gene) concentration of the template introduced with a single aptamer (ssDNA) in one strand (sense or antisense) or duplex aptamers (two ssDNA aptamers) in both strands of the double-stranded plasmid (Figure S1, Figure S2) exhibited no obvious difference compared with the template with no aptamer being introduced 761

DOI: 10.1021/acssynbio.6b00391 ACS Synth. Biol. 2017, 6, 758−765

Letter

ACS Synthetic Biology

Figure 4. Effect of the aptamer-induced DNA bubble on the expression of ecaA gene. (a) Effect of thrombin concentration on carbonic anhydrase (CA) activity of the duplex aptamers-modified ecaA gene (DBDAP = 12 bp). (b) Relationship between the multiple of mRNA amplification and the enzymatic activity. A linear relationship can be built: the multiple of mRNA amplification n = 0.82929 × CA activity/(U/mg) + 0.05727, (R2 = 0.93816).

tration of T7 promoter (Figure 2c),18,19 Km and kcat for the three kinds of templates were calculated and shown in Figure 2c. Compared with that of the template without aptamer being introduced, Km of the template introduced with duplex aptamers 12bp exhibited a significant decrease while that of the template introduced with a single aptamer 12bp showed no obvious difference, demonstrating that the introduction of duplex aptamers 12bp upstream to the T7 promoter can significantly enhance the recognition capability of RNAP to the promoter. On the other side, kcat of the template introduced with duplex aptamers significantly increased while that of the template introduced with a single aptamer exhibited an undistinguished difference compared with that of the template without aptamer being introduced. Liu et al.16 have proposed a strategy for photoregulation of gene expression with the azobenzene-tethered DNA. Azobenzene is introduced into the promoter region, and the transcription reaction by RNAP is photoregulated by trans−cis isomerization of the incorporated azobenzene. Their study found that tethering an azobenzene at the specific recognition region of a promoter can strongly enhance the binding of RNAP without changing the transcription rate constant (NTP), but tethering an azobenzene at the specific unwinding region enhanced the NTP without changing the affinity of RNAP to promoter. In this study, the recognition region of the promoter contained in the DNA bubble can be more easily recognized and bound by RNAPs and the separation of the unwinding region of the promoter contained in the DNA bubble can also be accelerated (corresponding to a larger NTP), leading to the enhanced expression of the target gene at the transcriptional level. The advantage of our strategy is that the recognition of RNAP to promoter and separation of the unwinding region can be enhanced at the same time, demonstrated by the decreased Km value and increased kcat value. The effect of the gene regulation system on the expression of the gf p gene was also investigated for the cases of no aptamer, a single aptamer, and duplex aptamers 33bp (DBDAP = 33 bp) being introduced upstream to the T7 promoter, the results showed that the Km value for the template introduced with duplex aptamers 33bp was decreased, while the kcat values of all the three kinds templates

were substantially the same (Figure S13), indicating that the introduction of duplex aptamers 33bp upstream to the T7 promoter can enhance the recognition of RNAP to the promoter, but does not affect the separation efficiency of the unwinding region. To clarify the effect of DBDAP on gene expression, kinetic assays were performed on templates introduced with duplex aptamers of different DBDAPs. Figure 3a demonstrated that when DBDAP is less than 9 bp, a relatively larger Km would be obtained since the recognition region of the promoter is sequestered by the bound thrombins. When DBDAP is larger than 9 bp, the recognition region starts to be exposed from the bound thrombins, and can be completely exposed when DBDAP = 12 bp, at which, the Km value reaches the minimum. Afterward, with a further increase in DBDAP, Km slightly increases with the recognition region moving out from the induced DNA bubble gradually until DBDAP = 39 bp, at which, no sequence of the recognition region is still contained in the induced DNA bubble. Figure 3b showed that when DBDAP < 33 bp, the kcat value increases with a decrease in DBDAP, meaning that the separation efficiency of the unwinding region can be improved by decreasing DBDAP. At these cases, partial sequences of the unwinding region can be contained in the induced DNA bubble while a smaller DBDAP indicates that more sequences would be contained. Figure 3c demonstrated the variation of GFP concentration for templates introduced with duplex aptamers at different DBDAPs in the presence of 1.2 μM thrombins. When DBDAP > 39 bp, no sequence of the promoter is contained in the induced DNA bubble, the expression of gf p gene would not be affected by the induced DNA bubble, resulting in a very low GFP concentration. When 12 bp < DBDAP < 39 bp, GFD concentration increases with a decrease in DBDAP, indicating that the enhancing effect of the induced DNA bubble on the expression of gf p gene becomes stronger at a smaller DBDAP. At this case, the sequences of the recognition region and the unwinding region start to be contained in the induced DNA bubble when DBDAP = 39 and 33 bp, respectively. The improvement of both the recognition of RNAP to the promoter and the separation efficiency of DNA strands with the unwinding region bring about the pronounced 762

DOI: 10.1021/acssynbio.6b00391 ACS Synth. Biol. 2017, 6, 758−765

Letter

ACS Synthetic Biology enhancement of gf p gene expression. While when DBDAP < 12 bp, the recognition region starts to be sequestered by the bound thrombins; the expression of gf p gene decreases sharply with a further decrease in DBDAP. When DBDAP < 9 bp, the recognition region of the promoter is completely sequestered by the bound thrombins, the expression of gf p gene reaches the lowest level, indicated by the minimum GFP concentration. In general, the optimum DBDAP for the DNA bubble-mediated gene regulation system to enhance the expression of gf p gene under T7 promoter is 12 bp. Under this condition, Km of the corresponding template introduced with the duplex aptamers in the presence of 1.2 μM of thrombin is 0.21, which is much lower than that without introducing the aptamer (0.74). On the other hand, kcat of the corresponding template introduced with the duplex aptamers in the presence of 1.2 μM of thrombin is 0.38, much higher than that without introducing the aptamer (0.26). To evaluate the reliability of the constructed gene regulation system, the effect of the induced DNA bubble on the expression of ecaA gene was also studied by using enzyme assays. The results in Figure 4a showed that with the existence of 1.2 μM thrombins, the activity of carbonic anhydrase (CA) encoded by the ecaA gene introduced with duplex aptamers is almost 4-fold higher than that without introducing the aptamer, confirming that the proposed DNA bubble-mediated gene regulation system is capable of enhancing gene expression. However, gene expression can be controlled at both the transcriptional and the translational level;20 moreover, when the translational level of gene expression is improved by the induced DNA bubble, the amount of proteins would also increase even if the conversion process from DNA to mRNA is not regulated.21−23 Therefore, the mechanism for the induced DNA bubble to affect gene expression should be further clarified. Accordingly, the template containing the duplex aptamer-introduced ecaA gene was investigated at nine different concentrations of thrombins, in which, the expression of the ecaA gene was evaluated by using enzyme assays and the mRNA level was analyzed by using the real-time quantitative PCR. The results in Figure 4b showed that, at different concentrations of thrombins, the multiple of ecaA mRNA amplification is different as well as the activity of ecaA gene, which is almost proportional to the multiple of ecaA mRNA amplification. These observations confirmed that the induced DNA bubble gene enhances the gene expression at the transcriptional level. In conclusion, a novel regulation system to enhance gene expression was constructed by symmetrically introducing duplex aptamers upstream to the T7 promoter in the sense and antisense strands of a double-stranded plasmid, which leads to the formation of a DNA bubble. With the presence of thrombins, the DNA bubble would be enlarged around the duplex aptamers due to the binding of aptamers with thrombins. Consequently, once the recognition region and the unwinding region of promoter are contained in the DNA bubble, the recognition region can be more easily recognized and bound by RNAP, and the separation efficiency of the unwinding region can also be significantly improved, leading to the enhanced expression of the target gene at the transcriptional level. The DNA bubble in the proposed gene regulation system is induced through the noncomplementary sequences of DNA aptamer and enlarged due to the binding of aptamers with thrombins.15 The method is generic and can be extended to

other DNA aptamers and corresponding ligands. For example, the unwinding of the two functional regions of promoter (recognition region and unwinding region) could be potentially achieved by using helicases and dCas9, especially dCas9 to an activator domain, because the RNA-guided DNA-binding protein dCas9 is an excellent alternative candidate for unwinding a specific region of DNA.28 In this case, the noncomplementary sequences of ligand-bound aptamers do not need to be constructed as dCas9-based activator domain would induce the separation of complementary dsDNA without the need of using any fusion. Javaherian et al.27 has introduced a method for developing specific aptamers for target proteins in crude cell lysate and purification of the target proteins from the cell lysate using the obtained aptamers. Through their approach, we can develop aptamers corresponding to specific proteins in future works, and construct duplex aptamer-introduced plasmids containing specific genes in living cells. Consequently, the metabolic flux of target products can be improved by overexpressing the key genes and binding the side-effect proteins24−26 using the gene regulation system developed in this study.

■

METHODS Construction of Plasmids. To establish the aptamer-based gene regulation system, three kinds of double-stranded plasmids were constructed according to the standard techniques by cloning gf p gene with duplex DNA aptamers upstream to T7 promoter, a single DNA aptamer upstream to T7 promoter and a fully double-stranded T7 promoter without introducing aptamer into pET28a(+) vector backbones, respectively (Figure S1). Two double-stranded plasmids were constructed according to the standard techniques by cloning ecaA gene with duplex DNA aptamers upstream to T7 promoter and a fully double-stranded T7 promoter without introducing aptamer into pET28a(+) vector backbones, respectively. (Figure S1). All the plasmids, primers and strains used in this study are list in Table S1. The 5 fragments of the modified gf p or ecaA genes were obtained through the method of overlap PCR (Figure S3Figure S12). Their backbones were generated by digesting pET28a(+) plasmid with XbaI and XhoI endonucleases, then the 5 products were isolated and purified. The backbone and 5 products were joined respectively using T4 ligase to give 5 parent plasmids: pET28aNgfp (no aptamer modified gpf gene as a control), pET28aSgfp, pET28aDgfp, pET28aNecaA (no aptamer modified gpf gene as a control) and pET28aDecaA. The aptamer-introduced gene regulation system was tested by incubating the DNA templates with thrombins along with 0.01% Tween-20 for 1 h at room temperature, followed by the addition of the cell extract. The used thrombins (purchased from MP Biomedicals, USA) were prepared by diluting the stock solution (in 50% glycerol) into 10 mM of Tris-HCl pH 7.5 and 50 mM of KCl to ensure that the final glycerol concentration in the cell-free protein synthesis reactions is less than 0.5%. The thrombin DNA aptamer sequences in the study used to enhance gene expression is 5′GGTTGGTGTGGTTGG-3′ by referring to the study of Iyer and Doktycz.15 Cell-Free Protein Synthesis Experiments and GFP assays. The Promega S30 T7 High-Yield Expression System kit (Promega TM306) was used for the cell-free protein synthesis experiments. The S30 premix and the cell extract were mixed in the proportion recommended by the manufacturer, 763

DOI: 10.1021/acssynbio.6b00391 ACS Synth. Biol. 2017, 6, 758−765

ACS Synthetic Biology

■

and templates were used at 25 ng/μL concentration per reaction. DNA concentrations used were based on Karig’s method.29 Reactions were set up in Corning CLS3820 plates following manufacturer’s instructions. For the GFP assays, the synthesis reactions of cell-free proteins were prepared first, incubated at 30 °C with shaking for 6 h, and fluorescence measurements (485/20 nm excitation, 528/20 nm emission) were made every 7 min in a Biotek Synergy 2 plate reader. Values indicated in the graphs representing GFP concentrations were obtained after 6 h of synthesis reactions. Thrombindependent gene enhancement system was tested by incubating the DNA templates with thrombins along with 0.01% Tween20 for 1 h at room temperature, followed by the addition of the cell extract. The used thrombins (purchased from MP Biomedicals, USA) were prepared by diluting the stock solution (in 50% glycerol) into 10 mM Tris-HCl pH 7.5 and 50 mM KCl to ensure that the final glycerol concentration in the cell-free protein synthesis reactions is less than 0.5%. Michaelis−Menten plots. The operating conditions for the T7 RNAP reaction are as follows: [T7 RNAP (from TaKaRa)] = 50 unit in 20 μL (corresponding to 0.15 μM), [[α-32P]ATP] = 2 μCi in 20 μL, [each NTP] = 0.5 mM, [each strand of the promoter] = 2.0 μM, and [spermidine] = 2 mM. Tris-HCl buffer (40 mM, pH 8.0) containing dithiothreitol (5 mM), MgCl2 (24 mM), and NaCl (2 mM). First, a mixture of template and nontemplate strands were annealed in 10 mM of Tris-HCl buffer (pH 8.0) with 10 mM of NaCl by heating at 95 °C for 3 min and cooling to 37 °C for 30 min. Subsequently, the mixture was cooled in ice, and the stock solution involving NTPs and [α-32P] ATP was added. After T7 RNAP was added, the reaction mixture was incubated at 37 °C for 6 h to achieve the transcription. During the reaction, the mixture was sampled at a desired interval, and the transcription was stopped by adding dye solution containing 80% formamide, 50 mM EDTA, and 0.025% bromophenol blue (with the same volume as the sampled reaction mixture). For the Michaelis−Menten analysis, the transcription was carried out by changing promoter concentration. The ATP concentration was measured every 10 min by Microbial ATP ELISA Kit. In this experiment, the concentration of T7 RNA polymerase was kept constant at 0.20 μM. mRNA Analysis and Enzyme Assays. Messenger RNA analysis was performed by RT-PCR and real-time PCR. Total RNA was isolated using an E.Z.N.A total RNA kit I (Omega); 10 μg of total RNA was reversely transcribed with reverse transcriptase (Vazyme) to obtain the cDNA. Real-time PCR was performed in an ICycler (BioRad) monitoring doublestranded DNA assays continuously with SYBR-Green (Invitrogen). The fragments of the target gene in the diluted cDNA were amplified in a PCR with AmpliTaq Gold by the primer sets: CA-RT-up, CA-RT-down; The Ct values of the target gene were compared to those of ecaA gene contained in the pET28a(+) plasmid to test the expression of the target gene on the transcriptional level. To analyze enzyme activities, the reaction liquid was centrifuged at 10 000g at 4 °C for 20 min and the resulted supernatant was used for enzyme activity assays. CA activity was expressed in Wilbur- Anderson units per mg of protein and was calculated using the formula [(t0/t − 1) × 10]/mg protein, where t0 and t represent the time required for the pH to change from 8.0 to 7.0 in a buffer control and cell extracts, respectively.30

Letter

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00391. All the plasmids, primers, and strains used in this study are listed in Table S1, plasmids construction are shown in Figure S1. The five fragments of the modified gf p or ecaA genes were obtained through the method of overlap PCR (Figures S2−S12). Effect of the introduction of aptamer(s) 33 bp upstream to the promoter on the expression of gf p gene (Figure S13) (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-23-65106053. E-mail: [email protected]. *E-mail: [email protected]. Address: 3137 MoSE, Georgia Institute of Technology, North Avenue, Atlanta, GA, 30332. ORCID

Lichun Dong: 0000-0002-9876-0133 Author Contributions #

J.W. and L.Y. contributed equally to this work. X.C. and Z.Z. revised the manuscript, L.C.D. and N.Z.G. designed the work, analyzed the data, and drafted the manuscript. All authors read and approved the final manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work is financially supported by the National Science Foundation of China (51272296) and the graduate scientific research and innovation foundation of Chongqing, China (CYS16014).

■

REFERENCES

(1) Nielsen, J., Fussenegger, M., Keasliing, J., Lee, S. Y., Liao, J. C., Prather, K., and Palsson, B. (2014) Engineering synergy in biotechnology. Nat. Chem. Biol. 10, 319−322. (2) Geiss, G. K., Bumgarner, R. E., Birditt, B., Dahl, T., Dowidar, N., Dunaway, D. L., Dell, H. P., Ferree, S., George, R. D., Grogan, T., et al. (2008) Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat. Biotechnol. 26, 317−325. (3) Wang, J., Yang, L., Wang, D., Dong, L. C., and Chen, R. (2016) Enhanced Succinic Acid Productivity by Expression of mgtCB gene in Escherichia coli mutant. J. Ind. Microbiol. Biotechnol. 43, 505−516. (4) Khalil, A. S., and Collins, J. J. (2010) Synthetic biology: applications come of age. Nat. Rev. Genet. 11, 367−379. (5) Wang, J., Qin, D. D., Zhang, B. Y., Li, Q., Li, S., Zhou, X. H., Dong, L. C., and Wang, D. (2015) Fine-tuning ecaA and pepc gene expression by rbSSR library in Escherichia coli mutant for enhancing succinic acid production. Appl. Microbiol. Biotechnol. 99, 8575−8586. (6) Politz, M. C., Copeland, M. F., and Pfleger, B. F. (2013) Artificial repressors for controlling gene expression in bacteria. Chem. Commun. 49, 4325−4327. (7) Salis, H. M., Mirsky, E. A., and Voigt, C. A. (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946−950. (8) Callura, J. M., Dwyer, D. J., Isaacs, F. J., Cantor, C. R., and Collins, J. J. (2010) Tracking, tuning, and terminating microbial physiology using synthetic riboregulators. Proc. Natl. Acad. Sci. U. S. A. 107, 15898−15903. (9) Cox, R. S., Surette, M. G., and Elowitz, M. B. (2007) Programming gene expression with combinatorial promoters. Mol. Sys. Biol. 3, 145.

764

DOI: 10.1021/acssynbio.6b00391 ACS Synth. Biol. 2017, 6, 758−765

Letter

ACS Synthetic Biology (10) Weisburg, W. G., Barns, S. M., Pelletier, D. A., and Lane, D. J. (1991) 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173, 697−703. (11) Ellington, A. D., and Szostak, J. W. (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818−822. (12) Bayer, T. S., and Smolke, C. D. (2005) Programmable ligandcontrolled riboregulators of eukaryotic gene expression. Nat. Biotechnol. 23, 337−343. (13) Rodrigo, G., Landrain, T. E., and Jaramillo, A. (2012) De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells. Proc. Natl. Acad. Sci. U. S. A. 109, 15271− 15276. (14) Tuerk, C., and Gold, L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505−510. (15) Iyer, S., and Doktycz, M. J. (2014) Thrombin mediated transcriptional regulation using DNA aptamers in DNA based cell free protein synthesis. ACS Synth. Biol. 3, 340−346. (16) Liu, M. Z., Hiroyuki, A., and Makoto, K. (2006) AzobenzeneTethered T7 Promoter for Efficient Photoregulation of Transcription. J. Am. Chem. Soc. 128, 1009−1015. (17) Skinner, G. M., Baumann, C. G., Quinn, D. M., Molloy, J. E., and Hoggett, J. G. (2004) Promoter Binding, Initiation, and Elongation By Bacteriophage T7 RNA Polymerase: a Single-Molecule View of the Transcription Cycle. J. Biol. Chem. 279, 3239−3244. (18) McClure, W. R. (1985) Mechanism and control of transcription initiation in prokaryotes. Annu. Rev. Biochem. 54, 171−204. (19) Martin, C. T., and Coleman, J. E. (1987) Kinetic Analysis of T7 RNA Polymerase-Promoter Interactions with Small Synthetic Promoters. Biochemistry 26, 2690−2696. (20) Bell, S. D., and Jackson, S. P. (2000) Mechanism of Autoregulation by an Archaeal Transcriptional Repressor. J. Biol. Chem. 275, 12934−12940. (21) Crawford, L. V., Cole, C. N., Smith, A. E., Paucha, E., Tegtmeyer, P., Rundell, K., and Berg, P. (1978) Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorageindependent growth in simian virus 40-transformed and revertant mouse cell lines. Proc. Natl. Acad. Sci. U. S. A. 75, 117−121. (22) White, K. A., and Nagy, P. D. (2004) Advances in the molecular biology of tombusviruses: gene expression, genome replication, and recombination. Prog. Nucleic Acid Res. Mol. Biol. 78, 187−226. (23) Dever, T. E. (2002) Gene-specific regulation by general translation factors. Cell 108, 545−556. (24) Tong, A. H. Y., Evangelista, M., Parsons, A. B., Xu, H., Bader, G. D., Page, N., Robinson, M., Raghibizadeh, S., Hogue, C. W. V., Bussey, H., Andrews, B., Tyers, M., and Boone, C. (2001) Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364−2368. (25) Benner, S. A., and Sismour, A. M. (2005) Synthetic biology. Nat. Rev. Genet. 6, 533−543. (26) Zhang, X. L., Jantama, K., Moore, J. C., Jarboe, L. R., Shanmugam, K. T., and Ingram, L. O. (2009) Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 106, 20180−20185. (27) Javaherian, S., Musheev, M. U., Kanoatov, M., Berezovski, M. V., and Krylov, S. N. (2009) Selection of aptamers for a protein target in cell lysate and their application to protein purification. Nucleic Acids Res. 37, e62. (28) Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z. R., Brar, G. A., Torres, S. E., Stern-Ginossar, N., Brandman, O., Whitehead, E. H., Doudna, J. A., Lim, W. A., Weissman, J. S., and Qi, L. S. (2013) CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell 154, 442−451. (29) Karig, D. K., Iyer, S., Simpson, M. L., and Doktycz, M. J. (2012) Expression optimization and synthetic gene networks in cell-free systems. Nucleic Acids Res. 2012 (40), 3763−3774. (30) Wibur, K. M., and Anderson, N. G. (1948) Electrometric and colorimetric determination of carbonic anhydrase. J. Biol. Chem. 1948 (176), 147−154. 765

DOI: 10.1021/acssynbio.6b00391 ACS Synth. Biol. 2017, 6, 758−765