Engineering and characterization of new intrinsic transcriptional terminators in Bacillus subtilis 168

Jean Paul Sinumvayo, Sen Yang, Jian Chen,2, Guocheng Du,2, and Zhen Kang,2

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Engineering and characterization of new intrinsic transcriptional terminators in168

Jean Paul Sinumvayo1, Sen Yang1, Jian Chen1,2, Guocheng Du1,2, and Zhen Kang1,2

1 The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China 2 Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, Jiangsu,China

Terminators as regulatory signals are typically placed behind the last coding sequence to block the transcription of DNA to RNA and release the transcript. In the present study, the hairpin and the U-rich sequence of the bacteriophage λtoterminator were first modified to investigate their effects on termination efficiency and mRNA stability in168. Compared with the native λtoterminator, the terminator variants M3, M11 and M12 showed higher termination efficiency values. Moreover, the variantsM3, M4 and M11 showed significant positive effects on the mRNA stability of the upstreamgene. Additionally, insertion of RNase site also increased the mRNA stability. The results of this study suggested that the composition of the hairpin loop is not required for effective intrinsic termination in. Our results also showed that the terminator could also be used as a potential tool for increasing mRNA stability and the corresponding enzyme production in.

synthetic biology, transcription terminator, λtoterminator, mRNA stability,

Introduction

Terminators are usually located at the end of a gene or an operon where they terminate the transcription of DNA to RNA and release the transcript[1]. Terminators fall into two categories: 1) rho-independent (or intrinsic) terminators and 2) rho-dependent terminators[2-3]. A majority of previous studies focused on intrinsic terminators, due to their function in the dissociation of transcription complexes without the assistance of auxiliary factors. In bacteria, the intrinsic terminator contains a stable GC-rich hairpin stem loop and a U-stretch that required for disturbing the stable transcription elongation complexes[3-5]. The transcription termination process involves in dissociation of RNA polymerase and separation of the RNA:DNA hybrid[6].

For a long time, terminators have not received enough attention as genetic regulators[2]and most studies have focused on prediction and identification alone[2,7], as well as the mutation and evaluation of the effects of the GC-rich hairpin and the poly(U) sequence[4,8-12]. Recently, terminators have been demonstrated to be highly important not only on downstream gene protection against transcript read-through but also for its positive effect on the stabilization of upstream mRNA transcripts[8,13]. In this regard, Chen. built a library of 582 natural and synthetic terminators. After calculation and comparison, they found that in addition to receiving the greatest contribution from the U-tract, the base content of the hairpin stem also correlated with the terminator strength. Specially, they found that strong terminators usually have higher GC content at the bottom of the hairpin stem. In contrast, no or weak correlations were observed between the hairpin stability and its length[14]. Similarly, Cambray. characterized 61 natural and synthetic terminators and attested their roles in the quantitative modeling of transcription termination for the optimization of synthetic genetic systems[8]. More recently, Liquantified the relationship between terminator position and terminator efficiency and provided a simple method for fine tuning termination efficiency without changing terminator sequences[11]. In addition to these fundamental studies, many studies have extended the usability of terminators in the construction of genetic systems (such as Logic gates[15-16]and genetic bandpass filter[17-18]), pathway optimization[19]and enzyme expression[9,13,20], suggesting the potential powerful applications of terminators in synthetic biology.

To date, most studies have concentrated onstrains and only a small fraction of literatures are focused on other microorganisms[21], such as[1,22-23]. In fact, the generally recognized safe strainhas been widely used as a powerful cell factory for multiple applications, including enzyme production, pathway engineering and synthetic biology[24-27]. Hence, engineering and characterization of rho-independent terminators inis of great significance. In the present study, the bacteriophage λtoterminator was engineered and investigated with an experimental system using. The results would have important applications in synthetic biology.

1 Materials and methods

1.1 Bacterial strains and plasmids

The strains, plasmids and primers used in this study are listed in Table 1 and Table 2, respectively.strain JM109 (e14?(mcrA), endA1, recA1, hsdR17 (rk?, mk+), (lac-proAB) lacIqZM15, relA1) used for plasmid construction and molecular manipulation of all genetic parts andstrain TOP10 (F–mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ (ara-leu) 7697 galE15 galK16 rpsL (StrR) endA1 λ), were purchased from Invitrogen. The pUC57-simple (Ampr, cloning vector in, isolated fromstrain DH5α, MCS) that harbored thegene was obtained from Takara. To perform the assay on the genetic system in this study, samples of168 (trpC2) obtained from BGSC and pP43NMK (Ampr, Kanr, shuttle expression-secretion vector) were used as expression host and vector, respectively.

1.2 Construction of characterization devices

The development of devices that allow the characterization of our new BioBrick terminator variants was based on construction of the GFP/RFP dual fluorescent system being with the inputs/outputs to the system controlled by the P43 promoter. We started by introducing several additional restriction enzyme recognition sites into the multiple cloning site (MCS) of the pP43NMK expression vector. Firstly, areporter gene fragment was amplified using primers GFP-F/GFP-R (Table 2). The resulting PCR product was digested withⅠ andⅠ and ligated intramolecularly to generate plasmid pP43NMK-, which contained successiveⅠ,Ⅰ,Ⅰ andⅠ recognition sites downstream. Secondly, a fragment of thereporter gene was amplified from the pUC57-simple plasmid using primers RFP-F/RFP-R and digested withⅠ andⅠ. The resulting PCR product was ligated in pP43NMK-plasmid digested with the same enzymes (Ⅰ andⅠ) to generate a control device pP43NMK--. The next step was the insertion of a testing terminator genetic sequence which was synthesized and amplified using primers λto-F/λto-R to generate pP43NMK--λto-. The construction of an insulated terminator was performed by insertion of new genetic devices (RNase site and strong hairpin) in the testing plasmid by means of amplification of pUC57-simple using primers RNase F/RNase R. The resulting PCR product was immediately mixed with 1 U ofⅠ to digest the remaining circular plasmid and the product was digested withⅠ then self-ligated to generate the plasmid pUC57-RNase site-λto-RNase site-strong hairpin. From the resulting plasmid, we amplified the fragment harboring the RNase site, λtoterminator and strong hairpin with primers λto-F/λto-R. The resulting PCR fragment was digested withⅠ andⅠ, then ligated downstream of thereporter in pP43NMK-digested with the same enzymes to generate the testing device pP43NMK--RNase site-λto-RNase site-strong hairpin-. Furthermore, we attained our characterization construct model by constructing terminator variants M1, M2, M3, M4, M5, M6, M7, M8, M9, M10 and M11 using primers M1-F/λto-R, M2–F/λto-R, M3-F/λto-R, M4-F/λto-R, M5-F/λto-R, M6-F/λto-R, M7-F/λto-R, M8-F/λto-R, M9-F/λto-R, M10-F/λto-R and M11-F/λto-R to generate recombinant plasmids named pP43NMK--M1-, pP43NMK--M2-, pP43NMK--M3-, pP43NMK--M4-, pP43NMK--M5-, pP43NMK-- M6-, pP43NMK--M7-, pP43NMK--M8-, pP43NMK--M9-, pP43NMK--M10-and pP43NMK--M11-, respectively. Recombinant bacteria harboring controls and testing devices were isolated, and the entire resulting devices were confirmed by sequencing. We further continued by transforming all confirmed plasmids into168 competent cells, and finally we obtained a control strain168 GFP-RFP and recombinant test strains168-M1,168-M2,168-M3,168-M4,168-M5,168-M6,168-M7,168-M8,168-M9,168-M10,168-M11 and168 GFP-RNase site-λto-RNase site-strong hairpin-which were treated and subjected to the characterization experiment.

Table 1 Strains and plasmids used in this study

Table 2 Primers used in this study

1.3 Media and culture conditions

JM109, TOP10 and168 were typically cultured in LB broth (per liter: 10.0 g tryptone, 5.0 g yeast extract, and 10.0 g NaCl) at 37 ℃ with shaking speed of 220 r/min. Solid media were prepared by adding 1.5 g/Lagar to the respective media. Unless otherwise stated, the antibiotic concentrations were 100 mg/Lampicillin for, and 100 mg/Lkanamycin for168. To evaluate the expression of our genetic devices, all plasmids harboring genetic sequences were cultured in LB medium supplemented with antibiotic and flask cultivations were performed at 37 ℃ with a rotational speed of 220 r/min for 12 hours where samples were collected and treated every 2 hours and subjected to fluorescence measurements.

1.4 Analytical methods

1.4.1 GFP and RFP intensity quantification

For the GFP and RFP determination, the culture broth was centrifuged at 10 000×for 10 min, washed with 0.1 mol/L phosphate-buffered saline (pH 7.4), and diluted in the same buffer. Therefore, 200 μL of each sample was pipetted, added to the 96-well plates and immediately subjected to fluorescence determination. The GFP and RFP fluorescence intensity produced by the characterization devices were measured by using a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek) at an excitation/emission wavelength of 490/530 nm and 555/584 nm respectively.

1.4.2 Analysis of GFP mRNA transcript level

Samples taken from the culture media between 7 and 8 hours were thawed and centrifuged for 2 min at～11 000×at 4 ℃. The supernatant was discarded, and the pellet was immediately frozen at ?80 ℃. Cell pellets were suspended in Tris-EDTA (TE) buffer (10 mmol/L Tris-HCl, pH 8.0, 1 mmol/L EDTA) with lysozyme to a final concentration of 0.4 mg/ml. Total RNA was extracted with RNAprep Pure Kit (Tiangen biotech, CHINA) and RNA concentration was determined with the NanoDrop1000 (Thermo Fisher Scientific, MA, USA). Furthermore, cDNA was synthesized by Takara Prime Script Reverse Transcriptase with gDNA eraser and quantitative-PCR (Q-PCR) was carried out using TaKaRa SYBR Premix Dimer Eraser.

2 Results and discussion

2.1 Structures of the λtoterminator and its variants

The DNA sequences as well as the free energies of the λtoterminator[28]and its variants are shown in Table 3. In particular, both the RNA hairpin structures and their corresponding free energies were predicted by[29]The loop and the adjacent C-G base pair were examined and mutated since each contributed to the terminator strength[9].Generally, nucleotides mismatches occur very rarely in strong terminators, thus, the nucleotides mismatches in the stem of the hairpin were eliminated. In addition, the effects of the poly (T) tail was also analyzed. Specifically, the TGCC loop in variants M2, M3, M4, M5, M6, M7 and M8 were replaced with TTCG and GAAA which are considered to increase terminator efficiency and mRNA stability in many prokaryotes[7]. In variants M1, M2, M3 and M4, A was substituted with T to increase the number of T in the poly (T) tract but in variants M9, M10 and M11, the TGCC loop was substituted with GAAC, AAATC and AAAA, respectively. Additionally, to eliminate the mismatched nucleotide pair, T at positions 22 and 26 in the G+C rich stem were substituted with C in the variants M6, M7, M8, M9, M10 and M11 (Fig. 1).

Table 3 Wild type and terminator variant sequences used in this study

Fig. 1 Secondary structures of the λtO terminator and its variants. The predicted stabilities of the RNA hairpin structures were determined by the Kinefold web server. The termination positions for all engineered functional λtO variants were highlighted in red.

Fig. 2 Structures of the expression cassettes with two reporter genes gfp and rfp. (a) The control without terminator. (b) Terminator was inserted between two fluorescent protein encoding genes. (c) RNase sites and strong hairpin were inserted followed by rfp gene. RBS: ribosome-binding site.

2.2 Termination efficiencies of the constructed terminator variants

To assess the effects of the loops, poly (T) tract and mismatched nucleotide pair on transcription termination, as well as the termination efficiencies of the variants were determined by measuring the input and output fluorescence of the reporter genes (and) in the constructed system (Fig. 2). As shown in Fig. 3b, the insertion of the wild-type strong λtoterminator and its variants resulted in low expression of, suggesting the strong termination efficiency of all the terminators. After further quantification of the GFP fluorescence (Fig. 3a) and calculation[18], terminator variants M3, M4 and M11 showed higher termination efficiency values compared with the wild type λtoand the remaining variants (Fig. 3c). Interestingly, the inclusion of RNase sites in the variant M12 (Fig. 3c), showed a considerable termination efficiency of about 98% which prove its positive effect on the termination efficiency. The results clearly showed that the loop causes no significant effect on transcription termination although both tetra loops GAAA and TTCG are frequently found in many prokaryotic and eukaryotic RNAs[2-7].Meanwhile, the results also suggested that the introduction of mutations to enhance the RNA stem stability did not contribute to the termination efficiency, which are different from that in[22].

Fig. 3 Comparison of termination efficiency and mRNA stability of the λto terminator variant. (a) The GFP fluorescence of the native terminator (control) and the variants. (b) The RFP fluorescence of the native terminator and its variants. (c) Termination efficiencies of native (λtO) and its variants. (d) The mRNA levels of the gfp gene with the λtO terminator and its variants. Error bars are standard deviations calculated from three independent experiments.

2.3 Insertion of terminator increased upstream gene expression

In addition to evaluating the termination efficiency, the effect of the terminators on the upstream gene was also investigated and compared. Furthermore, the lowexpression revel obtained by measuring the negative control compared with other studied terminator variants (Fig. 3a), explains the role of terminators on the upstream gene. Interestingly, the GFP fluorescence was significantly increased when inserting all the terminator variants (Fig. 3a), indicating its positive effect on the RNA stability of the upstream, which is similar to previous studies in[10,30]and[9,13]. To further confirm this phenomenon, the mRNA ofin all the constructs were analyzed. Compared with the control, most of the constructs with terminator variants (especially M2, M3, M4, M9, M11 and M12) yielded a higher level ofmRNA (Fig. 3d). Recently, it has been reported that the addition of RNase sites surrounding the target terminator can impact the expression of reporter genes in[8,31]. Thus, two RNase sites and one strong hairpin were inserted between the reporter genesand(Fig. 2c). As expected, the expression ofwas significantly increased (Fig. 3a).

3 Conclusions

In this study, the hairpin and the U-rich sequence of the strong bacteriophage λtoterminator were first engineered and investigated in168. Compared with the wild-type strong λtoterminator, all the variants showed a higher termination efficiencies. The results suggested that the hairpin loop is not a critical factor for strong terminator. More importantly, the results first demonstrated that terminator insertion can significantly increase the expression of upstream gene in. As a result, these rho-independent terminators should be used as a potential tool for synthetic biology research in. In addition, the constructed terminators should also be used for increasing mRNA stability and the corresponding enzyme production in.

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(本文責(zé)編 郝麗芳)

枯草芽孢桿菌168新型轉(zhuǎn)錄終止子的構(gòu)建與表征

Jean Paul Sinumvayo1，楊森1，陳堅(jiān)1,2，堵國(guó)成1,2，康振1,2

1 江南大學(xué)工業(yè)生物技術(shù)教育部重點(diǎn)實(shí)驗(yàn)室，江蘇無(wú)錫 214122 2 江南大學(xué)食品安全與營(yíng)養(yǎng)協(xié)同創(chuàng)新中心，江蘇無(wú)錫 214122

Jean Paul Sinumvayo, 楊森, 陳堅(jiān), 等. 枯草芽孢桿菌168新型轉(zhuǎn)錄終止子的構(gòu)建與表征. 生物工程學(xué)報(bào), 2017, 33(7): 1091?1100.Jean Paul Sinumvayo, Yang S, Chen J, et al. Engineering and characterization of new intrinsic transcriptional terminators in Bacillus subtilis 168. Chin J Biotech, 2017, 33(7): 1091?1100.

轉(zhuǎn)錄終止子作為一種位于終止密碼子后的調(diào)控信號(hào)，負(fù)責(zé)終止DNA的轉(zhuǎn)錄和RNA的釋放。文中首次改造并分析了來(lái)源于噬菌體的λto終止子的發(fā)卡結(jié)構(gòu)與富含尿嘧啶的序列對(duì)枯草芽孢桿菌168中基因轉(zhuǎn)錄終止效率以及mRNA穩(wěn)定性的影響。結(jié)果表明，相對(duì)于野生型的λt0終止子，突變體M3、M11和M12表現(xiàn)出了更高的轉(zhuǎn)錄終止效率，突變體M3、M4和M11更有利于上游綠色熒光蛋白mRNA的穩(wěn)定。另外，我們發(fā)現(xiàn)插入RNase作用位點(diǎn)同樣提高了mRNA的穩(wěn)定性。研究結(jié)果表明終止子中的發(fā)卡環(huán)對(duì)轉(zhuǎn)錄終止不是必需的，同時(shí)，結(jié)果也證明了轉(zhuǎn)錄終止子可以作為一種潛在的工具用于提高枯草芽孢桿菌中mRNA的穩(wěn)定性以及相應(yīng)酶的 表達(dá)。

合成生物學(xué)，轉(zhuǎn)錄終止子，λto終止子，mRNA穩(wěn)定性，枯草芽孢桿菌

December 16, 2016; Accepted: February 8, 2017

Supported by:National Natural Science Foundation of China (No. 31670092), Key Technologies R&D Program of Jiangsu Province, China (No. BE2014607), Program for Changjiang Scholars and Innovative Research Team in University (no. IRT_15R26), Natural Science Foundation of Jiangsu Province (No. BK20141107).

Zhen Kang. Tel: +86-510-85918307; E-mail: zkang@jiangnan.edu.cn Guocheng Du. Tel: +86-510-85918307; Fax: +86-510-85918309; E-mail: gcdu@jiangnan.edu.cn

國(guó)家自然科學(xué)基金 (No. 31670092)，江蘇省科技攻關(guān)項(xiàng)目(No. BE2014607)，長(zhǎng)江學(xué)者與創(chuàng)新團(tuán)隊(duì)發(fā)展計(jì)劃(no. IRT_15R26)，江蘇省自然科學(xué)基金(No. BK20141107) 資助。