Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology

Transcription

1 Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology Sen Yang a, Qingtao Liu a, Yunfeng Zhang a, Guocheng Du a,b, Jian Chen a,b*, Zhen Kang a,b* a The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi , China b Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu , China S Supporting Information * Corresponding authors, Zhen Kang, Phone: Fax: , zkang@jiangnan.edu.cn Jian Chen, Phone: Fax: , jchen@jiangnan.edu.cn

2 MATERIALS AND METHODS Strains, Plasmids and Cultivation Conditions. Strains and plasmids used in this study are listed in Supplementary Table S1. Escherichia coli JM109 was typically cultured in Luria Bertani (LB) broth (per liter: 10.0 g tryptone, 5.0 g yeast extract, and 10.0 g NaCl) at 37 C for plasmid construction. Solid media were prepared by adding 2.0 g/l agar to the media. To construct recombinant B. subtilis and S. cerevisiae strains, transformed cells were spread onto LB plates and yeast nitrogen base without Amino Acids (YNB) plates (per liter: 1.74 g YNB, 5.0 g (NH 4 ) 2 SO 4, and 20.0 g glucose) for screening. When required, ampicillin (100 mg/l) and kanamycin (50 mg /L) were added to the media. Saccharomyces cerevisiae harboring plasmid py26tef-gpd and its derivates was cultivated in YNB medium supplemented with 50 mg/l leucine, 50 mg/l histidine, and 50 mg/l tryptophan, while S. cerevisiae harboring plasmid pebs and its derivates was cultivated in YNB medium supplemented with 50 mg/l leucine, 50 mg/l histidine, 50 mg/l tryptophan, 50 mg/l uracil, and 600 mg/l geneticin (G418). To quantitatively evaluate the strength of P bs, a QPix 420 microbial colony picker (Molecular Devices, Sunnyvale, CA, USA) was used to pick E. coli and B. subtilis GFP-expressing recombinants from LB plates into LB broth in 96-well black plates (3603, Corning, Corning, NY, USA), followed by culturing at 37 C with shaking at 900 rpm. Saccharomyces cerevisiae recombinants were picked into YNB medium in 96-well black plates and cultivated at 30 C with shaking at 900 rpm. The relative fluorescence intensity and biomass of the cultures were detected every 2 h.

3 Plasmid Construction. The designed broad-spectrum promoter P bs was synthesized and amplified using the primers P bs F1/P bs R1 (Supplementary Table S2). The PCR product was fused with the GFP-encoding gene gfp (amplified using primers gfpf1/gfpr1), and the resulting P bs -gfp expression cassette was inserted between the BamHI/SacII sites of plasmid py26tef-gpd and the BamHI/PstI sites of plasmid pbsxph to produce py26-p bs -gfp and pucp-p bs -gfp, respectively. Subsequently, substitutions of P bs in py26-p bs -gfp with the promoters P min and P J23119 were performed by PCR using py26-p bs -gfp as the template and the primers P min F/P min R and P J23119 F/P J23119 R. The PCR products were phosphorylated and self-ligated to generate the plasmids py26-p min -gfp and py26-p J gfp, respectively. The promoter P cdd from the B. subtilis 168 genome (amplified using primers P cdd F/P cdd R) was fused with gfp (amplified using primers gfpf2/gfpr1), and the resulting P cdd -gfp expression cassette was inserted between the BamHI/PstI sites of plasmid pbsxph, resulting in plasmid pucp-p cdd -gfp. The open reading frame of the gfp gene was amplified using primers gfpf3/gfpr3 and inserted between the BamHI/XhoI sites of plasmid py26tef-gpd to generate py26-p GPD -gfp. To construct the E. coli, B. subtilis, and S. cerevisiae shuttle plasmid, the PCR product of P bs (amplified using primers P bs F2/P bs R2) was fused with the geneticin assistance gene kanr and the terminator T TEF from pug6 (amplified using primers kanrf/t TEF R). The resulting PCR product P bs -kanr-t TEF was assembled with the pamb replicon (amplified from pbsxph using the primers repef/reper), the repu replicon (amplified from pbsxph using primers repbf/repbr), and the 2-µm

4 replicon (amplified from py26tef-gpd using primers repsf/repsr) to generate the shuttle plasmid pebs1. Subsequently, the prokaryotic terminator T o was inserted between kanr and T TEF in plasmid pebs1 by PCR using pebs1 as the template and the T o F/T o R primers. The PCR product was phosphorylated and self-ligated to generate plasmid pebs. Then, the B. subtilis replicon and S. cerevisiae replicon were removed separately from pebs by PCR using the primers P bs F1/repER2 and repef2/t TEF R2, respectively. The PCR products were phosphorylated and self-ligated to generate the corresponding E. coli B. subtilis shuttle plasmid peb and the E. coli S. cerevisiae shuttle plasmid pes, respectively. The P bs -gfp-t ADH1 expression cassette (amplified from py26-p bs -gfp using the primers P bs F3/T ADH1 R) was inserted into the XhoI/KpnI sites of pebs, resulting in pebs-p bs -gfp. The broad-spectrum promoter library was obtained by PCR using the primers P bs F4/T ADH1 R and py26-p bs -gfp as the template. The PCR product containing the synthetic P bsx -gfp-t ADH1 cassette was inserted into the XhoI and KpnI sites of plasmid pebs to generate pebs-p bsx -gfp. Fluorescence and Biomass Measurement. To analyze the relative fluorescence intensity and biomass of the GFP0-expressing recombinants, 96-well black plates that contained the cultures were subjected to relative fluorescence intensity and cell optical density determination using a Multi-Mode Microplate Reader (Cytation 3; BioTek, Winooski, VT, USA). To determine the relative fluorescence intensity, an excitation wavelength of 490 nm and an emission wavelength of 530 nm with a gain value 60 were used, and the cell optical density was measured at a wavelength of 600

5 nm. Single-cell fluorescence was analyzed with the MoFlo XDP AY45019 (Beckman Coulter, Brea, CA, USA) flow cytometer. GFP was excited at a wavelength of 488 nm, and the fluorescence signal was recovered with a 529(28)-nm band pass filter. The E. coli, B. subtilis and S. cerevisiae recombinants were cultured overnight and then inoculated (1% inoculum size) into the corresponding media. After culturing to the mid-exponential phase (6 h of growth for E. coli and B. subtilis and 8 h of growth for S. cerevisiae), the cells were washed twice in phosphate-buffered saline and diluted 1:100 with 0.01 M phosphate-buffered saline. For each assay, 120,000 cells were recovered. Data processing was performed using Summit software (version 5.4). The arithmetic mean of each distribution was used as the fluorescence value. qrt-pcr Analysis. Cultures used for RNA extraction were incubated in 500-ml flasks containing 50 ml of LB or YNB medium. After inoculation with 1% of overnight cultures, the E. coli, B. subtilis, and S. cerevisiae recombinants were cultured to the mid-exponential phase (E. coli and B. subtilis for 6 h, S. cerevisiae for 8 h). Then, the cells were concentrated and frozen immediately in liquid nitrogen. The total RNA of all the recombinants strains was extracted using the RNAprep Pure Kit (Tiangen Biotech, Beijing, China) according to the manufacturer s instructions. The quantity and purity of the RNA were determined by optical density measurements at 260 and 280 nm using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Subsequently, cdna, which was obtained by reverse transcribing the total RNA using the PrimeScriptTM RT-PCR Kit (Takara, Dalian,

6 China), was used as the template for qrt-pcrs. The gfp mrna level was measured by qrt-pcr, and the gapa mrna in E. coli, the gapa mrna in B. subtilis, and the ACT1 mrna in S. cerevisiae were selected as internal standards. The sequences of the gapaef/gapaer, gapabf/gapabr, ACT1F/ACTR, and gfpf5/gfpr5 primers are listed in Table 2. Gene expression analysis via qrt-pcr was performed in a 96-well plate with a total reaction volume of approximately 20 µl using SYBR Premix Ex Taq TM (Takara) according to the manufacturer s specifications. The reactions were conducted with a LightCycler 480 II Real-time PCR instrument (Roche Applied Science, Mannheim, Germany). The PCR conditions were as follows: an initial denaturation at 95 C for 30 s, followed by 40 cycles of denaturation at 95 C for 5 s, and annealing and extension at 55 C for 20 s.

7 Table S1. Strains and plasmids used in this study Strains and plasmids Description Source Strains Escherichia coli JM109 e14 - (mcra), enda1, reca1, hsdr17 (rk -,mk + ), (lac-proab) Invitrogen laciqzm15, rela1 Bacillus subtilis 168 trpc2 BGSC S. cerevisiae CEN.PK2-1C MATa, ura3-52, trp1-289, leu2-3,112, his3 1, MAL2-8 C, SUC2 Lab stock Plasmids py26tef-gpd E. coli/s. cerevisiae shuttle vector, ScURA3, 2 µm, Amp R Li et al. 1 py26-p bs -gfp py26tef-gpd derivate, P GPD, P TEF, P bs -gfp This work pbsxph E. coli/b. subtilis shuttle vector,amp R, Kan R Yang et al. 2 pucp-p bs -gfp pbsxph derivate, P bs -gfp This work py26-p min -gfp py26tef-gpd derivate, P GPD, P TEF, P min -gfp This work py26-p J gfp py26tef-gpd derivate, P GPD, P TEF, P J gfp This work pucp-p cdd -gfp pbsxph derivate, P cdd -gfp This work py26-p GPD -gfp py26tef-gpd derivate, P TEF, P GPD -gfp, This work pug6 E. coli plasmid, Amp R, loxp-kanr-t TEF -loxp Güldener et al. 3 pebs E. coli-b. subtilis-s. cerevisiae shuttle vector, G418 R This work peb E. coli-b. subtilis shuttle vector, G418 R This work pes E. coli- S. cerevisiae shuttle vector, G418 R This work pebs-p bs -gfp pebs derivate, P bs -gfp This work

8 Table S2. Primers used in this study Primers P bs F1 P bs R1 gfpf1 gfpr1 P min F P min R P J23119 F P J23119 R P cdd F P cdd R gfpf2 gfpf3 gfpr3 P bs F2 P bs R2 kanrf T TEF R repef reper repbf repbr repsf repsr T o F T o R reper2 repef2 T TEF R2 P bs F3 T ADH1 R P bs F4 gapaef gapaer gapabf gapabr ACT1F ACT1R gfpf4 gfpr4 Sequence(5-3 ) ACTGGATCCGGCGCGCCCCTCCTTGACACTGAATTTAGC GTGAAAAGTTCTTCTCCCTTACCCATTTTTTTTCCTCCTTTTTTCGATGC GCATCGAAAAAAGGAGGAAAAAAAATGGGTAAGGGAGAAGAACTTTTCAC GCTTCTGCAGCCGCGGTTATTTGTATAGTTCATCCATGCCATGT CATGTGATTAATTAACTTGTAATATTCTACCCAAGCTTATAAAAGAGCACTGTTG CTAAAATTTCAGTTTCAAGGAGGGGCGCGCCGGATCCCCCGGGCTGCAGG GACTGAGCTAGCTGTCAAGGATCCCTCGAGTCATGTAATTAGTTATGTCACGCTTACAT CTAGGTATAATACTAGTAAAAGGAGGAAAAAAAATGGGTAAGGGAGAAGAACTTTTCAC ACTGGATCCTGATAGGTGGTATGTTTTCGCTTGAACTTTT TGAAAAGTTCTTCTCCCTTACCCATTTTTTTTCCTCCTTTTCTCGAGGGTACCGC GCGGTACCCTCGAGAAAAGGAGGAAAAAAAATGGGTAAGGGAGAAGAACTTTTCA CTAGTGGATCCAAAAAAGCATCGAAAAAAGGAGGAAAAAAAATGGGTAAG TCGACCTCGAGTTATTTGTATAGTTCATCCATGCCATGTG TAGCTGAATAAGAACGGTGCTCTCGGATCCGGCGCGCCCCTCCTTGACAC GAAACGTGAGTCTTTTCCTTACCCATTTTTTTTCCTCCTTTTTTCGATGCTTTTTTC GAAAAAAGCATCGAAAAAAGGAGGAAAAAAAATGGGTAAGGAAAAGACTCACGTTTC TTGATTTCGGTTTCTTTGAAATTTTTTTGGTACCATTAAGGGTTCTCGAGAGCTCGTTT CTAAATACATTCAAATATGTATCCGCCTGCAGAAGCTTGGCGTAATCATGGTCATAGCT TCTGTACGTTCCTTAAGGAATTAATTCTGTCAGACCAAGTTTACTCATAT ATATGAGTAAACTTGGTCTGACAGAATTAATTCCTTAAGGAACGTACAGA GTGTCAAGGAGGGGCGCGCCGGATCCGAGAGCACCGTTCTTATTCAGCTA AAACGAGCTCTCGAGAACCCTTAATGGTACCAAAAAAATTTCAAAGAAACCGAAATCAA AGCTATGACCATGATTACGCCAAGCTTCTGCAGGCGGATACATATTTGAATGTATTTAG AACGCTCGGTTGCCGCCGGGCGTTTTTTATGCACTGACAATAAAAAGATTCTTGTTTT CTGAATTAATTAATCATCGCGACTGCAGAGATCTACTGATTAGAAAAACTCATCGAGC TTAATTCTGTCAGACCAAGTTTACTCATAT CTGCAGAAGCTTGGCGTAATCATGGTCATAGCTG TGGTACCATTAAGGGTTCTCGAGAGCTCGTTT GCTCTCGAGGGCGCGCCCCTCCTTGACACTGAATTTAGC CTTAATGGTACCGCTATTACGCCAGCTGAATTGGAGCGACCTCAT GCTCTCGAGGGCGCGCCNNNNNTTGACANNNNNNNTAGCATGTGATATAATWWWNWW WWWWWWCTACCCAAGCTTATAAAAGAGCACT TGAATTAGATGGTGATGT TAAGAGTAGTGACAAGTG GATGTGTTTACGAGCAGTT GACGAAGTTGGTGTTGAC AACAGTTGTCATCATACC TTATCTCTAACGCATCTTG ATGATGGAGTTGTAAGTAG TGCTCAATCTTCTTCAAT

9 Figure S1. Characterization of the broad-spectrum promoter P bs in E. coli, B. subtilis, and S. cerevisiae. Comparison of GFP expression in E. coli/py26-p bs -gfp and E. coli/py26-p J gfp (A), B. subtilis/pucp-p bs -gfp and B. subtilis/pucp-p cdd -gfp (B), and S. cerevisiae/ py26-p bs -gfp, S. cerevisiae/py26-p min -gfp, and S. cerevisiae/ py26-p GPD -gfp (C). Data are presented as the mean ± standard deviation (n = 3).

10 Figure S2. Maps of the shuttle plasmids. The E. coli B. subtilis shuttle plasmid peb (4.0 kb) (A), the E. coli S. cerevisiae shuttle plasmid pes (4.2 kb) (B), and the E. coli B. subtilis S. cerevisiae shuttle plasmid pebs (5.8 kb) (C) were constructed by assembling the P bs -kanr-t o -T TEF kanamycin and geneticin resistance protein expression cassette with the corresponding replicons. T o and T TEF are prokaryotic and eukaryotic terminators, respectively. pebs-p bs -gfp-t ADH1 (D) was constructed by inserting the P bs -gfp-t ADH1 GFP expression cassette into pebs.

11 REFERENCES 1. Li, A., Liu, Z., Li, Q., Yu, L., Wang, D., and Deng, X. (2008) Construction and characterization of bidirectional expression vectors in Saccharomyces cerevisiae. FEMS Yeast Res 8, Yang, S., Du, G., Chen, J., and Kang, Z. (2017) Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis. Appl Microbiol Biotechnol 101, Güldener, U., Heck, S., Fielder, T., Beinhauer, J., and Hegemann, J. H. (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24, 2519.

12