Supplementary Materials
1. Construction of plasmids
Owing to small size and rich clone sites, pBBR1MCS was chosen as a basic plasmid to construct some derivative plasmids. Primers for amplification and check of positive colonies were listed in Table 2. Some promoters with high scores from T. versutus SOB306 genome were selected by online promoter prediction software (http://www.fruitfly.org/seq_tools/promoter.html) (see Supplementary Table 4). The region containning T3 and lac promoters between upstream of multiple cloning sites and downstream of catA gene of pBBR1MCS was substituted for terminator rrnB by primers FKpbbrF/R to aviod interference to next experiments. Terminator rrnB amplified using primers rrnBF/R from pTrcHisA plasmid. The gene strAB without promoter encoding streptomycin (sm) was amplified from pKT230 by PCR using the primers HX-smF and HX-smR. Furhter, promoterless strAB was inserted into the Hind III/Xba I cloning sites to construct a novel plasmid pBBR-sm for detecting the desired promoters. The plasmid was named pBBR-sm used as probe identifying promoters. The promoterless gene encoding red fluorescence protein (RFP) was amplified from pET30a-mcherry using primers R1-F and R1-R; and then a terminator T1 predicted by online software (http://www.softberry.com) was amplified from T. versutus SOB306 genome using primers R2-F and R2-R. The rfp gene and T1 was linked by fusion PCR using primers R1-F and R2-R. The fusing fragment was inserted into pBBR1MCS after double digestion with Hind III and BamH I, and then a sm fragments with native promoter amplified using primers X-smF and X-smR, was inserted into the Xba I cloning sites of pBBR1MCS after single digestion. The plasmid named pBBR-rfp was used as a probe to identify promoters and evaluate their strength. All linkage products were transformed into DH5α competent cells, coated plates, and selected monoclonal colony PCR. We amplified vgb using primers Vgb F/R, digested the amplicon with Hind III and BamH I and cloned it into Hind III and BamH I sites of plasmid pBBR-rfp-pN.
2.The recipe of medium
The recipe of TD medium is as follows: Na2S2O3·5H20 19.92 g l-1, NaHCO3 58.8 g l-1, NaOH 5 g l-1, NH4Cl 0.268 g l-1, KNO3 0.505 g l-1, K2HPO4·3H2O 2 g l-1, MgCl2·6H2O 0.1 g l-1, trace elements 2 ml l-1 (1 liter solution contains: ), pH is natural. The working concentration of antibiotics was as following: chloramphenicol (Cmr) 34 μg ml-1, streptomycin (Smr) 100 μg ml-1, Kanamycin (Kmr) 50 μg ml-1. Modified TD contains 35.1 g l-1 NaCl substituting for NaHCO3 and NaOH in TD medium, 5 g l-1 yeast extract and other ingredients same to those of TD medium.
3. Plasmid stability test
Individual T. versutus SOB306 clones with different plasmids were transferred from selection plates to liquid TD medium supplemented with the appropriate antibiotic and grown beyond log phase. The cultures were serially subcultured into fresh antibiotic-free TD medium at 1 % dilution for a total of five times (more than 50 generations). The final cultures were diluted and plated on solid TD media with and without antibiotic. After five days, the number of clones recovered were counted and the percentage of clones carrying the plasmid conferring antibiotic resistance was computed using equation 1.
Supplementary Table 1 Strains and plasmids used in this study
Strain or plasmid / Phenotype or genotype / Source or referenceE. coli
sm10 / Kmr thi-1 thr leu tonA lacY supE recA::RP4-2–Tc::M / (Liu et al. 2007)
s17 / Tpr Smr recA thi pro hsdR negative hsdM positive RP4::2-Tc::Mu::Km Tn7 pir lysogen / (Tan et al. 2014)
DH5α / F- SupE44ΔlacU169(φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 / (Hanahan. 1983)
T. versutus SOB306 / A mutant strain of D301 by ultraviolet mutagenesis / This study
Plasmids
pBBR1MCS / CmR , pBBR1 replicon, mob+ / (Tan et al. 2014)
pKT230 / Smr Kmr mob+ / (Bagdasarian et al. 1981)
pBBR-smr / Cmr, Smr, the derivative plasmid of broad-host-range pBBR1MCS.
pBBR-rfp / the derivative plasmid of broad-host-range pBBR1MCS, Smr，rfp，T1 terminator. / This study
pBBR-rfp-pN / the derivative plasmid of broad-host-range pBBR-rfp, insert promoter p1/2/3/4/5/6/32/tac between KpnI and HindIII. / This study
pBBR-sm / Cmr, streptomycin-resistance gene without promoter, the derivative plasmid of broad-host-range pBBR1MCS. / This study
pBBR-sm-pN / the derivative plasmid of broad-host-range pBBR-sm, insert promoter p1/2/3/4/5/6/32/tac into pBBR-sm between KpnI and HindIII / This study
pBBR-Tvgb / Smr, tac, vgb / This study
Supplementary Table 2 The primers used in this paper
Primers / Sequences (5’to 3’)P1 / F: CGGGGTACC AGTTTCGGGCATTGAGGTACT
R: CCCAAGCTT GATATCGTGTTCGGTCGCGTT
P2 / F: CGGGGTACC GGAAACCCACCTGAAGATGAT
R: CCCAAGCTT CGCAGCAGCAGACTTCAC
P3 / F: CGGGGTACC AGTTGCTACCGTTGCTCCCTT
R: CCCAAGCTT ACCTTCGCGATTTTCGAGTTC
P4 / F: CGGGGTACC GCTGCTTGGTCACCACGACT
R: CCCAAGCTT GCCGTTCAGTTGGTTCTGTCG
P5 / F: CGGGGTACC CAGGGTTGATCGCGGTCAGT
R: CCCAAGCTT GCAGACATTGCGAATCGAATC
P6 / F: CGGGGTACC CTGGTGTTGGATCGAACGTGA
R: CCCAAGCTT CTTCTTGTCGGATGGGTCCTT
P32 / F: CGGGGTACC CCTCCTCATCCTCTTCATCCTC
R: CCCAAGCTT GGCGAGCTCGAATTACGAATT
Ptac / F: CGGGGTACC CTTTCCTGGCTTTGCTTCCA
R: CCCAAGCTT CCTCCTGCAGTGTTTCCTGT
R1 / F: CCCAAGCTT ATGGTGAGCAAGGGCGAGGA
R: GTTAATCCCAGCTCAACGCC TTAGCCGGCCTTGTACAGCTC
R2 / F: GAGCTGTACAAGGCCGGCTAA GGCGTTGAGCTGGGATTAAC
R: CGCGGATCC GTACACTCCATGCCACGGGTT
FKpbbr / F: TGGGCGCATGCATAAAAACT
R: GAACAAAAGCTGGGTACCGG
X-sm / F: TGCTCTAGA TCAATGGCTTATTTTCCTGCT
R: TGCTCTAGA GTGTTCCCAGGGGATAGGAG
HX-sm / F: CCCAAGCTT GAAGGAACCTCCATTGAATCG
R: TGCTCTAGA GTGTTCCCAGGGGATAGGAG
rrnB / F: ACGCAGAAGCGGTCTGATA
R: AGAAACGCAAAAAGGCCATC
M13 / F: CACACAGGAAACAGCTATGACCATG
R: CGTTGTAAAACGACGGCCAGTG
Vgb / F: CCCAAGCTTATGTTAGACCAGCAAACCATTAAC
R: CGCGGATCCTTATTCAACCGCTTGAGCGTA
Supplementary Table 3 Statistic of transfer frequency and plasmid stability
Donors / Plasmids / Recipient / Transfer Frequency (6h) / StabilityE. coli
Sm10 / pBBR-smr / T. versutus SOB306 / (1.33±0.33) x10-7 / 43%±8
pKT230 / (8.82±1.12) x10-7 / 59%±5
E. coli
S17 / pBBR-smr / (3.39±0.89) x10-5 / -
pKT230 / - / -
Supplementary Table 4 Information of predicted promoters from SOB306 strain.
Promoters / Relative proteins / ScoresP1 / pyruvate dehydrogenase / 0.87
P2 / OmAp porin protein / 0.93
P3 / peroxiredoxin / 0.61
P4 / glutamate synthase / 0.93
P5 / NADH dehydrogenase / 1
P6 / thioredoxin reductase / 1
Supplementary Fig. 1 Check of conjugative transfer between E. coli and T. versutus SOB306.(a) Exploration of different conjugative time. (b) Identification of positive colonies from selective plates
Supplementary Fig. 2 Expression of rfp gene promoted by different promoters in SOB306 strain. (I) Color of SOB306 strain and its two recombinant strain on plates. (II) Fluorescence microscope photos of SOB306 strain and its two recombinant strains at 550nm
Supplementary Fig. 3 The apparent condition of bacterial liquid of two kinds of strains at (a)30 h and (b) 40 h
Reference
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Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol (166) 557-580
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Tan D, Wu Q, Chen JC, Chen GQ (2014) Engineering Halomonas TD01 for the low-cost production of polyhydroxyalkanoates. Metab Eng 26: 34-47
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