Supplementary information harboring CBB genes Non ...7Sangmin Leea, Yang-Hoon Kim*cand Jiho Min*b. 8...
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1 1 Supplementary information 2 3 Non-photosynthetic CO 2 bio-mitigation by Escherichia coli 4 harboring CBB genes † 5 6 Soo Youn Lee ‡a , Young Su Kim ‡b , Woo-Ri Shin ‡c , Jaeyoung Yu b , Jiye Lee a , 7 Sangmin Lee a , Yang-Hoon Kim *c and Jiho Min *b 8 9 a Gwangju Bio/Energy R&D Centre, Korea Institute of Energy Research, Gwangju 61003, 10 Korea. E-mail: [email protected], [email protected], [email protected] 11 b Graduate School of Semiconductor and Chemical Engineering, Jeonbuk National University, 12 Jeonju 54896, Korea. E-mail: [email protected] 13 c School of Biological Sciences, Chungbuk National University, Cheongju 28644, Korea. E- 14 mail: [email protected] 15 16 * Correspondence: [email protected]; [email protected] 17 ‡ These authors contributed equally. 18 †Electronic supplementary information (ESI) available. See DOI: 00.0000/0000000000 19 20 List of contents 21 Table S1. Nucleotide lengths of the rCBB genes and their molecular weights after translation 22 Table S2. Bacterial strains and plasmids used in this study 23 Table S3. Oligonucleotide used in this study 24 Table S4. Calculation of the specific CO 2 -fixtation rate of E. coli expressing whole rCBB 25 (CBB I/II ) after 24 h of anaerobic cultivation in 5% CO 2 26 Fig. S1. Schematic diagram of the plasmids. 27 Fig. S2. Growth profiles of the E. coli control (Mock) and E. coli expressing CBB I and CBB II . 28 Fig. S3. Relative CO 2 release during bacterial cultivation. 29 Fig. S4. Transmission electron microscope images of bacteria. 30 Fig. S5. Schematic illustration of the bioelectrochemical reactor for the electrochemical 31 analysis of E. coli strains. 32 Fig. S6. Cyclic voltammograms of E. coli control (Mock, black lines) and E. coli expressing 33 whole rCBB (CBB I / II , red lines) 34 Fig. S7. Photograph of the CO 2 bio-mitigation process in this work. 35 Electronic Supplementary Material (ESI) for Green Chemistry. This journal is © The Royal Society of Chemistry 2020
Transcript of Supplementary information harboring CBB genes Non ...7Sangmin Leea, Yang-Hoon Kim*cand Jiho Min*b. 8...
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1 Supplementary information
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3 Non-photosynthetic CO2 bio-mitigation by Escherichia coli 4 harboring CBB genes†
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6 Soo Youn Lee‡a, Young Su Kim‡b, Woo-Ri Shin‡c, Jaeyoung Yu b, Jiye Leea, 7 Sangmin Lee a, Yang-Hoon Kim*c and Jiho Min*b
89 a Gwangju Bio/Energy R&D Centre, Korea Institute of Energy Research, Gwangju 61003,
10 Korea. E-mail: [email protected], [email protected], [email protected] b Graduate School of Semiconductor and Chemical Engineering, Jeonbuk National University, 12 Jeonju 54896, Korea. E-mail: [email protected] c School of Biological Sciences, Chungbuk National University, Cheongju 28644, Korea. E-14 mail: [email protected] * Correspondence: [email protected]; [email protected]
17 ‡ These authors contributed equally.
18 †Electronic supplementary information (ESI) available. See DOI: 00.0000/0000000000
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20 List of contents
21 Table S1. Nucleotide lengths of the rCBB genes and their molecular weights after translation22 Table S2. Bacterial strains and plasmids used in this study23 Table S3. Oligonucleotide used in this study24 Table S4. Calculation of the specific CO2-fixtation rate of E. coli expressing whole rCBB 25 (CBBI/II) after 24 h of anaerobic cultivation in 5% CO2
26 Fig. S1. Schematic diagram of the plasmids.27 Fig. S2. Growth profiles of the E. coli control (Mock) and E. coli expressing CBBI and CBBII.28 Fig. S3. Relative CO2 release during bacterial cultivation.29 Fig. S4. Transmission electron microscope images of bacteria.30 Fig. S5. Schematic illustration of the bioelectrochemical reactor for the electrochemical 31 analysis of E. coli strains.32 Fig. S6. Cyclic voltammograms of E. coli control (Mock, black lines) and E. coli expressing 33 whole rCBB (CBBI/II, red lines)34 Fig. S7. Photograph of the CO2 bio-mitigation process in this work.35
Electronic Supplementary Material (ESI) for Green Chemistry.This journal is © The Royal Society of Chemistry 2020
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1 Table S1. Nucleotide lengths of the rCBB genes and their molecular weights after translation
Operon Gene Nucleotide length (bps)
Molecular weight of protein (kDa)
cbbF 1,002 36.7prkA 1,080 39.6cfxA 837 30.7cbbL 1,461 53.6
cbbI
cbbS 390 14.3
fbpB 996 36.5prkB 879 32.2tklB 1,974 72.4gapB 1,002 36.7cfxB 1,065 39.1
cbbII
rbpL 1,380 50.6
2345 Table S2. Bacterial strains and plasmids used in this study
Strains and plasmids Characteristics a Source or reference
StrainsR. sphaeroides 2.4.1 Wild-type KCTC 1434S. cerevisiae s2805 Wild-type, MATa pep4::HIS3 pro 1-8.6 can 1
his3-2000 ura3-52Korea Research Institute of Bioscience and Biotechnology,Korea
E. coli BL21 F- ompT hsdSB (rB- mB
-) gal dcm (DE3) Novagen, Madison, WI, USA
E. coli control E. coli BL21, pET-21a and pET-28b (Mock strain)
This study
E. coli (CBBI/II) E. coli BL21, pMBTLY-I/pMBTLY-II (recombinant strain)
This study
E. coli (CBBI) E. coli BL21, pMBTLY-I (recombinant strain) This studyE. coli (CBBII) E. coli BL21, pMBTLY-II (recombinant strain) This study
PlasmidspET-28b KmR, IPTG inducible T7 polymerase dependent
expression vectorNovagen, Madison, WI, USA
pET-21a AmR, IPTG inducible T7 polymerase dependent expression vector
Novagen, Madison, WI, USA
pMBTLY-I KmR, pET-28b derivative carrying cbbF-cbbS gene (cbbI)
This study
pMBTLY-II AmR, pET-21a derivative carrying fbpB-rbpL gene (cbbII)
This study
6 a Km, kanamycin; Am, ampicillin
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1 Table S3. Oligonucleotide used in this study
Oligo name a Target gene Sequence (5'-3') b Note b
cbbI operoncbbI-1F cbbF taatctagagtgaagccctttcccacc XbaIcbbI-1R cbbF taacatatgtcagctccggaacaggcc NheIcbbI-2F prkA-cfxA gggcatatgagcaagaagcatcccatc NheIcbbI-2R prkA-cfxA taagctagctcaggcggctttggcggt NdeIcbbI-3F cbbL-cbbS tttgctagcatggataccaacaccacc NdeIcbbI-3R cbbL-cbbS aataagctttcagcggacgatgctgtg HindIII
cbbII operoncbbII-1F fbpB-prkB taatctagaatggccatcgagctggaggac XbaIcbbII-1R fbpB-prkB taacatatgtctccgtctgtctgtcgc NheIcbbII-2F tklB-gapB ttacatatgaaggacattggagccgcg NheIcbbII-2R tklB-gapB attgctagctcagagaagccggcccat NdeIcbbII-3F cfxB-rbpL aaggctagcatggcactcatcacgctt NdeIcbbII-3R cfxB-rbpL aataagctttcaggccgcgcgatgcag HindIII
2 a F, forward; R, reverse3 b Underline, sites for restriction enzymes
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7 Table S4. Calculation of the specific CO2-fixtation rate of E. coli expressing whole rCBB 8 (CBBI/II) after 24 h of anaerobic cultivation in 5% CO2 as only the carbon source a
Consumed CO2(mmol L-1)
CO2-fixation rate(mg L-1 h-1)
Biomass(g DCW L-1)
Specific CO2-fixation rate(mg g DCW h-1) b
21.4 ± 0.2 39.2 ± 0.3 0.5 ± 0.0 78.4 ± 0.6
9 a Gas composition was CO2/H2/Ar (5:60:35) in 20 mL of M9 minimal medium10 b Calculated by the CO2-fixation rate in the unit of mg L-1 h-1 divided by the biomass concentration in 11 the g DCW L-1
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2
3456
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pMBTLY-I(10.3 kb)
cbbFprkAcfxAcbbLcbbS HindIII
NdeI
NheIXbaI
gapBcfxB
prkB
rbpL
fbpB
pMBTLY-II(12.7 kb)
tklB
HindIII
NdeI
NheIXbaIA) B)
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9 Fig. S1. Schematic diagram of the plasmids. (A) The genes on cbbI operon (cbbF, prkA, cfxA,
10 cbbL and cbbS) from R. sphaeroides 2.4.1 were cloned into cloned into pET-28b for the
11 inducible expression of CBBI enzymes in E. coli BL21 (DE3). (B) The genes on cbbII operon
12 (fbpB, prkB, tklB, gapB, cfxB and rbpL) from R. sphaeroides 2.4.1 were cloned into cloned into
13 pET-21a for the inducible expression of CBBII enzymes in E. coli BL21 (DE3).
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5 Fig. S2. Growth profiles of the E. coli control (Mock) and E. coli expressing CBBI and CBBII.
6 Heterogeneous expressions of whole rCBB (CBBI/II), CBBI-only or CBBII-only in E. coli were
7 induced by supplementation of IPTG (1 mM) at 3-h after inoculation (arrow).
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Time (h)0 5 4 24
Rel
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e C
O2 r
elea
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CBBI/II CBBI/II(I)
T / h0 2 4 24
Rel
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O2 r
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CBBII CBBII(I)
Time (h)0 2 4 24
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O2 r
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MockMock(I)
T / h0 2 4 24
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O2 r
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CBBI
CBBI(I)
A) B)
C) D)
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4 Fig. S3. Relative CO2 release during bacterial cultivation. (A) E. coli control (Mock). (B) E.
5 coli expressing whole rCBB (CBBI/II). (C) E. coli expressing CBBI-only. (D) E. coli expressing
6 CBBII-only. Opened symbols indicate non-induced cells. Closed symbols indicate 1 mM IPTG-
7 supplemented cells (I) for overexpression of target genes. The ratio of relative CO2 release was
8 calculated that the peak area of CO2 in gas chromatogram at T0 in each case was 1.
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2 Fig. S4. Transmission electron microscope images of bacteria. Black arrows indicate the
3 periplasmic spaces. White arrows indicate inclusion bodies.
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Convection(Magnetic stirring)
CE WE RE N2 purging
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4 Fig. S5. Schematic illustration of the bioelectrochemical reactor for the electrochemical
5 analysis of the E. coli strains. Bacterial cells attached to the glass carbon electrode (GCE) as
6 the working electrode (WE). The reference (RE) and counter (CE) were silver/silver chloride
7 (Ag/AgCl, NaCl saturated) and platinum wire, respectively. 0.1 mM neutral red was suspended
8 to mediate electron transfer from WE to bacteria. M9 minimal medium with 0.4 g L-1 glucose
9 was used as electrolyte. Electrochemical experiments, cyclic voltammetry and
10 chronoamperometry were performed at 37 oC and 100 rpm.
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2 E / V vs. Ag/AgCl-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8
J /
A cm
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4 Fig. S6. Cyclic voltammograms of the E. coli control (Mock, black lines) and E. coli expressing
5 whole rCBB (CBBI/II, red lines) without (dotted lines) and with 0.1 mM neutral red (NR) before
6 conducting chronoamperometry experiment (T(i, 0) in Figure 5a). 1 mM IPTG was
7 supplemented with NR. Inset, NR only (blue line). Scan rate, 10 mV s-1.
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6 Fig. S7. Photograph of the CO2 bio-mitigation process in this work. Double vessels were linked
7 with gas-line for CO2 recycling. Left, culture vessel for yeast fermentation; right, culture vessel
8 for E. coli expressing whole rCBB (CBBI/II) growth.
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