Engineering bacteria to catabolize the carbonaceous component of sarin
Supplementary Information for
Engineering bacteria to catabolize the carbonaceous component of sarin: teaching E. coli to eat isopropanol Margaret E. Brown, Aindrila Mukhopadhyay, and Jay D. Keasling
Supplementary Results Abbreviations
S2
Figure S1. Lysate reactivity of E. coli expressing ACX complex
S3
Figure S2. Promoter and sequence modification for ACX protein complex expression
S4
Table S1. Plasmids used in this study
S5
Table S2. Plasmid depictions for ACX operon construction
S6
Figure S3. SDS-PAGE analysis of alcohol dehydrogenase expression and solubility
S7
Figure S4. Purification of recombinant ACX complex from E. coli DH1(DE3)
S8
Figure S5. Kinetic assays surveying activity of the recombinant ACX
S9
Figure S6. Plasmid system for growth of E. coli on IPA
S10
Figure S7. HPLC analysis of linearity in IPA and acetone detection
S10
Figure S8. Growth of plasmid-bearing E. coli BW25113(DE3) on IPA
S11
Figure S9. Increasing IPTG induction concentrations leads to lower IPA-dependent growth
S12
Figure S10. Expanding the IPA degradation system to include overexpression of the carbonic anhydrase
S13
Figure S11. Supplementation with thiamine or extra metals show no growth benefit
S14
Figure S12. Solubility of ACX protein as induced with increasing concentrations of IPTG inducer
S15
Figure S13. Purification of MMS-grown ACX
S16
Table S3. Oligonucleotides used for plasmid construction
S17
Table S4. G-blocks used for gene assembly
S19
Literature cited
S25
Abbreviations ADH, alcohol dehydrogenase ACX, acetone carboxylase AACS, acetoacetyl-CoA synthetase AACT, acetoacetyl-CoA thiolase PTE, phosphotriesterase PDE, phosphodiesterase PHN, phosphonate degradation cluster CA, carbonic anhydrase XAacx, acetone carboxylase from Xanthobacter autotrophicus PY2 AAacx, acetone carboxylase from Aromatoleum aromaticum PY2 PFadh (PF), alcohol dehydrogenase from Pseudomonas fluorescens CBadh (CB), alcohol dehydrogenase from Clostridium beijerinckii GSadh2 (GS), alcohol dehydrogenase from Gordonia sp. str. TY-5 TBadh (TB), alcohol dehydrogenase from Thermoanaerobacter brockii RNaacs (RN), acetoacetyl-CoA synthetase from Rattus norvegicus ECaact (EC), acetoacetyl-CoA thiolase from E. coli IPA, isopropanol IPTG, isopropyl β-D-1-thiogalactopyranoside HPLC, high-performance liquid chromatography TPP, thiamine pyrophosphate TMP, thiamine monophosphate T, thiamine MM, minimal media MMS, minimal media with supplements LB, lysogeny broth RFP, red fluorescent protein Glc, glucose aTet, anhydrotetracycline
2
Figure S1. Lysate reactivity of E. coli expressing ACX complex. HPLC traces of ACX reactivity with acetone shows the production of acetoacetate only in the presence of the ACX and acetone. +ACX, ACX-expressing lysate; -ACX, control lysate.
3
Figure S2. Promoter and sequence modification for ACX protein complex expression. A) Codon-modified acx sequences transcribed from different promoters, comparing the original expression off of the Trc-encoded pTrc33 and the BglBrick plasmid promoters (Trc, lacUV5, T7, ara).1 Modification of promoter shows little alteration in expression levels. B) Use of the native acx sequence in expression plasmids shows an increase in protein content when expressed from the T7 promoter of the pBbE7c vector. Lane 1, ACX; 2, ACXn; 3, ACXnT; 4, ACXnT-rbs; 5, ACXnT-dT7; 6, ACXnT-dT7-ssC; 7, ACXnT-dT7-v3; 8, RFP). Final modification (ACXnT-dT7v3) placing the native C subunit in front of the native A subunit for expression yields expression levels of all three subunits similar to that found in acetone-grown Xanthobacter. Blue arrows indicate the 3 subunits. C) Densitometric analysis of the 3 subunits of ACXnT-dT7-v3 as determined by ImageJ. Density, intensity measurement of band; size, molecular weight of subunit (Da).
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Table S1. Plasmids used in this study. Strain a
1 a 2 a 3 a 4 a 5 a 6 a 7 a 8 a 9 a 10 a 11 a 12 a 13 a 14 a 15 a 16 a 17 a 18 a 19 a 20 21 22 23 24 25 26 27 28 29 30 31 32 Plasmid pTrc33-* pET16b-* pBbE1c-* pBbE5c-* pBbE7c-* pBbE8c-* pBbS5a-* pBbS2a-*
E. coli host DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) BW25113(DE3) BW25113(DE3) BW25113(DE3)
Plasmid 1 pTrc33-empty** pTrc33-XAacx pTrc33-AAacx pET16b-RFP pET16b-PFadh pET16b-CBadh pET16b-GSadh2 pET16b-TBadh pET16b-ECaact pET16b-RNaacs pBbE1c-XAacx pBbE5c-XAacx pBbE7c-XAacx pBbE8c-XAacx pBbE7c-XAacxn pBbE7c-XAacxnT pBbE7c-XAacxnT-rbs pBbE7c-XAacxnT-dT7 pBbE7c-XAacxnT-dT7-ssC pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3
Plasmid 2 pBbS5a-RFP pBbS5a-PF pBbS5a-CB pBbS5a-PF-CA pBbS5a-CB-CA pBbS5a-CB-CA-RN-EC pBbS2a-RFP pBbS2a-CB-CA pBbS2a-CB-CA-RN-EC pBbS5a-RFP pBbS5a-PF pBbS5a-CB
Promoter Trc T7 Trc lacUV5 T7 arabinose lacUV5 Tet
Origin of Replication p15a ColE1 ColE1 ColE1 ColE1 ColE1 SC101 SC101
Antibiotic resistance CmR CbR CmR CmR CmR CmR CbR CbR
a
* = multiple constructs, ** = no insert, = permanent storage in DH10B-T1
Strain number JBEI-14106 JBEI-14107 JBEI-14108 JBEI-14102 JBEI-14103 JBEI-14104 JBEI-14105 JBEI-14101 JBEI-14100 JBEI-14098 JBEI-14096 JBEI-14097 JBEI-14113 JBEI-14112 JBEI-14118 JBEI-14206 JBEI-14205 JBEI-14117 JBEI-14116 JBEI-14132 JBEI-14133 JBEI-14127 JBEI-14128 JBEI-14129 JBEI-14130 JBEI-14131 JBEI-14123 JBEI-14124 JBEI-14125 JBEI-14126 JBEI-14121
R
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Table S2. Plasmid depictions for ACX operon construction.
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Figure S3. SDS-PAGE analysis of alcohol dehydrogenase expression and solubility. Expression of the four candidate alcohol dehydrogenases in E. coli DH1(DE3) using 500 µM IPTG. For ADHs: TB, Thermoanaerobacter brockii; CB, Clostridium beijerinckii; PF, Pseudomonas fluorescens; GS, Gordonia sp. str. TY-5. T, total lysate; S, soluble lysate.
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Figure S4. Purification of recombinant ACX complex from E. coli DH1(DE3). ACX was expressed in 1 L of both LB and MM-0.8%glc using 500 µM IPTG as the inducing agent. A) FPLC traces for each purification using 5 mL DEAE resin. B) FPLC traces showing further separation of pooled fractions using size-exclusion chromatography (SEC). C) SDS-PAGE analyzing purity of pooled fractions from each purification stage. Red bars indicate pooled fractions of purification and analysis. T, total cell lysate; S, soluble lysate; P1, DEAE purification results; P2, SEC purification results. Blue arrows indicate the 3 subunits. D) Densitometric analysis of the purified ACX as determined by ImageJ. Density, intensity measurement of band; size, molecular weight of subunit (Da). MM, minimal medium.
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Figure S5. Kinetic assays surveying activity of the recombinant ACX. Enzymatic data is in triplicate. Reactions monitored the oxidation of NADH at 340 nm. A) Using an acetoacetate standard, a coupled kinetic assay using the NADH-dependent 3-hydroxybutyrate dehydrogenase to monitor acetoacetate presence was verified. B) The activity of purified ACX (from growth in LB) against acetone to produce acetoacetate was monitored using this coupled kinetic assay at 340 nm. C) Monitoring the effect of adding thiamine analogues (1 mM) to the reactions of ACX against acetone. SA, specific activity; -, no additional supplement; T, thiamine; TMP, thiamine monophosphate; TPP, thiamine pyrophosphate.
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Figure S6. Plasmid system for growth of E. coli on IPA.
Figure S7. HPLC analysis of linearity in IPA and acetone detection. Concentrations used include 500 mM, 100 mM, 10 mM, and 2.5 mM. Black squares, IPA; red points, acetone; lines are best fit.
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Figure S8. Growth of plasmid-bearing E. coli BW25113(DE3) on IPA. A) BW25113(DE3) carrying pBbE7c-XAacxnT-dT7-v3 and either an alcohol dehydrogenase (JBEI-14126, JBEI14121) or red fluorescent protein (JBEI-14125). Growth in supplemented minimal media in the presence or absence of isopropanol. B) HPLC analysis of day 6 isopropanol concentrations in extracellular medium. RFP, red fluorescent protein; PF, PFadh; CB, CBadh; IPA, 0.75% isopropanol; glc, 0.2% glucose.
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Figure S9. Increasing IPTG induction concentrations leads to lower IPA-dependent growth. A) Growth of E. coli DH1(DE3) carrying the pBbE7c-XAacxnT-dT7-v3 + CBadh plasmid system (JBEI-14127) with increasing inducer concentrations of IPTG. B) HPLC analysis of day 4 isopropanol concentrations in extracellular medium. C) HPLC analysis of day 4 acetone concentrations in extracellular medium. IPA, 0.75% isopropanol; glc, 0.2% glucose. D) Daily analysis of IPA consumption using no cell, IPA-only media as control. Transparent bars show IPA concentrations used to determine fraction of IPA remaining.
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Figure S10. Expanding the IPA degradation system to include overexpression of the carbonic anhydrase. A) Growth curves of E. coli DH1(DE3) carrying the pBbE7c-XAacxnTdT7-v3 + alcohol dehydrogenase plasmids with (JBEI-14128, JBEI-14129) and without (JBEI14133, JBEI-14127) the carbonic anhydrase. B) HPLC analysis of day 4 isopropanol concentrations in extracellular medium. PF, PFadh; CB, CBadh; CA, carbonic anhydrase; IPA, 0.75% isopropanol; glc, 0.2% glucose.
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Figure S11. Supplementation with thiamine or extra metals show no growth benefit. A) Growth of E. coli DH1(DE3) carrying the pBbE7c-ACXnT-dT7-v3 + pBbS5a-CB-CA plasmids (JBEI-14129). Induction of system at either 10 or 20 µM IPTG and supplementing with extra thiamine (T, 0.05 µg/mL). B) Growth of E. coli DH1(DE3) carrying the pBbE7c-ACXnT-dT7-v3 + pBbS5a-CB-CA-RN-EC plasmids (JBEI-14130) induced with 20 µM IPTG and supplementing with extra metals (concentrations below). CB, CBadh; CA, carbonic anhydrase; IPA, 0.75% isopropanol; glc, 0.2% glucose; MM, minimal medium; MMS, supplemented minimal medium; T, thiamine.
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Figure S12. SDS-PAGE analysis of ACX protein solubility as induced with increasing concentrations of IPTG inducer. T, total lysate; S, soluble lysate. Blue arrows denote the 3 subunits of ACX.
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Figure S13. Purification of MMS-grown ACX. ACX purification from 1 L of MMS-0.8%glc using 10 µM IPTG induction. A) FPLC trace for the purification with DEAE resin. B) FPLC trace showing further separation using size-exclusion chromatography. C) SDS-PAGE analyzing purity of each purification stage. Red bars indicate pooled fractions for next purification step. T, total cell lysate. S, soluble lysate. P1, DEAE purification results. P2, SEC purification results. Blue arrows indicate the 3 subunits. D) Kinetic activity analysis and comparison of all purified ACX protein on acetone. SA, specific activity. E) Kinetic analysis of MMS ACX activity against acetone with supplemental thiamine analogs. -, no supplement; T, thiamine; TMP, thiamine monophosphate; TPP, thiamine pyrophosphate; SEC, size-exclusion chromatography.
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Table S3. Oligonucleotides used for plasmid construction.
Name
Sequence
TBadh-F1 TBadh-R1 TBadh-F2 TBadh-R2 CBadh-F1 CBadh-R1 CBadh-F2 CBadh-R2 PFadh-F1 PFadh-R1 PFadh-F2 PFadh-R2 AAaas-R3 GSadh2-F1 GSadh2-R1 GSadh2-F2 GSadh2-R2 XAacxA-F1 XAacxA-R1 XAacxA-F2 XAacxA-R2 XAacxA-F3 XAacxA-R3 XAacxB-F1 XAacxB-R1 XAacxB-F2 XAacxB-R2 XAacxB-F3 XAacxB-R3 XAacxB-F4 XAacxB-R4 XAacxC-F1 XAacxC-R1 AAacxA-F1 AAacxA-R1 AAacxA-F2 AAacxA-R2 AAacxA-F3 AAacxA-R3 AAacxB-F1 AAacxB-R1 AAacxB-F2 AAacxB-R2 AAacxB-F3 AAacxB-R3 AAacxB-F4 AAacxB-R4
ctagaaataattttgtttaactttaagaaggagatataCCatgaagggattcgctatg cagtagcgccataatactttgccgcatcaacacataccgg ccggtatgtgttgatgcggcaaagtattatg ttaagccaggatgaccactggtttaatcagg ctagaaataattttgtttaactttaagaaggagatataCCatgaaaggctttgcgatg cacgcggtccacacctttgccgttagtaagtttcattacttgg agtaatgaaacttactaacggcaaaggtgtggac USE AAaas-R3 ctagaaataattttgtttaactttaagaaggagatataCCatggctacgatgaaagc catcaacaccgataatggtgcttgcaccctttaaac gcacgtttaaagggtgcaagcaccattatcggtgttgatgctgtcgc USE AAaas-R3 cctttcgggctttgttagcagccggatcctcgagCATATGtta ctagaaataattttgtttaactttaagaaggagatataCCatgaaggcgctggtgtttc ccagaatctcttcacgccaaccagatgatgtcgagacaac gttgtctcgacatcatctggttggcgtgaa ttatgccgacatgatcacctttaaggctttgg gataacaatttcacacgagctcggtaccaggagatataCCatgaacgttccggtc gagtctggcgagaaggttccgctgcataaccttccagaat attctggaaggttatgcagcggaaccttct accaccgtaactgaagcagacgaaggatg ctactctccagcatccttcgtctgcttcagttacggtggtgccggaccagtacata tttggtcaacggtcacattcatggtatatctcctTCTAGAttaaacctcacggagatgaaagaggcgatgcc gaaggagatataccatgaatgtgaccgttgaccaaagtaccttagcag accccgcgatgcgggtacgctcgtctaacatccaatagcg cgctattggatgttagacgagcgtaccc acattcaaagctattaactgcgtgaatctcgtcgtattggttgaagc ccaatacgacgagattcacgcagttaatagctttg ttcacctgcgaagcgttccaagacataaccaccgttgata tatcaacggtggttatgtcttggaacgctt cgggtatatgccatggtatatctcctttactcttcaacaaattgcacg aagagtaaaggagatataccatggcatatacccgttcgaa atccgccaaaacagccaagcttgcatgcctgcaggtcgactta gataacaatttcacacgagctcggtaccaggagatataCCatggaagccctgcaagc cgatcgctcaccttcttcagggtctggcgagaaggttctg cagaaccttctcgccagaccctgaagaag gaagcccaccccttcggtatagccgtaagtatgcaccggt accggtgcatacttacggctataccgaag tcagcatattcatggtatatctccttctagattatttcacttctttcaggtgaaacaggc agtgaaataaTCTAGAaggagatataccatgaatatgctgaccaataaagaagttg tgccttcccaaaaaatcggcacgatggtagcgatgtcaca tgtgacatcgctaccatcgtgccgatttttt tggtgagtgcatgatgctgtccagtttggcgaatgcattg caatgcattcgccaaactggacagcatca caatcactattcatataaccattgcccatgaaaaacatggtcca ccatgtttttcatgggcaatggttatatgaatagtgattggggtatg cgtatacgttgacatggtatatctcctttattcttctacctggaccaccgtg
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AAacxC-F1 AAacxC-R1 XAacx_F XAacx_R pBbEXC_F pBbEXC_R E1C_A-Bridging E1C_B-Bridging E5C E7C E8C_A-Bridging E8C_B XAacxAn-F6 XAacxAn-R4 XAacxBn-F5 XAacxBn-R5 XAacxCn-F2 XAacxCn-R4 XAacxAn-R5 XAacxBn-F6 XAacxBn-R6 XAacxCn-F3 XAacxAn-R6 XAacxAn-R7 XAacxAn-R8 XAacxBn-R6 XAacxBn-F7 XAacxAn-F8 XAacxAn-R9 XAacxBn-F8 XAacxBn-R7 XAacxCn-F4 XAacxCn-R5 PFadh-F5 PFadh-F6 PFadh-R5 CBadh-F3 CBadh-R3 CA-F1 CA-R4 CA-F4 CA-F5 pTet-F1 ECaat-F3 ECaat-R8 RNaas-R6 RNaas-F7
ggtagaagaataaaggagatataccatgtcaacgtatacgaacgaacaggtggc atccgccaaaacagccaagcttgcatgcctgca /5phos/atgaacgttccggtcgga /5phos/ttacgcatccgcacgttc /5phos/ggatccaaactcgagtaagga /5phos/atgtatatctccttcttaaaagatcttttga caatttcagaattcaaaagatcttttaagaaggagatatacatatgaacgttccggtcggacac ggaacgtgcggatgcgtaaggatccaaactcgagtaaggatctcca use E1C A&B use E1C A&B gggaattcaaaagatcttttaagaaggagatatacatatgaacgttccggtcggacac use E1C_B aattcaaaagatcttttaagaaggagatatacatatgaacgttcccgtgggacacctgc agtcacgttcatggtatatctccttctagatcacacctcgcgcaggtggaacag gtgtgatctagaaggagatataccatgaacgtgactgttgaccagagcaccc tcgagcgggtataggccatggtatatctccttcattcctccacgaactgcacggtgtg gaggaatgaaggagatataccatggcctatacccgctcgaagatcgtcg ttgatgcctggagatccttactcgagtttggatcctcaggcgtcggcccgctcgg aacagtcacgttcatattgattcctcctttgagttcgttgactcggattgagcgtgc aacgaactcaaaggaggaatcaatatgaacgtgactgttgaccagagcaccc taggccatggtatatctcctattgatttttgttttccaaggaaaaaatgggcggcagcg tggaaaacaaaaatcaataggagatataccatggcctatacccgctcgaagatcgtcg cgctcacaattcccctatagtgagtcgtattattgagttcgttgactcggattgagcgtg tactaagatggggaattgttatccgctcacaattcccctatagtgagtcgtattattgag ctcctcttatacttaactaatatactaagatggggaattgttatccgctcacaattcccc taggccatggtatatctcctattgatttttgttttccaaggaaaaaatgggcggcagcg tatattagttaagtataagaggaggaatcaatatgaacgtgactgttgaccagagcaccc tgaagatcttataagaaggaggtattcatatgaacgttcccgtgggacacctgc gggtgctctggtcaacagtcacgttcat atgaacgtgactgttgaccagagcaccc gcctggagatccttactcgagtttggatcctcattcctccacgaactgcacggtgtg caaaagatcttttaagaaggagatatacatatggcctatacccgctcgaagatcgtcg catatgaatacctccttcttataagatcttcaggcgtcggcccgctcgg taacaatttcatttaagaaggagatatacatatggctacgatgaaagcagccat acaatttcagaattcaaaagatcttttaagaaggagatatacatatggctacg tgcctggagatccttactcgagtttggatccttacggagtaatcgccactttcatcaca gaattcaaaagatcttttaagaaggagatatacatatgaaaggctttgcgatgctgg ctggagatccttactcgagtttggatccttataaaatcaccactgctttaatcaggtctt aaattaagaaggagatataccatgaaagacatagatacactcatca agatccttactcgagtttggatccttatttgtggttggcgtgtttc aagtggcgattactccgtaaggatccaaattaagaaggagatataccatg aagcagtggtgattttataaggatccaaattaagaaggagatataccatg cagtgatagagaaaagaattcaaaagatcttttaagaaggagatatacat caagacttttaaagatctaggagataatccatgaaaaattgtgtcatcgtcagtgcggta cctggagatccttactcgagttaattcaaccgttcaatcaccatc gattatctcctagatctttaaaagtcttgtaattccggaatatcgcgatagagatccagg aataaggatccaaactcgagaattattataagaaggaggtataccatgtccaaactcgca
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Table S4. G-blocks used for gene assembly.
Name
Sequence
TBadh -G1
aagaaggagatataccATGAAGGGATTCGCTATGCTGAGTATTGGCAAGGTGGGTTGGATCGA GAAAGAAAAACCCGCGCCCGGTCCGTTTGATGCCATCGTACGTCCACTCGCCGTTGCCC CGTGCACCTCGGACATTCATACAGTTTTCGAAGGCGCGATCGGTGAACGCCATAACATG ATTCTGGGGCATGAAGCGGTAGGAGAGGTGGTTGAAGTCGGCTCGGAAGTGAAAGATT TTAAACCGGGTGATCGTGTTGTGGTCCCAGCTATTACCCCAGACTGGCGTACCTCTGAA GTGCAACGCGGTTACCATCAGCATTCGGGTGGTATGTTAGCCGGCTGGAAATTTTCGAA CGTGAAGGATGGCGTGTTTGGGGAATTTTTCCACGTCAATGACGCCGACATGAACCTGG CACACCTGCCGAAAGAAATTCCATTAGAGGCAGCGGTGATGATTCCTGATATGATGACC ACCGGCTTCCATGGCGCGGAGCTCGCGGATATTGAGCTGGGTGCGACTGTTGCAGTTC TTGGTATCGGCCCGGTAGGTCTGATGGCGGTGGCCGGCGCTAAGCTGCGCGGTGCGG GCCGTATTATTGCCGTGGGTTCACGCCCGGTATGTGTTGATGCGGCAAAGTATT CCGGTATGTGTTGATGCGGCAAAGTATTATGGCGCTACTGATATCGTTAACTACAAAGAC GGTCCGATTGAAAGCCAGATTATGAATCTCACGGAGGGCAAAGGAGTCGACGCAGCCAT TATCGCTGGCGGTAATGCTGATATTATGGCAACAGCGGTCAAGATTGTGAAACCTGGCG GAACCATTGCCAATGTGAACTATTTTGGCGAGGGCGAAGTGTTGCCAGTCCCCCGCCTG GAATGGGGCTGCGGCATGGCACACAAGACCATTAAAGGTGGTCTGTGCCCTGGCGGCC GTCTGCGTATGGAGCGTTTAATTGACCTGGTATTTTATAAACGCGTCGATCCGTCCAAAC TGGTCACCCACGTCTTTCGTGGCTTTGATAACATCGAAAAAGCATTTATGCTGATGAAAG ATAAACCGAAAGACCTGATTAAACCAGTGGTCATCCTGGCTcatatgctcgaggatccggctgctaac aaa
TBadh -G2
CBadh -G1
CBadh -G2
PFadh -G1
aagaaggagatataccATGAAAGGCTTTGCGATGCTGGGTATTAATAAGCTGGGCTGGATCGA AAAAGAACGTCCCGTTGCTGGTTCGTATGATGCGATTGTTCGCCCCCTGGCGGTCAGCC CGTGTACCAGCGATATCCATACCGTCTTTGAAGGTGCCCTGGGCGATCGTAAAAACATG ATTTTAGGCCATGAAGCGGTGGGCGAAGTCGTGGAAGTTGGGAGCGAAGTTAAAGACTT CAAACCAGGCGATCGCGTGATTGTCCCATGCACGACCCCCGACTGGCGTAGTCTGGAG GTGCAAGCGGGGTTTCAACAACACAGTAACGGCATGCTGGCCGGTTGGAAATTTAGCAA CTTTAAAGACGGCGTGTTCGGTGAATACTTTCATGTGAACGATGCTGACATGAACCTGGC AATCTTGCCGAAGGACATGCCTTTGGAAAATGCAGTAATGATTACGGATATGATGACGAC GGGTTTTCATGGCGCTGAACTGGCGGATATTCAGATGGGCTCAAGCGTTGTTGTTATCG GAATTGGTGCGGTAGGTCTGATGGGTATTGCTGGTGCCAAATTGCGCGGCGCCGGCCG TATCATTGGTGTAGGATCTCGTCCGATTTGCGTCGAGGCTGCAAAATTCTACGGGGCAA CTGATATTCTCAACTATAAAAACGGGCACATTGTCGACCAAGTAATGAAACTTACTAACG GCAAAGGTGTG AGTAATGAAACTTACTAACGGCAAAGGTGTGGACCGCGTGATCATGGCCGGGGGCGGA AGTGAGACTTTGTCGCAAGCTGTATCCATGGTTAAGCCGGGCGGTATCATTAGTAATATT AACTACCACGGGAGCGGCGATGCCCTGTTAATTCCGCGTGTGGAGTGGGGTTGTGGCA TGGCGCATAAGACTATTAAGGGTGGTCTGTGTCCGGGTGGCCGCTTGCGCGCCGAAAT GCTGCGTGATATGGTGGTATATAACCGTGTTGACTTAAGTAAGTTAGTAACCCATGTGTA TCACGGTTTTGATCATATCGAGGAAGCACTTCTGCTGATGAAAGACAAACCAAAAGACCT GATTAAAGCAGTGGTGATTTTATAAcatatgctcgaggatccggctgctaacaaa aagaaggagatataccATGGCTACGATGAAAGCAGCCATTTTCGTCGAAAAAAATCGTATCGTG CTGGATGACAAACCGATTCCGGAAGTTGGTCCGTTAGACGCTTTGATTCGCATTACCACT ACGACCATTTGCGGTACCGACGTTCATATTCTGCGCGGTGAGTATCCGGTTGCGAAAGG TCTTACAGTGGGTCACGAGCCAGTGGGTATTATCGAACGTCTGGGTAGCCAAGTTCGCG GGTTTGTTGAAGGACAGCGCGTGATTGCAGGCGCTATCACCCCGTCTGGTCAATCTTAT GCCTGCTTATGCGGCTGTGGAAGTCAGGATGGACCAGACACACGTCATGGCTTTCGCG CGATCGGTGGCTGGAAGTTCGGCAACACGATCGATGGTTGTCAGGCAGAATATGTTCTT GTTCCGGATGCCCTGGCAAATCTGTGCCCAATTCCAGATGGCCTGAGCGATGAACAAGT GTTAATGTGTCCAGATATCATGTCTACTGGTTTTTCAGGTGCAGAACGTGGTGAAGTGAG TATTGGTGATAGCGTGGCTGTGTTTGCTTTAGGTCCCATTGGATTGTGCGCAGTTGCGG GTGCACGTTTAAAGGGTGCAAGCACCATTATCGGTGTTGATGCTGTCGC
19
PFadh -G2
AGCACCATTATCGGTGTTGATGCTGTCGCGGAACGTATGACCGTGGCCCGCCGTCTGG GTGCGACACATGTCGTGAACTTTCGTGAGGGTGATGTGGTCGAACAAATCATGGCCTTG ACCGATGGTCGTGGCGTCGATGTGGCGATTGAAGCGCTGGGCACGCAGGGCACTTTCG AAAGTGCACTGCGTGTGCTGCGTCCGGGAGGCCGTTTATCTTCACTGGGTGTCTATTCA TCGGATCTCCGTATCCCGTTAGATGCGTTCGCAGCAGGTTTAGGTGACTACAGCATTGT GACCACCCTTTGCCCGGGTGGAAAAGAGCGTATGCGTCGCCTGATGGCAGTCGTACAG AGTGGAATGGTGGACCTGTCTCCCCTGGTTACTCATCGCTTCAAATTGGACGATATCGAA GCAGCGTACGATCTGTTCGCACACCAACGCGACGGTGTGATGAAAGTGGCGATTACTCC GTAAcatatgctcgaggatccggctgctaacaaa
GSadh -G1
aagaaggagatataccATGAAGGCGCTGGTGTTTCATGGGCCTGGTCAGAAAGCCTGGGAAGA TGTTCCGGACCCGAAGCTGTCCGAACCGACTGACGTAATTGTCAAAATGGATACGACCA CTATTTGTGGTACGGACCTGCACATTCTGAAAGGCGATGTTCCAGCGGTTACCCCAGGT CGCATCCTGGGTCATGAAGGTGTCGGTACTATCACGGAAGTGGGAGATGCAGTTACTAC CCTGGCAGTTGGCGACCAGGTGATCCTGTCTTGTATTTCAAGTTGCGGAAAATGCTCCTT CTGCAAGCAGGGTGTCTACTCGCATTGCCTCGGTAGCGAGGGTACGTCAGGTATTGGTT GGATCTTCGGTCACTTAATCGACGGTACGCAAGCGGAGTACGTACGCGTTCCGTACGCT GAAAACAGTGTACATAAACTGCCGAGTGCCGTCTCACCGGAGCAGGGCACCCTGCTGA GCGATATCCTGCCAACAGGTCATGAAATTGGTGTGCGTCACGGTGCAGTGAAACCTGGT GATGTGGTTGCGGTGATTGGCACTGGTCCCGTTGGCTTAGCTGCCATCGCAACGGCTG GACTGTATGGACCGTCTCGCGTCATTGCTGTCGACATTGACGCCAATCGCGTGGAACAA GCACGTCGCTTTGGTGCTACTGACGGGGTTGTCTCGACATCATCTGGTTGGCGTGAA GTTGTCTCGACATCATCTGGTTGGCGTGAAGAGATTCTGGCGATGACCGATGGCTTAGG CGTGGACGTCGCCATTGAAGCAGTGGGCATCCCAGACACTTTCCAAATGTGCCTGGACA TTGTTCGTCCAGCAGGACACGTGGCTAATGTCGGTGTGCACGGCAAACCTGTCGAATTA CCGATCCAGGATCTGTGGATTAGTAATATCGTGATGTCTATGGGCCTGGTGAACACCAAT ACACTGGGTACACTGCTGAAACTTGTGGCACAGCGTCGCATTGATCCGGAGCCATTTAT TAGCCATCGCTTTGGACTGGGTGAAATTATCGATGCCTATGACGTCTTTAGCCGCGCTG CCGAGACCAAAGCCTTAAAGGTGATCATGTCGGCAcatatgctcgaggatccggctgctaacaaa
GSadh -G2
AAaas -G1
AAaas -G2
gaaggagatataccATGAACTCGTACATGCCACAGGGATATGAATGGCGTACTACCCGTCTTG CACAGTTTATCGCCCGTCACGGCTATCGCGATCTGGATGATCTGCGTGAACGTGCAGCA CGCGAACCTGAACGCTTCTGGGACCAGGTCTTAGAGGCTATTGGCCTTGACTGGGCAAC TCCTTACCGTCAGACCCTGGACTTGACTGATGGTGTTATGTGGCCAAAATGGTTCGTTG GTGGACGTCTGGATTTAGTGGATAACTTGGTTGGTAAACATGCACGTGCCACCCCTGCG AAAATTGCGGTGCGTTGGGAGGGTGATGGAGGCGAACTGCGTACCTTAAGTTACGCAG CGCTCGCTGGTGAGGTCGAACGTGTTGCAGCAGGCCTTCGTGCGCTGGGAGTATCTGA AGGCGAGCGCATTGCGTTATATCTGCCGATGGTTGCCGAAGCAGTGGTGACAATGTTGG CAGCCACCCGTATCGGTGCGGTATTCATTCCATTTTTTTCAGGCTATGGTGCAGATTCTG TTGCGCAGCGTGTTGCGGATTGCGAAGCACGTGTGCTGGTTTGCGCGAATGGTTATTTC CGTCGTGGTAAACGCGTCCCCATGCTGGCAGACGCACGTGCAGCGGCTCGTGCCTGCC CATCTTTACGTCATCTGGTGGTGGTCGATC CATCTTTACGTCATCTGGTGGTGGTCGATCGTCTTGGTCTGGGTCAGCTGGCAGCCAAC GATGGTGCCGCAGAGAGCTTTGCCGAGGTGGATTTTCGTGCCTTGCAGGCAGCGTCTC CTGATCGTGGACCAGCAAGCGCTTGGCCAGCAGATCAAACTTTAATGCTTATTTATACCA GCGGTACCACCGGTAAACCGAAAGGGGTGGTGCACACCCACGCAGGATTTCCGGTGAA GGCAGCACAAGACTTACTCATGGCATTCGACCTGCGTGCGGACGACACGCTCATGTGG GTTACCGATATGGGTTGGCTGATGGGTCCATGGATGGTGTATGGTGGTCTTATGCTGGG TGCCACACTGGTGCTGTATGAAGGGACCCCGGATTATCCGGATGCAGGTCGTTTGTGGC AAGTGGTTGAACGCCACGGTGTGACCCACTTTGGTCTGTCTCCGACACTGGTGCGTCTC CTGATGGCGAACGACGCCTCACTGCCAGCTCCGGGTGCGCTGGATACTTTACGCGTATT TGGCAGCACAGGCGAAGCATGGAACGAAGCACCGTGGTTGTGGTTATTTGAACAAGTTG GGCAGAGTCGTCGTCCCATCATTAACTACTCTGGTGGCACAGAAATTGGTGGTGGCATC CTGGCGTGTTTTCCCGGTCTGCCACAAAAGCCTTGTGCGTTTGACGGTCCAAT
20
AAaas -G3
CAAAAGCCTTGTGCGTTTGACGGTCCAATTCCGGGCATGGCCGTCGAGGTCCTGGATTC GGATGGTCGTCCTGTGCGCGGTAGCGTAGGGGAACTGGTCATTCGCCAGCCGTGGCCA GGTATGACCCATGGCTTCTGGCGTGACACCGAACGCTACCGTGAAGCATATTGGAGCGT CTGGCCAGATGTGTGGGTCCATGGAGATTGGGCACGTGTGGACGCAGATGGCTATTGG TTCGTGCATGGTCGCAGCGATGATACCATTAAAATTGCAGGGAAGCGCGTTGGTCCGGC AGACTTTGAAAGTGCTTTGGTCTCACATCCATTGGTGGTAGAGGCTGTTGCGGTGGGTG TACCAGATGAGTTAAAAGGCGAAACGGCAGTATGCTTTGTGACCGTTGCCGACAACGAA GCATTGGCAGCACGTCCGTGGCAAGCCTGGGAAGCTGAACTCGTGGATCACGTTGGTC GCCAGTTAGGTAAACCTTTACGTCCAGCTCATGTGCATCGCGTTGAAGCCATCCCTAAAA CCCGTAATGGTAAAACGGTACGTCGCGTAATGCGTAATGCCTATTTGGGGACCGCGCTG GGTGACCTTAGTTCGATTGAAAACCCAGCAGCTATCGATGCTGTGAGCGCTCTGCGTCC GTCTGCACAGGCAGTTCCCGACGCGATTGGCACTcatatgctcgaggatccggctgctaacaaa
RNaas -G1
gaaggagatataccATGTCCAAACTCGCACGCTTGGAGCGTGAAGAGATTATGGAATGTCAAG TGATGTGGGAACCGGACTCTAAAAAAGACACCCAGATGGATCGTTTTCGTGCAGCAGTA GGCACAGCCTGTGGGTTGGCCCTGGGAAACTATGACGATTTATACCATTGGAGCGTGCG CTCATATAGCGATTTTTGGGCAGAATTCTGGAAATTCTCGGGTATTGTGTGCTCACGCAT GTATGATGAGGTCGTGGATACCTCCAAGGGGATTGCTGACGTTCCTGAGTGGTTTCGTG GGTCGCGTTTAAACTACGCCGAAAATCTGCTGCGCCACAAAGAAAACGATCGCGTTGCC CTCTACGTGGCACGTGAGGGTCGCGAAGAGATTGCCAAAGTGACCTTCGAGGAACTGC GTCAACAGGTTGCCCTCTTCGCAGCAGCTATGCGCAAAATGGGTGTCAAGAAAGGTGAT CGCGTGGTCGGATACTTACCCAACTCTGCCCATGCGGTCGAAGCGATGCTGGCAGCAG CAAGCATCGGTGCGATTTGGAGCTCAACCAGTCCGGATTTCGGCGTTAATGGCGTATTA GACCGCTTCTCGCAGATTCAGCCGAAACTGATTTTCTCTGTTGAAGCAGTGGTCTACAAC GGCAAAGAACATGGACACCTGGAGAAACTGCAGCGTGTGGTCAAAGGCCTCCCAGATC TGCAACGCGTCGTATTGATC TCCCAGATCTGCAACGCGTCGTATTGATCCCGTACGTGTTGCCACGCGAAAAGATCGAC ATCTCGAAGATTCCGAATAGCATGTTTCTGGATGATTTTCTGGCTTCTGGCACGGGTGCC CAAGCACCTCAGCTGGAATTTGAACAACTGCCCTTCAGTCATCCGCTCTTCATCATGTTT TCTTCTGGTACCACCGGTGCACCGAAATGCATGGTGCACTCTGCAGGCGGAACACTGAT CCAGCACCTGAAAGAGCACGTCCTGCATGGTAACATGACCAGCAGCGATATCTTACTTT ACTATACCACTGTGGGCTGGATGATGTGGAACTGGATGGTCAGTGCTTTAGCAACCGGT GCCAGTTTAGTCTTATACGATGGGTCGCCTCTGGTGCCGACTCCGAACGTCCTCTGGGA TTTAGTCGATCGCATTGGTATCACCATCTTAGGGACCGGTGCGAAATGGCTGAGTGTTCT GGAAGAGAAGGACATGAAACCGATGGAAACCCACAACCTGCACACACTCCATACAATCC TCTCAACTGGCTCTCCGTTAAAAGCCCAAAGCTATGAATATGTTTATCGCTGTATTAAATC TACCGTGCTCCTGGGTTCTATTTCGGGTGGTACAGATATTATCTCATGCTTTATGGGTCA AAACAGTAGCATTCCAGTCTACAAAGGAGAGATCCAAGCACGTAATC TCTACAAAGGAGAGATCCAAGCACGTAATCTGGGTATGGCAGTGGAAGCCTGGGATGAA GAAGGCAAGACCGTATGGGGTGCATCTGGCGAACTCGTTTGCACCAAACCTATCCCGTG TCAGCCGACTCATTTTTGGAACGATGAGAATGGGTCTAAATATCGTAAGGCGTACTTTAG CAAGTATCCAGGCGTTTGGGCACATGGTGATTACTGTCGCATCAACCCGAAAACGGGTG GTATTGTGATGTTGGGTCGTTCGGACGGGACCTTAAACCCCAACGGTGTTCGTTTTGGC TCCAGCGAAATCTATAACATCGTGGAAGCTTTCGACGAAGTCGAGGATTCATTGTGCGT GCCACAATATAATCGTGATGGTGAAGAACGCGTTGTGCTGTTTCTCAAAATGGCGTCCG GTCATACCTTTCAGCCGGATTTGGTGAAACATATCCGCGATGCTATTCGTCTGGGTCTGT CCGCTCGCCACGTTCCATCCTTAATTTTGGAGACACAGGGGATTCCGTATACGATCAATG GCAAAAAAGTGGAGGTTGCAGTGAAGCAGGTAATCGCAGGCAAAACTGTTGAACATCGT GGTGCTTTTTCCAACCCTGAGAGCCTGGATCTCTATCGCGATATTCCGGAATTACAAGAC TTTcatatgctcgaggatccggctgctaacaaa
RNaas -G2
RNaas -G3
21
XAacx A-G1
XAacx A-G2
XAacx A-G3
XAacx B-G1
taccaggagatataccATGAACGTTCCGGTCGGACACCTGCGCAATGTCCAAGTTCTCGGAATC GATGCAGGTGGTACGATGACCGACACGTTTTTTGTCGACCAAGATGGCGATTTTGTAGTT GGGAAAGCTCAGTCCACTCCACAGAATGAAGCGCTTGGTCTCATTGCATCCTCCGAAGA CGGTCTGGCCAACTGGGGAATGAGCCTGCACGAAGCGTTAGCCCAATTGCAGACCGGT GTTTACTCTGGCACTGCGATGCTGAATCGCGTAGTTCAGCGCAAAGGTTTAAAATGCGG TCTGATTGTGAACCGTGGTATGGAAGACTTTCATCGCATGGGTCGTGCGGTTCAGTCTC ACTTAGGCTATGCTTATGAAGATCGCATCCATCTTAATACCCACCGCTACGACCCGCCGT TAGTTCCGCGTCATTTGACGCGTGGTGTGGTCGAGCGCACCGACATGATTGGTACCCAA GTAATTCCACTTCGTGAAGACACCGCACGCGACGCAGCACGTGATCTCATTGCTGCTGA TGCGGAAGGCATCGTAATCAGCTTGCTCCACTCGTATAAAAACCCTGAAAATGAACGCC GTGTTCGCGATATTGTTCTGGAAGAGGTGGAAAAAAGTGGGAAAAAGATTCCGGTTTTC GCCTCTGCGGACTATTATCCTGTACGTAAAGAAACCCATCGTACCAACACGACCATTCTG GAAGGTTATGCAGCGGAACCTTCT ATTCTGGAAGGTTATGCAGCGGAACCTTCTCGCCAGACTCTGTCAAAAATTAGTAACGCG TTTAAAGAACGTGGCACAAAATTCGACTTCCGTGTCATGGCAACTCATGGAGGCACTATC TCATGGAAAGCGAAAGAACTGGCACGCACCATCGTATCGGGTCCGATTGGTGGAGTCAT CGGTGCAAAATACCTGGGTGAAGTTTTGGGTTACAAGAACATTGCGTGTAGCGATATTG GTGGCACCAGCTTCGATGTCGCGTTGATTACACAAGGAGAAATGACCATTAAAAACGAC CCAGATATGGCTCGTTTGGTACTTTCTTTACCGCTCGTGGCAATGGACAGCGTCGGTGC AGGTGCGGGAAGTTTCATTCGCTTGGACCCGTATACCCGTGCGATCAAACTGGGTCCCG ACTCGGCTGGTTACCGTGTGGGTGTATGTTGGAAGGAGTCTGGCATTGAAACGGTTACC ATTTCAGACTGTCATATGGTGCTCGGATATCTCAATCCGGATAACTTCCTGGGTGGTGCG GTGAAACTGGATCGCCAGCGTTCTGTTGATGCTATTAAAGCCCAGATTGCAGATCCGCT GGGCTTATCCGTGGAAGATGCGGCAGCAGGTGTAATTGAATTACTCGATTCGGATTTAC GTGATTACTTACGCTCCATGATTAGTGGCAAAGGCTACTCTCCAGCATCCTTCGTCTGCT TCAGTTACGGTGGTGCCGGACCAGTACATA TCAGTTACGGTGGTGCCGGACCAGTACATACCTATGGCTACACAGAAGGTCTGGGCTTT GAAGACGTTATTGTGCCTGCGTGGGCTGCTGGCTTCAGTGCGTTCGGGTGCGCAGCTG CAGACTTCGAATATCGTTATGATAAAAGCCTGGATATTAATATGCCGACCGAAACCCCTG ACACCGATAAAGAAAAAGCTGCAGCAACGTTACAGGCAGCGTGGGAAGAGCTGACCAA GAATGTCCTGGAAGAATTTAAATTGAATGGCTACTCCGCAGACCAGGTTACCCTGCAACC TGGTTATCGCATGCAGTATCGTGGTCAGCTGAATGATCTGGAAATTGAAAGCCCACTGG CCCAAGCCCACACCGCAGCCGACTGGGACCAGCTTACCGATGCGTTCAATGCTACTTAT GGACGTGTGTACGCAGCAAGTGCGCGTTCTCCAGAACTCGGATACAGCGTCACAGGTG CGATTATGCGTGGCATGGTGCCTATCCCGAAACCGAAAATCCCAAAAGAACCGGAAGAA GGCGAAACACCTCCGGAGAGCGCTAAAATTGGTACCCGTAAATTTTACCGTAAAAAACG CTGGGTGGATGCCCAGTTGTACCATATGGAATCTTTACGTCCAGGCAACCGTGTTATGG GTCCTGCTGTTATTGAAAGCGACGCGACGACATTTGTCGTACCGGACGGCTTTGAAACT TGGTTGGATGGGCATCGCCTCTTTCATCTCCGTGAGGTTTAA tctagaaggagatataccatgATGAATGTGACCGTTGACCAAAGTACCTTAGCAGGCGCTACTCGT GGGATTGTTCGTGGTGGTGAAACCCTGAAAGAGCATCGTGACCGTCTGATGGCAGCAAC CAAGGCGACCGGACGCTATGCAGGGTTAAAAACCCTTGAACTTCGTGAACGCGAACCCA TCCTGTACAATAAGCTGTTTAGCCGTCTCCGTGCAGGTGTTGTTGACGCTCGCGAAACG GCTAAGAAGATTGCAGCGTCTCCGATTGTGGAACAGGAAGGGGAACTGTGTTTCACGCT TTATAACGCTGCTGGAGATTCCTTACTGACCTCCACGGGAATCATCATCCACGTGGGGA CGATGGGTGCAGCGATCAAGTACATGATCGAGAACAACTGGGAGGCTAATCCGGGTGT CCATGATAAAGATATCTTCTGTAACAATGATAGCCTTATCGGCAATGTCCACCCGTGCGA TATTCACACAATCGTACCGATTTTTTGGGAAGGTGAGTTGATCGGCTGGGTAGGTGGTG TGACCCATGTAATTGATACTGGTGCAGTTGGACCAGGTTCAATGGCTACCGGTCAGGTC CAACGCTTCGGTGACGGCTATTCCATTACCTGTCGTAAGGTAGGTGCGAATGATACGCT GTTCCGTGATTGGCTGCACGAAAGCCAGCGCATGGTACGCACCACCCGCTATTGGATGT TAGACGAGCGTACCC
22
XAacx B-G2
XAacx B-G3
XAacx B-G4
CGCTATTGGATGTTAGACGAGCGTACCCGCATCGCGGGGTGTCACATGATCCGCAAACT GGTGGAAGAAGTGGTTGCGGAAGAGGGTATTGAGGCGTACTGGAAATTCGCGTATGAA GCGGTGGAACATGGTCGTTTAGGGCTGCAAGCGCGTATTAAAGCAATGACCATCCCTGG CACATACCGTCAAGTTGGTTTCGTAGATGTTCCTTACGCTCATGAGGATGTTCGCGTGCC GTCGGACTTTGCGAAACTGGATACGATTATGCATGCACCGTGCGAGATGACAATCCGCC GTGATGGCACCTGGCGTCTCGACTTCGAAGGGTCCTCGCGTTGGGGTTGGCACACCTA CAATGCCCATCAGGTATCCTTTACGTCTGGTATTTGGGTTATGATGACGCAGACTCTGAT TCCGAGCGAAATGATTAACGACGGTGCAGCCTATGGCACCGAATTTCGTCTGCCAAAAG GCACTTGGATGAATCCGGATGATCGTCGTGTGGCTTTTTCCTATTCCTGGCATTTCCTGG TGAGTGCGTGGACCGCTCTGTGGCGTGGTCTTTCACGCTCCTATTTTGGTCGTGGGTAC TTAGAGGAGGTTAATGCAGGGAACGCGAACACTAGCAATTGGCTGCAAGGTGGTGGCTT CAACCAATACGACGAGATTCACGCAGTTAATAG CCAATACGACGAGATTCACGCAGTTAATAGCTTTGAATGTGCTGCGAACGGCACTGGTG CGACTGCCGTTCAGGATGGTTTGAGCCACGCTGCTGCTATTTGGAATCCGGAAGGAGAC ATGGGTGATATGGAGATCTGGGAACTTGCCGAACCGCTGGTTTATCTGGGTCGTCAGAT TAAAGCAAGTTCTGGTGGAAGCGGTAAATATCGTGGAGGCTGTGGTTTTGAGTCACTCC GCATGGTGTGGAACGCGAAGGATTGGACCATGTTTTTTATGGGGAACGGTCATATTTCAT CGGATTGGGGTCTGATGGGTGGCTACCCTGCAGCTTCTGGTTATCGCTTTGCTGCCCAC AAGACTAATCTCAAGGAATTGATTGCGAGCGGTGCTGAAATCCCGTTAGGTGGTGATAC CGATCCGGAAAACCCGACCTGGGACGCCATGCTGCCAGATGCGCAGATCAAGCGTGAT AAGCAAGCCATTACGACCGAGGAGATGTTCAGTGATTACGATCTCTATCTGAACTATATG CGTGGTGGTCCAGGGTTTGGCGATCCGCTGGATCGTGAACCGCAAGCGGTTGCGGATG ATATCAACGGTGGTTATGTCTTGGAACGCTT TATCAACGGTGGTTATGTCTTGGAACGCTTCGCAGGTGAAGTCTATGGTGTTGTTGTGC GTAAAGGTGCTGACGGTCAGTACGGTGTCGATGAAGCAGGTACTGCAGCAGCACGTGC GCAAATTCGCAAAGATCGTTTAGCCAAAAGCGTTCCGGTTAGCGAATGGATGAAAGGTG AGCGTGAAAAAATTCTCGCCAAGGATGCAGGGACCCAAGTTCGCCAGATGTTCGCTGCA AGCTTCAAGCTCGGTCCTCGTTTTGAAAAAGATTTTCGTACCTTTTGGAGCTTACCGGAT AGCTGGACTCTGCCTGAAGAAGAAATCGGTGTGCCCACGTACGGTTCGCGCTATAGTAT GGACATTTCCGAGTTACCGGACGTGCACACCGTGCAATTTGTTGAAGAGTAAaggagatata ccatggcg
XAacx C-G1
ataaaggagatataccATGGCATATACCCGTTCGAAAATTGTTGACCTGGTTGACGGTAAAATC GATCCGGATACCTTACATCAGATGTTATCTACACCGAAAGATCCGGAACGCTTTGTGACG TATGTGGAAATTCTGCAAGAACGCATGCCGTGGGATGATAAAATCATTCTTCCCCTGGGT CCGAAGCTGTTTATCGTTCAGCAGAAAGTTAGCAAAAAATGGACAGTACGCTGTGAATGT GGCCATGATTTTTGCGATTGGAAAGACAATTGGAAACTCTCTGCACGCGTACACGTGCG TGACACCCCACAAAAGATGGAGGAAATTTATCCGCGTCTGATGGCTCCAACTCCGAGCT GGCAGGTAATTCGTGAATACTTTTGTCCCGAGTGTGGCACGCTCCATGACGTCGAAGCA CCGACTCCGTGGTACCCAGTAATCCACGATTTTAGCCCTGACATTGAGGGCTTTTATCAG GAATGGCTTGGTCTGCCAGTTCCGGAACGTGCGGATGCGTAAgtcgacctgcaggcatgcaagctt
AAacx A-G1
accaggagatataccATGGAAGCCCTGCAAGCGTCCCAAGTGCAGGTCATGGGCATTGATGC TGGAGGCACGATGACTGACACTTTTTTCGTCCGTGAAGATGGTCGTTTCGTTGTAGGCA AAGCGCAGAGCAACCCAGCGGATGAGTCTTTGGCGATCTTTAATAGCAGCCAGGATGCG CTGGCACATTGGGAACGTGATGTGGATGAAGTTTATCCGGAACTGGTTACCTGTGTGTA TTCCGGTACTGCCATGTTAAACCGTATCCTGATGCGCAAGGGTCTGGATGTGGGCTTAA TCTGTAACCGTGGATTCGAACAAATCCATAGCATGGGTCGTGCACTGCAGAGCTACTTA GGTTATGCGCTGGAAGATCGCATTCATTTGAATACACATCGTTATGACGAGCCTCTGGTT CCGGTTTCTCGTACGCGTGGTGTGGCTGAACGTACGGATGTACAGGGTAAAGTCGTGAT TCCGCTTCGCGAAGACGAAGTCCGTCAGGCCACCCGTGAGCTTGTAGAAGCAGGTTCT CAAGCAGTGGTGATTTGCCTGTTACAGAGTCATAAAAACGAAACCTCAGAACAACGTGCA CGCGATGTGGTCAAAGATGAATTGAAAAAACTGAAGGTAGACATTCCCGTTTTTGCTTCT GTGGACTATTACCCGAGTCGTAAAGAATCGCACCGTATGAACACGACAATTTTGGAAGC GTACGGAGCAGAACCTTCTCGCCAGACCCTGAAGAAG
23
AAacx A-G2
AAacx A-G3
AAacx B-G1
AAacx B-G2
AAacx B-G3
CAGAACCTTCTCGCCAGACCCTGAAGAAGGTGAGCGATCGCTTCAAGAAACATGGAGCC AAGTTTGATCTCCGTGTAATGGCTACGCACGGTGGTACGATCAGCTGGAAAGCCAAAGA GCTTGCACGTACCATCGTGAGCGGTCCGATCGGTGGCGTGATCGGTAGTAAATTACTTG GCGAATATCTGGGTGATGAGAACATTGCGTGCAGCGATATTGGAGGTACCAGCTTCGAT GTTGCTCTGATCACCAAAGGGTCGTTCGCGATCAAAAGCGATCCCGATATGGCACGCCT GGTACTGAGTCTGCCGTTGGTAGCAATGGACTCTGTTGGCGCTGGTGCAGGTTCGTTTG TGCGTCTGGATCCGTATAGTAAAAGCATTAAACTGGGACCGGATAGCGCAGGATATCGC GTGGGCACGTGTTGGCCAGAATCTGGGCTGGACACCGTATCCGTGAGTGACTGCCACG TGGTTCTGGGATACCTGAATCCGGATAATTTTCTGGGTGGAGCGATCAAATTAGATGTAC AGCGTGCACGCGACCATATTAAAGTCCAGATTGCCGATCCTCTGGGTCTTAGCGTCGAG GATGCTGCGAGCGGTGTTATTGAACTGCTGGATCTGACGCTCTCGGAGTATCTCCGTGC AAATATTTCAGCGAAAGGCTATAATCCAGCGGAATTTACCTGCTTCTCGTATGGTGGTGC AGGACCGGTGCATACTTACGGCTATACCGAAG ACCGGTGCATACTTACGGCTATACCGAAGGGGTGGGCTTCAAAGATGTCGTGGTGCCTG CTTGGGCAGCGGGTTTCTCTGCGTTTGGCTGTGCATGTGCGGATTTCGAATATCGCTAC GATAAATCCGTTGACCTGGGTGTCGCACAATTCGCGTCCGACGAAGTGAAAGCAGCAGC GTGCGTCAGCCTGCAAGCAGCCTGGGAAGAATTAGCGGTAAAAGTAATTGACGAGTTCA TTATCAACGGTTACAAACCTGAAGATGTATTGCTTATCCCAGGCTATAAGATGCAGTATAT GGGCCAACTGAACGATCTGGAGATTGTATCTCCGGTGTCAACGGCAGCCACGGCAGGT GACTGGAATCAGATTATCGAGGCGTTCGAGACCACCTATAGTCGCGTTTACGCTAATAG CGCTCGTAGCCCTGAACTGGGCTTCTCTATTACTGGTGCTATCCTTCGTGGTATGGTTGT GACCCAGAAACCAGTTCTGCCTGAAGATGCTGACTGCGGTCCGACCCCACCAAAGGAA GCATATCTGGGAACCCGTCCGTTCTATCGTCATAAACGCTGGGTCGAAGCCACTCTGTG GAAAATGGAATCCCTCAAAGCAGGTAACCACATTGTCGGTCCTGCGATTATCGAATCTGA TGCAACCACCTTTGTGGTTCCGGATGGTTTCGAGACTACGGTAGACAAGCATCGCCTGT TTCACCTGAAAGAAGTGAAATAAtctagaaggagatatac tctagaaggagatataccATGAATATGCTGACCAATAAAGAAGTTGGGTTCGCCGATCTGTTAA AAAATGGGCAGACACTGAAACAATTCCGTGATGGTATTATCGAACGTACCAAAGCCTCTG GCTGCTATAATGGTATCGAAAAGCTGGAATTTCGCGACTCGGATCCGATTGGCTATGAAA AACTGTTTTCGAAACTGCGTGGTGGTTTAGTGCACGCACGTGAGACTGCGAAGAAAATC GCAGCGTCTCCGATTGTGGAGCAGGAAGGGGAACTGTGTTTTACCTTGTACAACGCAGC AGGCGATTGCGTGCTGACCTCGACTGGCATCATTATCCATGTGGGTACCATGGGTGCAG CGATCAAATACATGATTGAGAACAATTGGGAAGGAAACCCTGGCATCAACCCACGTGAC ATGTTCACCACGAACGATTGTGCTATCGGCAATGTTCATCCGTGTGACATCGCTACCATC GTGCCGATTT CGTGTGACATCGCTACCATCGTGCCGATTTTTTGGGAAGGCAAGTTAATTGGATGGGTG GGCGGTGTCACGCATGTGATCGACACGGGTGCAGTTACACCAGGCAGCATGTCGACGG GTCAGACCCAACGTTTTGGCGATGGCTATATGATTACGTGTCGTAAAACGGGTGTTAAC GATGAACCGCTGCGTGACTGGCTGCATGAATCCCAGCGTAGCGTTCGTACTCCGAAATA TTGGATCCTGGACGAGAAAACCCGTATTGCAGGATGCCATATGATTCGTGAATTAGTGG AAGAAGTTATCCGTGCGGATGGCCTTGATGCTTATGAGAAGTTTGGCTTTGAGGTTATTG AGGAAGGTCGTCGTGGATTGATGTCTCGTGTTAAAGCTATGACGCTTCCAGGAAAATATC GCAAAGTTAGCTTTGTTGACGTTCCCTTTAAGCATGAGGACGTACAAGTGAGCAATGCAT TCGCCAAACTGGACAGCATCA CAATGCATTCGCCAAACTGGACAGCATCATGCACTCACCAGTTGAGATGACCATCAAAC CCGATGGTAAGTGGCGTTTAGACTTTGAAGGTGCAAGCCGCTGGGGATGGCACACCTTC AACGCTCACCAGGTGGCCTTCACGAGCGGTATTTGGGTCATGATGTCCCAGACGCTGGT TCCGACTCAACGTATCAACGATGGAGCCTACTTTGCCACGGAATTTAAACTGCCAAAGG GTACATGGTGTAATCCTGACGATCGTCGCACTGGGCATGCGTACGCATGGCACTTTTTA GTGTCAGGTTGGGCTGCCCTGTGGCGTGGCCTTAGTCAATCTTACTTCTCTCGTGGCTA TCTGGAGGAAGTGAACGCAGGTAATGCTAACACGTCGAATTGGCTTCAAGGTGGTGGCA TCAATCAGGATGGTGAGATTCATGCCGTAAACTCTTTTGAGGCGTCCTCGTGTGGAACG GGAGCCTGTGCGGTGAAAGATGGCCTTAATCACGCAGCAGCGATTTGGAATCCGGAAG GCGATATGGGTGACATCGAAATCTGGGAGATGGCTGAACCGCTCTTATATCTGGGTCGC AACGTTAAAGCGAACTCTGGAGGCTACGGGAAATATCGTGGTGGCTGTGGCTTTGAAAC CCTGCGCATGGTCTGGAATGCACAAGACTGGACCATGTTTTTCATGGGCAATGGTTATAT GAATAGTGATTG
24
AAacx B-G4
CATGGGCAATGGTTATATGAATAGTGATTGGGGTATGATGGGTGGCTACCCTTCAGCGA CGGGTTATCGTTTTGAAGCCCATAAAACGGGTCTGGAACGTCGTATTGCCAATGGGGAG AGCCTGCCGTTAGGTGGCGATATTGACCCAGCGGCCCCTACCTACGAACGTAATATCGA TGCGACTAGCGTGGTCAAACGTGATAAACAGTGCATGACCACTGAAGACTGCTACGAAA ATCACGACCTGTATCTGAATTATTTGCGTGGAGGTCCAGGTTTTGGCGATCCGATTGATC GTGAACCGAAGGCCATCGAAAACGATTTGAACCAGAAATTCTTATTACCTGAATACGCCG AGAAAGTATATGGTGCGGTTATTACCCAAGATGCGAAAGGTGTATTTACCGTGGACGCA GCAAAAACCGCAGCACGTCGTGCGGAAGTCCGCAAAGAACGTCTGGCTCGTGCTGTCC CAACTCACGAATGGATGAAACAGGAGCGTGAACGCATCTTGATCAAAGATGCAGCTGTC CAAGTACAACACATGTTCGCGACGTCGTTTGGCCTGTCTGAAAAATTCACCAAAGAATTT AAAGCGTTTTGGTCACTTCCGGATGATTGGCAGCTGCTGGAAAGCGAGCTTAATGTCCC GACCTACGGAAGCAAACATCGTATGGATCTCTCGCTGTTACCGGATGTGACCACGGTGG TCCAGGTAGAAGAAtaaaggagat
AAacx C-G1
tataccATGTCAACGTATACGAACGAACAGGTGGCTCATCTGGTTGAAGGTTCACTTGATT GGGAAACGACTTTTCGCATGCTGTCAATGCCGAAAGACAATTCTCGCTTCGAACAGTATC TGAAAGCCCTGCAAGCGAAAGTGAGCTTTCCAGACAAAATTGTGCTCCCGTTGGGTCCG CACTTGTACATTGTCCAGTCGGCAAAAACCAAACAATGGGTCACCATGTGTGACTGTGG GCACGTGTTTGGGGATTATCGCGAAAACTGGAAACTGAACGCAGCAATCTATGTTCGCG ATACCGAGGAGGCAATGACCGAAGTTTATCCGAAGCTGATGGCTCCGGATACCCAATGG CAGGTTTATCGCGAATATTATTGCCCAACCTGCGGTACTATGCATGACGTGGAAGCACC GACCCCTTGGTATCCGGTTATTCATGATTTTGAACCGGACATCGAAGCGTTTTACCAGGA ATGGGTAGGACTGCCTGTTCCCGAACGTGCAGCATAAgtcgacctgcaggcatgcaagcttggctg ttttg
Literature Cited.
(1) Lee, T. S., Krupa, R. A., Zhang, F., Hajimorad, M., Holtz, W. J., Prasad, N., Lee, S. K., and Keasling, J. D. (2011) BglBrick vectors and datasheets: A synthetic biology platform for gene expression. J. Biol. Eng. 5, 1-14.
25
Engineering bacteria to catabolize the carbonaceous component of sarin: teaching E. coli to eat isopropanol Margaret E. Brown, Aindrila Mukhopadhyay, and Jay D. Keasling
Supplementary Results Abbreviations
S2
Figure S1. Lysate reactivity of E. coli expressing ACX complex
S3
Figure S2. Promoter and sequence modification for ACX protein complex expression
S4
Table S1. Plasmids used in this study
S5
Table S2. Plasmid depictions for ACX operon construction
S6
Figure S3. SDS-PAGE analysis of alcohol dehydrogenase expression and solubility
S7
Figure S4. Purification of recombinant ACX complex from E. coli DH1(DE3)
S8
Figure S5. Kinetic assays surveying activity of the recombinant ACX
S9
Figure S6. Plasmid system for growth of E. coli on IPA
S10
Figure S7. HPLC analysis of linearity in IPA and acetone detection
S10
Figure S8. Growth of plasmid-bearing E. coli BW25113(DE3) on IPA
S11
Figure S9. Increasing IPTG induction concentrations leads to lower IPA-dependent growth
S12
Figure S10. Expanding the IPA degradation system to include overexpression of the carbonic anhydrase
S13
Figure S11. Supplementation with thiamine or extra metals show no growth benefit
S14
Figure S12. Solubility of ACX protein as induced with increasing concentrations of IPTG inducer
S15
Figure S13. Purification of MMS-grown ACX
S16
Table S3. Oligonucleotides used for plasmid construction
S17
Table S4. G-blocks used for gene assembly
S19
Literature cited
S25
Abbreviations ADH, alcohol dehydrogenase ACX, acetone carboxylase AACS, acetoacetyl-CoA synthetase AACT, acetoacetyl-CoA thiolase PTE, phosphotriesterase PDE, phosphodiesterase PHN, phosphonate degradation cluster CA, carbonic anhydrase XAacx, acetone carboxylase from Xanthobacter autotrophicus PY2 AAacx, acetone carboxylase from Aromatoleum aromaticum PY2 PFadh (PF), alcohol dehydrogenase from Pseudomonas fluorescens CBadh (CB), alcohol dehydrogenase from Clostridium beijerinckii GSadh2 (GS), alcohol dehydrogenase from Gordonia sp. str. TY-5 TBadh (TB), alcohol dehydrogenase from Thermoanaerobacter brockii RNaacs (RN), acetoacetyl-CoA synthetase from Rattus norvegicus ECaact (EC), acetoacetyl-CoA thiolase from E. coli IPA, isopropanol IPTG, isopropyl β-D-1-thiogalactopyranoside HPLC, high-performance liquid chromatography TPP, thiamine pyrophosphate TMP, thiamine monophosphate T, thiamine MM, minimal media MMS, minimal media with supplements LB, lysogeny broth RFP, red fluorescent protein Glc, glucose aTet, anhydrotetracycline
2
Figure S1. Lysate reactivity of E. coli expressing ACX complex. HPLC traces of ACX reactivity with acetone shows the production of acetoacetate only in the presence of the ACX and acetone. +ACX, ACX-expressing lysate; -ACX, control lysate.
3
Figure S2. Promoter and sequence modification for ACX protein complex expression. A) Codon-modified acx sequences transcribed from different promoters, comparing the original expression off of the Trc-encoded pTrc33 and the BglBrick plasmid promoters (Trc, lacUV5, T7, ara).1 Modification of promoter shows little alteration in expression levels. B) Use of the native acx sequence in expression plasmids shows an increase in protein content when expressed from the T7 promoter of the pBbE7c vector. Lane 1, ACX; 2, ACXn; 3, ACXnT; 4, ACXnT-rbs; 5, ACXnT-dT7; 6, ACXnT-dT7-ssC; 7, ACXnT-dT7-v3; 8, RFP). Final modification (ACXnT-dT7v3) placing the native C subunit in front of the native A subunit for expression yields expression levels of all three subunits similar to that found in acetone-grown Xanthobacter. Blue arrows indicate the 3 subunits. C) Densitometric analysis of the 3 subunits of ACXnT-dT7-v3 as determined by ImageJ. Density, intensity measurement of band; size, molecular weight of subunit (Da).
4
Table S1. Plasmids used in this study. Strain a
1 a 2 a 3 a 4 a 5 a 6 a 7 a 8 a 9 a 10 a 11 a 12 a 13 a 14 a 15 a 16 a 17 a 18 a 19 a 20 21 22 23 24 25 26 27 28 29 30 31 32 Plasmid pTrc33-* pET16b-* pBbE1c-* pBbE5c-* pBbE7c-* pBbE8c-* pBbS5a-* pBbS2a-*
E. coli host DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) DH1(DE3) BW25113(DE3) BW25113(DE3) BW25113(DE3)
Plasmid 1 pTrc33-empty** pTrc33-XAacx pTrc33-AAacx pET16b-RFP pET16b-PFadh pET16b-CBadh pET16b-GSadh2 pET16b-TBadh pET16b-ECaact pET16b-RNaacs pBbE1c-XAacx pBbE5c-XAacx pBbE7c-XAacx pBbE8c-XAacx pBbE7c-XAacxn pBbE7c-XAacxnT pBbE7c-XAacxnT-rbs pBbE7c-XAacxnT-dT7 pBbE7c-XAacxnT-dT7-ssC pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3 pBbE7c-XAacxnT-dT7-v3
Plasmid 2 pBbS5a-RFP pBbS5a-PF pBbS5a-CB pBbS5a-PF-CA pBbS5a-CB-CA pBbS5a-CB-CA-RN-EC pBbS2a-RFP pBbS2a-CB-CA pBbS2a-CB-CA-RN-EC pBbS5a-RFP pBbS5a-PF pBbS5a-CB
Promoter Trc T7 Trc lacUV5 T7 arabinose lacUV5 Tet
Origin of Replication p15a ColE1 ColE1 ColE1 ColE1 ColE1 SC101 SC101
Antibiotic resistance CmR CbR CmR CmR CmR CmR CbR CbR
a
* = multiple constructs, ** = no insert, = permanent storage in DH10B-T1
Strain number JBEI-14106 JBEI-14107 JBEI-14108 JBEI-14102 JBEI-14103 JBEI-14104 JBEI-14105 JBEI-14101 JBEI-14100 JBEI-14098 JBEI-14096 JBEI-14097 JBEI-14113 JBEI-14112 JBEI-14118 JBEI-14206 JBEI-14205 JBEI-14117 JBEI-14116 JBEI-14132 JBEI-14133 JBEI-14127 JBEI-14128 JBEI-14129 JBEI-14130 JBEI-14131 JBEI-14123 JBEI-14124 JBEI-14125 JBEI-14126 JBEI-14121
R
5
Table S2. Plasmid depictions for ACX operon construction.
6
Figure S3. SDS-PAGE analysis of alcohol dehydrogenase expression and solubility. Expression of the four candidate alcohol dehydrogenases in E. coli DH1(DE3) using 500 µM IPTG. For ADHs: TB, Thermoanaerobacter brockii; CB, Clostridium beijerinckii; PF, Pseudomonas fluorescens; GS, Gordonia sp. str. TY-5. T, total lysate; S, soluble lysate.
7
Figure S4. Purification of recombinant ACX complex from E. coli DH1(DE3). ACX was expressed in 1 L of both LB and MM-0.8%glc using 500 µM IPTG as the inducing agent. A) FPLC traces for each purification using 5 mL DEAE resin. B) FPLC traces showing further separation of pooled fractions using size-exclusion chromatography (SEC). C) SDS-PAGE analyzing purity of pooled fractions from each purification stage. Red bars indicate pooled fractions of purification and analysis. T, total cell lysate; S, soluble lysate; P1, DEAE purification results; P2, SEC purification results. Blue arrows indicate the 3 subunits. D) Densitometric analysis of the purified ACX as determined by ImageJ. Density, intensity measurement of band; size, molecular weight of subunit (Da). MM, minimal medium.
8
Figure S5. Kinetic assays surveying activity of the recombinant ACX. Enzymatic data is in triplicate. Reactions monitored the oxidation of NADH at 340 nm. A) Using an acetoacetate standard, a coupled kinetic assay using the NADH-dependent 3-hydroxybutyrate dehydrogenase to monitor acetoacetate presence was verified. B) The activity of purified ACX (from growth in LB) against acetone to produce acetoacetate was monitored using this coupled kinetic assay at 340 nm. C) Monitoring the effect of adding thiamine analogues (1 mM) to the reactions of ACX against acetone. SA, specific activity; -, no additional supplement; T, thiamine; TMP, thiamine monophosphate; TPP, thiamine pyrophosphate.
9
Figure S6. Plasmid system for growth of E. coli on IPA.
Figure S7. HPLC analysis of linearity in IPA and acetone detection. Concentrations used include 500 mM, 100 mM, 10 mM, and 2.5 mM. Black squares, IPA; red points, acetone; lines are best fit.
10
Figure S8. Growth of plasmid-bearing E. coli BW25113(DE3) on IPA. A) BW25113(DE3) carrying pBbE7c-XAacxnT-dT7-v3 and either an alcohol dehydrogenase (JBEI-14126, JBEI14121) or red fluorescent protein (JBEI-14125). Growth in supplemented minimal media in the presence or absence of isopropanol. B) HPLC analysis of day 6 isopropanol concentrations in extracellular medium. RFP, red fluorescent protein; PF, PFadh; CB, CBadh; IPA, 0.75% isopropanol; glc, 0.2% glucose.
11
Figure S9. Increasing IPTG induction concentrations leads to lower IPA-dependent growth. A) Growth of E. coli DH1(DE3) carrying the pBbE7c-XAacxnT-dT7-v3 + CBadh plasmid system (JBEI-14127) with increasing inducer concentrations of IPTG. B) HPLC analysis of day 4 isopropanol concentrations in extracellular medium. C) HPLC analysis of day 4 acetone concentrations in extracellular medium. IPA, 0.75% isopropanol; glc, 0.2% glucose. D) Daily analysis of IPA consumption using no cell, IPA-only media as control. Transparent bars show IPA concentrations used to determine fraction of IPA remaining.
12
Figure S10. Expanding the IPA degradation system to include overexpression of the carbonic anhydrase. A) Growth curves of E. coli DH1(DE3) carrying the pBbE7c-XAacxnTdT7-v3 + alcohol dehydrogenase plasmids with (JBEI-14128, JBEI-14129) and without (JBEI14133, JBEI-14127) the carbonic anhydrase. B) HPLC analysis of day 4 isopropanol concentrations in extracellular medium. PF, PFadh; CB, CBadh; CA, carbonic anhydrase; IPA, 0.75% isopropanol; glc, 0.2% glucose.
13
Figure S11. Supplementation with thiamine or extra metals show no growth benefit. A) Growth of E. coli DH1(DE3) carrying the pBbE7c-ACXnT-dT7-v3 + pBbS5a-CB-CA plasmids (JBEI-14129). Induction of system at either 10 or 20 µM IPTG and supplementing with extra thiamine (T, 0.05 µg/mL). B) Growth of E. coli DH1(DE3) carrying the pBbE7c-ACXnT-dT7-v3 + pBbS5a-CB-CA-RN-EC plasmids (JBEI-14130) induced with 20 µM IPTG and supplementing with extra metals (concentrations below). CB, CBadh; CA, carbonic anhydrase; IPA, 0.75% isopropanol; glc, 0.2% glucose; MM, minimal medium; MMS, supplemented minimal medium; T, thiamine.
14
Figure S12. SDS-PAGE analysis of ACX protein solubility as induced with increasing concentrations of IPTG inducer. T, total lysate; S, soluble lysate. Blue arrows denote the 3 subunits of ACX.
15
Figure S13. Purification of MMS-grown ACX. ACX purification from 1 L of MMS-0.8%glc using 10 µM IPTG induction. A) FPLC trace for the purification with DEAE resin. B) FPLC trace showing further separation using size-exclusion chromatography. C) SDS-PAGE analyzing purity of each purification stage. Red bars indicate pooled fractions for next purification step. T, total cell lysate. S, soluble lysate. P1, DEAE purification results. P2, SEC purification results. Blue arrows indicate the 3 subunits. D) Kinetic activity analysis and comparison of all purified ACX protein on acetone. SA, specific activity. E) Kinetic analysis of MMS ACX activity against acetone with supplemental thiamine analogs. -, no supplement; T, thiamine; TMP, thiamine monophosphate; TPP, thiamine pyrophosphate; SEC, size-exclusion chromatography.
16
Table S3. Oligonucleotides used for plasmid construction.
Name
Sequence
TBadh-F1 TBadh-R1 TBadh-F2 TBadh-R2 CBadh-F1 CBadh-R1 CBadh-F2 CBadh-R2 PFadh-F1 PFadh-R1 PFadh-F2 PFadh-R2 AAaas-R3 GSadh2-F1 GSadh2-R1 GSadh2-F2 GSadh2-R2 XAacxA-F1 XAacxA-R1 XAacxA-F2 XAacxA-R2 XAacxA-F3 XAacxA-R3 XAacxB-F1 XAacxB-R1 XAacxB-F2 XAacxB-R2 XAacxB-F3 XAacxB-R3 XAacxB-F4 XAacxB-R4 XAacxC-F1 XAacxC-R1 AAacxA-F1 AAacxA-R1 AAacxA-F2 AAacxA-R2 AAacxA-F3 AAacxA-R3 AAacxB-F1 AAacxB-R1 AAacxB-F2 AAacxB-R2 AAacxB-F3 AAacxB-R3 AAacxB-F4 AAacxB-R4
ctagaaataattttgtttaactttaagaaggagatataCCatgaagggattcgctatg cagtagcgccataatactttgccgcatcaacacataccgg ccggtatgtgttgatgcggcaaagtattatg ttaagccaggatgaccactggtttaatcagg ctagaaataattttgtttaactttaagaaggagatataCCatgaaaggctttgcgatg cacgcggtccacacctttgccgttagtaagtttcattacttgg agtaatgaaacttactaacggcaaaggtgtggac USE AAaas-R3 ctagaaataattttgtttaactttaagaaggagatataCCatggctacgatgaaagc catcaacaccgataatggtgcttgcaccctttaaac gcacgtttaaagggtgcaagcaccattatcggtgttgatgctgtcgc USE AAaas-R3 cctttcgggctttgttagcagccggatcctcgagCATATGtta ctagaaataattttgtttaactttaagaaggagatataCCatgaaggcgctggtgtttc ccagaatctcttcacgccaaccagatgatgtcgagacaac gttgtctcgacatcatctggttggcgtgaa ttatgccgacatgatcacctttaaggctttgg gataacaatttcacacgagctcggtaccaggagatataCCatgaacgttccggtc gagtctggcgagaaggttccgctgcataaccttccagaat attctggaaggttatgcagcggaaccttct accaccgtaactgaagcagacgaaggatg ctactctccagcatccttcgtctgcttcagttacggtggtgccggaccagtacata tttggtcaacggtcacattcatggtatatctcctTCTAGAttaaacctcacggagatgaaagaggcgatgcc gaaggagatataccatgaatgtgaccgttgaccaaagtaccttagcag accccgcgatgcgggtacgctcgtctaacatccaatagcg cgctattggatgttagacgagcgtaccc acattcaaagctattaactgcgtgaatctcgtcgtattggttgaagc ccaatacgacgagattcacgcagttaatagctttg ttcacctgcgaagcgttccaagacataaccaccgttgata tatcaacggtggttatgtcttggaacgctt cgggtatatgccatggtatatctcctttactcttcaacaaattgcacg aagagtaaaggagatataccatggcatatacccgttcgaa atccgccaaaacagccaagcttgcatgcctgcaggtcgactta gataacaatttcacacgagctcggtaccaggagatataCCatggaagccctgcaagc cgatcgctcaccttcttcagggtctggcgagaaggttctg cagaaccttctcgccagaccctgaagaag gaagcccaccccttcggtatagccgtaagtatgcaccggt accggtgcatacttacggctataccgaag tcagcatattcatggtatatctccttctagattatttcacttctttcaggtgaaacaggc agtgaaataaTCTAGAaggagatataccatgaatatgctgaccaataaagaagttg tgccttcccaaaaaatcggcacgatggtagcgatgtcaca tgtgacatcgctaccatcgtgccgatttttt tggtgagtgcatgatgctgtccagtttggcgaatgcattg caatgcattcgccaaactggacagcatca caatcactattcatataaccattgcccatgaaaaacatggtcca ccatgtttttcatgggcaatggttatatgaatagtgattggggtatg cgtatacgttgacatggtatatctcctttattcttctacctggaccaccgtg
17
AAacxC-F1 AAacxC-R1 XAacx_F XAacx_R pBbEXC_F pBbEXC_R E1C_A-Bridging E1C_B-Bridging E5C E7C E8C_A-Bridging E8C_B XAacxAn-F6 XAacxAn-R4 XAacxBn-F5 XAacxBn-R5 XAacxCn-F2 XAacxCn-R4 XAacxAn-R5 XAacxBn-F6 XAacxBn-R6 XAacxCn-F3 XAacxAn-R6 XAacxAn-R7 XAacxAn-R8 XAacxBn-R6 XAacxBn-F7 XAacxAn-F8 XAacxAn-R9 XAacxBn-F8 XAacxBn-R7 XAacxCn-F4 XAacxCn-R5 PFadh-F5 PFadh-F6 PFadh-R5 CBadh-F3 CBadh-R3 CA-F1 CA-R4 CA-F4 CA-F5 pTet-F1 ECaat-F3 ECaat-R8 RNaas-R6 RNaas-F7
ggtagaagaataaaggagatataccatgtcaacgtatacgaacgaacaggtggc atccgccaaaacagccaagcttgcatgcctgca /5phos/atgaacgttccggtcgga /5phos/ttacgcatccgcacgttc /5phos/ggatccaaactcgagtaagga /5phos/atgtatatctccttcttaaaagatcttttga caatttcagaattcaaaagatcttttaagaaggagatatacatatgaacgttccggtcggacac ggaacgtgcggatgcgtaaggatccaaactcgagtaaggatctcca use E1C A&B use E1C A&B gggaattcaaaagatcttttaagaaggagatatacatatgaacgttccggtcggacac use E1C_B aattcaaaagatcttttaagaaggagatatacatatgaacgttcccgtgggacacctgc agtcacgttcatggtatatctccttctagatcacacctcgcgcaggtggaacag gtgtgatctagaaggagatataccatgaacgtgactgttgaccagagcaccc tcgagcgggtataggccatggtatatctccttcattcctccacgaactgcacggtgtg gaggaatgaaggagatataccatggcctatacccgctcgaagatcgtcg ttgatgcctggagatccttactcgagtttggatcctcaggcgtcggcccgctcgg aacagtcacgttcatattgattcctcctttgagttcgttgactcggattgagcgtgc aacgaactcaaaggaggaatcaatatgaacgtgactgttgaccagagcaccc taggccatggtatatctcctattgatttttgttttccaaggaaaaaatgggcggcagcg tggaaaacaaaaatcaataggagatataccatggcctatacccgctcgaagatcgtcg cgctcacaattcccctatagtgagtcgtattattgagttcgttgactcggattgagcgtg tactaagatggggaattgttatccgctcacaattcccctatagtgagtcgtattattgag ctcctcttatacttaactaatatactaagatggggaattgttatccgctcacaattcccc taggccatggtatatctcctattgatttttgttttccaaggaaaaaatgggcggcagcg tatattagttaagtataagaggaggaatcaatatgaacgtgactgttgaccagagcaccc tgaagatcttataagaaggaggtattcatatgaacgttcccgtgggacacctgc gggtgctctggtcaacagtcacgttcat atgaacgtgactgttgaccagagcaccc gcctggagatccttactcgagtttggatcctcattcctccacgaactgcacggtgtg caaaagatcttttaagaaggagatatacatatggcctatacccgctcgaagatcgtcg catatgaatacctccttcttataagatcttcaggcgtcggcccgctcgg taacaatttcatttaagaaggagatatacatatggctacgatgaaagcagccat acaatttcagaattcaaaagatcttttaagaaggagatatacatatggctacg tgcctggagatccttactcgagtttggatccttacggagtaatcgccactttcatcaca gaattcaaaagatcttttaagaaggagatatacatatgaaaggctttgcgatgctgg ctggagatccttactcgagtttggatccttataaaatcaccactgctttaatcaggtctt aaattaagaaggagatataccatgaaagacatagatacactcatca agatccttactcgagtttggatccttatttgtggttggcgtgtttc aagtggcgattactccgtaaggatccaaattaagaaggagatataccatg aagcagtggtgattttataaggatccaaattaagaaggagatataccatg cagtgatagagaaaagaattcaaaagatcttttaagaaggagatatacat caagacttttaaagatctaggagataatccatgaaaaattgtgtcatcgtcagtgcggta cctggagatccttactcgagttaattcaaccgttcaatcaccatc gattatctcctagatctttaaaagtcttgtaattccggaatatcgcgatagagatccagg aataaggatccaaactcgagaattattataagaaggaggtataccatgtccaaactcgca
18
Table S4. G-blocks used for gene assembly.
Name
Sequence
TBadh -G1
aagaaggagatataccATGAAGGGATTCGCTATGCTGAGTATTGGCAAGGTGGGTTGGATCGA GAAAGAAAAACCCGCGCCCGGTCCGTTTGATGCCATCGTACGTCCACTCGCCGTTGCCC CGTGCACCTCGGACATTCATACAGTTTTCGAAGGCGCGATCGGTGAACGCCATAACATG ATTCTGGGGCATGAAGCGGTAGGAGAGGTGGTTGAAGTCGGCTCGGAAGTGAAAGATT TTAAACCGGGTGATCGTGTTGTGGTCCCAGCTATTACCCCAGACTGGCGTACCTCTGAA GTGCAACGCGGTTACCATCAGCATTCGGGTGGTATGTTAGCCGGCTGGAAATTTTCGAA CGTGAAGGATGGCGTGTTTGGGGAATTTTTCCACGTCAATGACGCCGACATGAACCTGG CACACCTGCCGAAAGAAATTCCATTAGAGGCAGCGGTGATGATTCCTGATATGATGACC ACCGGCTTCCATGGCGCGGAGCTCGCGGATATTGAGCTGGGTGCGACTGTTGCAGTTC TTGGTATCGGCCCGGTAGGTCTGATGGCGGTGGCCGGCGCTAAGCTGCGCGGTGCGG GCCGTATTATTGCCGTGGGTTCACGCCCGGTATGTGTTGATGCGGCAAAGTATT CCGGTATGTGTTGATGCGGCAAAGTATTATGGCGCTACTGATATCGTTAACTACAAAGAC GGTCCGATTGAAAGCCAGATTATGAATCTCACGGAGGGCAAAGGAGTCGACGCAGCCAT TATCGCTGGCGGTAATGCTGATATTATGGCAACAGCGGTCAAGATTGTGAAACCTGGCG GAACCATTGCCAATGTGAACTATTTTGGCGAGGGCGAAGTGTTGCCAGTCCCCCGCCTG GAATGGGGCTGCGGCATGGCACACAAGACCATTAAAGGTGGTCTGTGCCCTGGCGGCC GTCTGCGTATGGAGCGTTTAATTGACCTGGTATTTTATAAACGCGTCGATCCGTCCAAAC TGGTCACCCACGTCTTTCGTGGCTTTGATAACATCGAAAAAGCATTTATGCTGATGAAAG ATAAACCGAAAGACCTGATTAAACCAGTGGTCATCCTGGCTcatatgctcgaggatccggctgctaac aaa
TBadh -G2
CBadh -G1
CBadh -G2
PFadh -G1
aagaaggagatataccATGAAAGGCTTTGCGATGCTGGGTATTAATAAGCTGGGCTGGATCGA AAAAGAACGTCCCGTTGCTGGTTCGTATGATGCGATTGTTCGCCCCCTGGCGGTCAGCC CGTGTACCAGCGATATCCATACCGTCTTTGAAGGTGCCCTGGGCGATCGTAAAAACATG ATTTTAGGCCATGAAGCGGTGGGCGAAGTCGTGGAAGTTGGGAGCGAAGTTAAAGACTT CAAACCAGGCGATCGCGTGATTGTCCCATGCACGACCCCCGACTGGCGTAGTCTGGAG GTGCAAGCGGGGTTTCAACAACACAGTAACGGCATGCTGGCCGGTTGGAAATTTAGCAA CTTTAAAGACGGCGTGTTCGGTGAATACTTTCATGTGAACGATGCTGACATGAACCTGGC AATCTTGCCGAAGGACATGCCTTTGGAAAATGCAGTAATGATTACGGATATGATGACGAC GGGTTTTCATGGCGCTGAACTGGCGGATATTCAGATGGGCTCAAGCGTTGTTGTTATCG GAATTGGTGCGGTAGGTCTGATGGGTATTGCTGGTGCCAAATTGCGCGGCGCCGGCCG TATCATTGGTGTAGGATCTCGTCCGATTTGCGTCGAGGCTGCAAAATTCTACGGGGCAA CTGATATTCTCAACTATAAAAACGGGCACATTGTCGACCAAGTAATGAAACTTACTAACG GCAAAGGTGTG AGTAATGAAACTTACTAACGGCAAAGGTGTGGACCGCGTGATCATGGCCGGGGGCGGA AGTGAGACTTTGTCGCAAGCTGTATCCATGGTTAAGCCGGGCGGTATCATTAGTAATATT AACTACCACGGGAGCGGCGATGCCCTGTTAATTCCGCGTGTGGAGTGGGGTTGTGGCA TGGCGCATAAGACTATTAAGGGTGGTCTGTGTCCGGGTGGCCGCTTGCGCGCCGAAAT GCTGCGTGATATGGTGGTATATAACCGTGTTGACTTAAGTAAGTTAGTAACCCATGTGTA TCACGGTTTTGATCATATCGAGGAAGCACTTCTGCTGATGAAAGACAAACCAAAAGACCT GATTAAAGCAGTGGTGATTTTATAAcatatgctcgaggatccggctgctaacaaa aagaaggagatataccATGGCTACGATGAAAGCAGCCATTTTCGTCGAAAAAAATCGTATCGTG CTGGATGACAAACCGATTCCGGAAGTTGGTCCGTTAGACGCTTTGATTCGCATTACCACT ACGACCATTTGCGGTACCGACGTTCATATTCTGCGCGGTGAGTATCCGGTTGCGAAAGG TCTTACAGTGGGTCACGAGCCAGTGGGTATTATCGAACGTCTGGGTAGCCAAGTTCGCG GGTTTGTTGAAGGACAGCGCGTGATTGCAGGCGCTATCACCCCGTCTGGTCAATCTTAT GCCTGCTTATGCGGCTGTGGAAGTCAGGATGGACCAGACACACGTCATGGCTTTCGCG CGATCGGTGGCTGGAAGTTCGGCAACACGATCGATGGTTGTCAGGCAGAATATGTTCTT GTTCCGGATGCCCTGGCAAATCTGTGCCCAATTCCAGATGGCCTGAGCGATGAACAAGT GTTAATGTGTCCAGATATCATGTCTACTGGTTTTTCAGGTGCAGAACGTGGTGAAGTGAG TATTGGTGATAGCGTGGCTGTGTTTGCTTTAGGTCCCATTGGATTGTGCGCAGTTGCGG GTGCACGTTTAAAGGGTGCAAGCACCATTATCGGTGTTGATGCTGTCGC
19
PFadh -G2
AGCACCATTATCGGTGTTGATGCTGTCGCGGAACGTATGACCGTGGCCCGCCGTCTGG GTGCGACACATGTCGTGAACTTTCGTGAGGGTGATGTGGTCGAACAAATCATGGCCTTG ACCGATGGTCGTGGCGTCGATGTGGCGATTGAAGCGCTGGGCACGCAGGGCACTTTCG AAAGTGCACTGCGTGTGCTGCGTCCGGGAGGCCGTTTATCTTCACTGGGTGTCTATTCA TCGGATCTCCGTATCCCGTTAGATGCGTTCGCAGCAGGTTTAGGTGACTACAGCATTGT GACCACCCTTTGCCCGGGTGGAAAAGAGCGTATGCGTCGCCTGATGGCAGTCGTACAG AGTGGAATGGTGGACCTGTCTCCCCTGGTTACTCATCGCTTCAAATTGGACGATATCGAA GCAGCGTACGATCTGTTCGCACACCAACGCGACGGTGTGATGAAAGTGGCGATTACTCC GTAAcatatgctcgaggatccggctgctaacaaa
GSadh -G1
aagaaggagatataccATGAAGGCGCTGGTGTTTCATGGGCCTGGTCAGAAAGCCTGGGAAGA TGTTCCGGACCCGAAGCTGTCCGAACCGACTGACGTAATTGTCAAAATGGATACGACCA CTATTTGTGGTACGGACCTGCACATTCTGAAAGGCGATGTTCCAGCGGTTACCCCAGGT CGCATCCTGGGTCATGAAGGTGTCGGTACTATCACGGAAGTGGGAGATGCAGTTACTAC CCTGGCAGTTGGCGACCAGGTGATCCTGTCTTGTATTTCAAGTTGCGGAAAATGCTCCTT CTGCAAGCAGGGTGTCTACTCGCATTGCCTCGGTAGCGAGGGTACGTCAGGTATTGGTT GGATCTTCGGTCACTTAATCGACGGTACGCAAGCGGAGTACGTACGCGTTCCGTACGCT GAAAACAGTGTACATAAACTGCCGAGTGCCGTCTCACCGGAGCAGGGCACCCTGCTGA GCGATATCCTGCCAACAGGTCATGAAATTGGTGTGCGTCACGGTGCAGTGAAACCTGGT GATGTGGTTGCGGTGATTGGCACTGGTCCCGTTGGCTTAGCTGCCATCGCAACGGCTG GACTGTATGGACCGTCTCGCGTCATTGCTGTCGACATTGACGCCAATCGCGTGGAACAA GCACGTCGCTTTGGTGCTACTGACGGGGTTGTCTCGACATCATCTGGTTGGCGTGAA GTTGTCTCGACATCATCTGGTTGGCGTGAAGAGATTCTGGCGATGACCGATGGCTTAGG CGTGGACGTCGCCATTGAAGCAGTGGGCATCCCAGACACTTTCCAAATGTGCCTGGACA TTGTTCGTCCAGCAGGACACGTGGCTAATGTCGGTGTGCACGGCAAACCTGTCGAATTA CCGATCCAGGATCTGTGGATTAGTAATATCGTGATGTCTATGGGCCTGGTGAACACCAAT ACACTGGGTACACTGCTGAAACTTGTGGCACAGCGTCGCATTGATCCGGAGCCATTTAT TAGCCATCGCTTTGGACTGGGTGAAATTATCGATGCCTATGACGTCTTTAGCCGCGCTG CCGAGACCAAAGCCTTAAAGGTGATCATGTCGGCAcatatgctcgaggatccggctgctaacaaa
GSadh -G2
AAaas -G1
AAaas -G2
gaaggagatataccATGAACTCGTACATGCCACAGGGATATGAATGGCGTACTACCCGTCTTG CACAGTTTATCGCCCGTCACGGCTATCGCGATCTGGATGATCTGCGTGAACGTGCAGCA CGCGAACCTGAACGCTTCTGGGACCAGGTCTTAGAGGCTATTGGCCTTGACTGGGCAAC TCCTTACCGTCAGACCCTGGACTTGACTGATGGTGTTATGTGGCCAAAATGGTTCGTTG GTGGACGTCTGGATTTAGTGGATAACTTGGTTGGTAAACATGCACGTGCCACCCCTGCG AAAATTGCGGTGCGTTGGGAGGGTGATGGAGGCGAACTGCGTACCTTAAGTTACGCAG CGCTCGCTGGTGAGGTCGAACGTGTTGCAGCAGGCCTTCGTGCGCTGGGAGTATCTGA AGGCGAGCGCATTGCGTTATATCTGCCGATGGTTGCCGAAGCAGTGGTGACAATGTTGG CAGCCACCCGTATCGGTGCGGTATTCATTCCATTTTTTTCAGGCTATGGTGCAGATTCTG TTGCGCAGCGTGTTGCGGATTGCGAAGCACGTGTGCTGGTTTGCGCGAATGGTTATTTC CGTCGTGGTAAACGCGTCCCCATGCTGGCAGACGCACGTGCAGCGGCTCGTGCCTGCC CATCTTTACGTCATCTGGTGGTGGTCGATC CATCTTTACGTCATCTGGTGGTGGTCGATCGTCTTGGTCTGGGTCAGCTGGCAGCCAAC GATGGTGCCGCAGAGAGCTTTGCCGAGGTGGATTTTCGTGCCTTGCAGGCAGCGTCTC CTGATCGTGGACCAGCAAGCGCTTGGCCAGCAGATCAAACTTTAATGCTTATTTATACCA GCGGTACCACCGGTAAACCGAAAGGGGTGGTGCACACCCACGCAGGATTTCCGGTGAA GGCAGCACAAGACTTACTCATGGCATTCGACCTGCGTGCGGACGACACGCTCATGTGG GTTACCGATATGGGTTGGCTGATGGGTCCATGGATGGTGTATGGTGGTCTTATGCTGGG TGCCACACTGGTGCTGTATGAAGGGACCCCGGATTATCCGGATGCAGGTCGTTTGTGGC AAGTGGTTGAACGCCACGGTGTGACCCACTTTGGTCTGTCTCCGACACTGGTGCGTCTC CTGATGGCGAACGACGCCTCACTGCCAGCTCCGGGTGCGCTGGATACTTTACGCGTATT TGGCAGCACAGGCGAAGCATGGAACGAAGCACCGTGGTTGTGGTTATTTGAACAAGTTG GGCAGAGTCGTCGTCCCATCATTAACTACTCTGGTGGCACAGAAATTGGTGGTGGCATC CTGGCGTGTTTTCCCGGTCTGCCACAAAAGCCTTGTGCGTTTGACGGTCCAAT
20
AAaas -G3
CAAAAGCCTTGTGCGTTTGACGGTCCAATTCCGGGCATGGCCGTCGAGGTCCTGGATTC GGATGGTCGTCCTGTGCGCGGTAGCGTAGGGGAACTGGTCATTCGCCAGCCGTGGCCA GGTATGACCCATGGCTTCTGGCGTGACACCGAACGCTACCGTGAAGCATATTGGAGCGT CTGGCCAGATGTGTGGGTCCATGGAGATTGGGCACGTGTGGACGCAGATGGCTATTGG TTCGTGCATGGTCGCAGCGATGATACCATTAAAATTGCAGGGAAGCGCGTTGGTCCGGC AGACTTTGAAAGTGCTTTGGTCTCACATCCATTGGTGGTAGAGGCTGTTGCGGTGGGTG TACCAGATGAGTTAAAAGGCGAAACGGCAGTATGCTTTGTGACCGTTGCCGACAACGAA GCATTGGCAGCACGTCCGTGGCAAGCCTGGGAAGCTGAACTCGTGGATCACGTTGGTC GCCAGTTAGGTAAACCTTTACGTCCAGCTCATGTGCATCGCGTTGAAGCCATCCCTAAAA CCCGTAATGGTAAAACGGTACGTCGCGTAATGCGTAATGCCTATTTGGGGACCGCGCTG GGTGACCTTAGTTCGATTGAAAACCCAGCAGCTATCGATGCTGTGAGCGCTCTGCGTCC GTCTGCACAGGCAGTTCCCGACGCGATTGGCACTcatatgctcgaggatccggctgctaacaaa
RNaas -G1
gaaggagatataccATGTCCAAACTCGCACGCTTGGAGCGTGAAGAGATTATGGAATGTCAAG TGATGTGGGAACCGGACTCTAAAAAAGACACCCAGATGGATCGTTTTCGTGCAGCAGTA GGCACAGCCTGTGGGTTGGCCCTGGGAAACTATGACGATTTATACCATTGGAGCGTGCG CTCATATAGCGATTTTTGGGCAGAATTCTGGAAATTCTCGGGTATTGTGTGCTCACGCAT GTATGATGAGGTCGTGGATACCTCCAAGGGGATTGCTGACGTTCCTGAGTGGTTTCGTG GGTCGCGTTTAAACTACGCCGAAAATCTGCTGCGCCACAAAGAAAACGATCGCGTTGCC CTCTACGTGGCACGTGAGGGTCGCGAAGAGATTGCCAAAGTGACCTTCGAGGAACTGC GTCAACAGGTTGCCCTCTTCGCAGCAGCTATGCGCAAAATGGGTGTCAAGAAAGGTGAT CGCGTGGTCGGATACTTACCCAACTCTGCCCATGCGGTCGAAGCGATGCTGGCAGCAG CAAGCATCGGTGCGATTTGGAGCTCAACCAGTCCGGATTTCGGCGTTAATGGCGTATTA GACCGCTTCTCGCAGATTCAGCCGAAACTGATTTTCTCTGTTGAAGCAGTGGTCTACAAC GGCAAAGAACATGGACACCTGGAGAAACTGCAGCGTGTGGTCAAAGGCCTCCCAGATC TGCAACGCGTCGTATTGATC TCCCAGATCTGCAACGCGTCGTATTGATCCCGTACGTGTTGCCACGCGAAAAGATCGAC ATCTCGAAGATTCCGAATAGCATGTTTCTGGATGATTTTCTGGCTTCTGGCACGGGTGCC CAAGCACCTCAGCTGGAATTTGAACAACTGCCCTTCAGTCATCCGCTCTTCATCATGTTT TCTTCTGGTACCACCGGTGCACCGAAATGCATGGTGCACTCTGCAGGCGGAACACTGAT CCAGCACCTGAAAGAGCACGTCCTGCATGGTAACATGACCAGCAGCGATATCTTACTTT ACTATACCACTGTGGGCTGGATGATGTGGAACTGGATGGTCAGTGCTTTAGCAACCGGT GCCAGTTTAGTCTTATACGATGGGTCGCCTCTGGTGCCGACTCCGAACGTCCTCTGGGA TTTAGTCGATCGCATTGGTATCACCATCTTAGGGACCGGTGCGAAATGGCTGAGTGTTCT GGAAGAGAAGGACATGAAACCGATGGAAACCCACAACCTGCACACACTCCATACAATCC TCTCAACTGGCTCTCCGTTAAAAGCCCAAAGCTATGAATATGTTTATCGCTGTATTAAATC TACCGTGCTCCTGGGTTCTATTTCGGGTGGTACAGATATTATCTCATGCTTTATGGGTCA AAACAGTAGCATTCCAGTCTACAAAGGAGAGATCCAAGCACGTAATC TCTACAAAGGAGAGATCCAAGCACGTAATCTGGGTATGGCAGTGGAAGCCTGGGATGAA GAAGGCAAGACCGTATGGGGTGCATCTGGCGAACTCGTTTGCACCAAACCTATCCCGTG TCAGCCGACTCATTTTTGGAACGATGAGAATGGGTCTAAATATCGTAAGGCGTACTTTAG CAAGTATCCAGGCGTTTGGGCACATGGTGATTACTGTCGCATCAACCCGAAAACGGGTG GTATTGTGATGTTGGGTCGTTCGGACGGGACCTTAAACCCCAACGGTGTTCGTTTTGGC TCCAGCGAAATCTATAACATCGTGGAAGCTTTCGACGAAGTCGAGGATTCATTGTGCGT GCCACAATATAATCGTGATGGTGAAGAACGCGTTGTGCTGTTTCTCAAAATGGCGTCCG GTCATACCTTTCAGCCGGATTTGGTGAAACATATCCGCGATGCTATTCGTCTGGGTCTGT CCGCTCGCCACGTTCCATCCTTAATTTTGGAGACACAGGGGATTCCGTATACGATCAATG GCAAAAAAGTGGAGGTTGCAGTGAAGCAGGTAATCGCAGGCAAAACTGTTGAACATCGT GGTGCTTTTTCCAACCCTGAGAGCCTGGATCTCTATCGCGATATTCCGGAATTACAAGAC TTTcatatgctcgaggatccggctgctaacaaa
RNaas -G2
RNaas -G3
21
XAacx A-G1
XAacx A-G2
XAacx A-G3
XAacx B-G1
taccaggagatataccATGAACGTTCCGGTCGGACACCTGCGCAATGTCCAAGTTCTCGGAATC GATGCAGGTGGTACGATGACCGACACGTTTTTTGTCGACCAAGATGGCGATTTTGTAGTT GGGAAAGCTCAGTCCACTCCACAGAATGAAGCGCTTGGTCTCATTGCATCCTCCGAAGA CGGTCTGGCCAACTGGGGAATGAGCCTGCACGAAGCGTTAGCCCAATTGCAGACCGGT GTTTACTCTGGCACTGCGATGCTGAATCGCGTAGTTCAGCGCAAAGGTTTAAAATGCGG TCTGATTGTGAACCGTGGTATGGAAGACTTTCATCGCATGGGTCGTGCGGTTCAGTCTC ACTTAGGCTATGCTTATGAAGATCGCATCCATCTTAATACCCACCGCTACGACCCGCCGT TAGTTCCGCGTCATTTGACGCGTGGTGTGGTCGAGCGCACCGACATGATTGGTACCCAA GTAATTCCACTTCGTGAAGACACCGCACGCGACGCAGCACGTGATCTCATTGCTGCTGA TGCGGAAGGCATCGTAATCAGCTTGCTCCACTCGTATAAAAACCCTGAAAATGAACGCC GTGTTCGCGATATTGTTCTGGAAGAGGTGGAAAAAAGTGGGAAAAAGATTCCGGTTTTC GCCTCTGCGGACTATTATCCTGTACGTAAAGAAACCCATCGTACCAACACGACCATTCTG GAAGGTTATGCAGCGGAACCTTCT ATTCTGGAAGGTTATGCAGCGGAACCTTCTCGCCAGACTCTGTCAAAAATTAGTAACGCG TTTAAAGAACGTGGCACAAAATTCGACTTCCGTGTCATGGCAACTCATGGAGGCACTATC TCATGGAAAGCGAAAGAACTGGCACGCACCATCGTATCGGGTCCGATTGGTGGAGTCAT CGGTGCAAAATACCTGGGTGAAGTTTTGGGTTACAAGAACATTGCGTGTAGCGATATTG GTGGCACCAGCTTCGATGTCGCGTTGATTACACAAGGAGAAATGACCATTAAAAACGAC CCAGATATGGCTCGTTTGGTACTTTCTTTACCGCTCGTGGCAATGGACAGCGTCGGTGC AGGTGCGGGAAGTTTCATTCGCTTGGACCCGTATACCCGTGCGATCAAACTGGGTCCCG ACTCGGCTGGTTACCGTGTGGGTGTATGTTGGAAGGAGTCTGGCATTGAAACGGTTACC ATTTCAGACTGTCATATGGTGCTCGGATATCTCAATCCGGATAACTTCCTGGGTGGTGCG GTGAAACTGGATCGCCAGCGTTCTGTTGATGCTATTAAAGCCCAGATTGCAGATCCGCT GGGCTTATCCGTGGAAGATGCGGCAGCAGGTGTAATTGAATTACTCGATTCGGATTTAC GTGATTACTTACGCTCCATGATTAGTGGCAAAGGCTACTCTCCAGCATCCTTCGTCTGCT TCAGTTACGGTGGTGCCGGACCAGTACATA TCAGTTACGGTGGTGCCGGACCAGTACATACCTATGGCTACACAGAAGGTCTGGGCTTT GAAGACGTTATTGTGCCTGCGTGGGCTGCTGGCTTCAGTGCGTTCGGGTGCGCAGCTG CAGACTTCGAATATCGTTATGATAAAAGCCTGGATATTAATATGCCGACCGAAACCCCTG ACACCGATAAAGAAAAAGCTGCAGCAACGTTACAGGCAGCGTGGGAAGAGCTGACCAA GAATGTCCTGGAAGAATTTAAATTGAATGGCTACTCCGCAGACCAGGTTACCCTGCAACC TGGTTATCGCATGCAGTATCGTGGTCAGCTGAATGATCTGGAAATTGAAAGCCCACTGG CCCAAGCCCACACCGCAGCCGACTGGGACCAGCTTACCGATGCGTTCAATGCTACTTAT GGACGTGTGTACGCAGCAAGTGCGCGTTCTCCAGAACTCGGATACAGCGTCACAGGTG CGATTATGCGTGGCATGGTGCCTATCCCGAAACCGAAAATCCCAAAAGAACCGGAAGAA GGCGAAACACCTCCGGAGAGCGCTAAAATTGGTACCCGTAAATTTTACCGTAAAAAACG CTGGGTGGATGCCCAGTTGTACCATATGGAATCTTTACGTCCAGGCAACCGTGTTATGG GTCCTGCTGTTATTGAAAGCGACGCGACGACATTTGTCGTACCGGACGGCTTTGAAACT TGGTTGGATGGGCATCGCCTCTTTCATCTCCGTGAGGTTTAA tctagaaggagatataccatgATGAATGTGACCGTTGACCAAAGTACCTTAGCAGGCGCTACTCGT GGGATTGTTCGTGGTGGTGAAACCCTGAAAGAGCATCGTGACCGTCTGATGGCAGCAAC CAAGGCGACCGGACGCTATGCAGGGTTAAAAACCCTTGAACTTCGTGAACGCGAACCCA TCCTGTACAATAAGCTGTTTAGCCGTCTCCGTGCAGGTGTTGTTGACGCTCGCGAAACG GCTAAGAAGATTGCAGCGTCTCCGATTGTGGAACAGGAAGGGGAACTGTGTTTCACGCT TTATAACGCTGCTGGAGATTCCTTACTGACCTCCACGGGAATCATCATCCACGTGGGGA CGATGGGTGCAGCGATCAAGTACATGATCGAGAACAACTGGGAGGCTAATCCGGGTGT CCATGATAAAGATATCTTCTGTAACAATGATAGCCTTATCGGCAATGTCCACCCGTGCGA TATTCACACAATCGTACCGATTTTTTGGGAAGGTGAGTTGATCGGCTGGGTAGGTGGTG TGACCCATGTAATTGATACTGGTGCAGTTGGACCAGGTTCAATGGCTACCGGTCAGGTC CAACGCTTCGGTGACGGCTATTCCATTACCTGTCGTAAGGTAGGTGCGAATGATACGCT GTTCCGTGATTGGCTGCACGAAAGCCAGCGCATGGTACGCACCACCCGCTATTGGATGT TAGACGAGCGTACCC
22
XAacx B-G2
XAacx B-G3
XAacx B-G4
CGCTATTGGATGTTAGACGAGCGTACCCGCATCGCGGGGTGTCACATGATCCGCAAACT GGTGGAAGAAGTGGTTGCGGAAGAGGGTATTGAGGCGTACTGGAAATTCGCGTATGAA GCGGTGGAACATGGTCGTTTAGGGCTGCAAGCGCGTATTAAAGCAATGACCATCCCTGG CACATACCGTCAAGTTGGTTTCGTAGATGTTCCTTACGCTCATGAGGATGTTCGCGTGCC GTCGGACTTTGCGAAACTGGATACGATTATGCATGCACCGTGCGAGATGACAATCCGCC GTGATGGCACCTGGCGTCTCGACTTCGAAGGGTCCTCGCGTTGGGGTTGGCACACCTA CAATGCCCATCAGGTATCCTTTACGTCTGGTATTTGGGTTATGATGACGCAGACTCTGAT TCCGAGCGAAATGATTAACGACGGTGCAGCCTATGGCACCGAATTTCGTCTGCCAAAAG GCACTTGGATGAATCCGGATGATCGTCGTGTGGCTTTTTCCTATTCCTGGCATTTCCTGG TGAGTGCGTGGACCGCTCTGTGGCGTGGTCTTTCACGCTCCTATTTTGGTCGTGGGTAC TTAGAGGAGGTTAATGCAGGGAACGCGAACACTAGCAATTGGCTGCAAGGTGGTGGCTT CAACCAATACGACGAGATTCACGCAGTTAATAG CCAATACGACGAGATTCACGCAGTTAATAGCTTTGAATGTGCTGCGAACGGCACTGGTG CGACTGCCGTTCAGGATGGTTTGAGCCACGCTGCTGCTATTTGGAATCCGGAAGGAGAC ATGGGTGATATGGAGATCTGGGAACTTGCCGAACCGCTGGTTTATCTGGGTCGTCAGAT TAAAGCAAGTTCTGGTGGAAGCGGTAAATATCGTGGAGGCTGTGGTTTTGAGTCACTCC GCATGGTGTGGAACGCGAAGGATTGGACCATGTTTTTTATGGGGAACGGTCATATTTCAT CGGATTGGGGTCTGATGGGTGGCTACCCTGCAGCTTCTGGTTATCGCTTTGCTGCCCAC AAGACTAATCTCAAGGAATTGATTGCGAGCGGTGCTGAAATCCCGTTAGGTGGTGATAC CGATCCGGAAAACCCGACCTGGGACGCCATGCTGCCAGATGCGCAGATCAAGCGTGAT AAGCAAGCCATTACGACCGAGGAGATGTTCAGTGATTACGATCTCTATCTGAACTATATG CGTGGTGGTCCAGGGTTTGGCGATCCGCTGGATCGTGAACCGCAAGCGGTTGCGGATG ATATCAACGGTGGTTATGTCTTGGAACGCTT TATCAACGGTGGTTATGTCTTGGAACGCTTCGCAGGTGAAGTCTATGGTGTTGTTGTGC GTAAAGGTGCTGACGGTCAGTACGGTGTCGATGAAGCAGGTACTGCAGCAGCACGTGC GCAAATTCGCAAAGATCGTTTAGCCAAAAGCGTTCCGGTTAGCGAATGGATGAAAGGTG AGCGTGAAAAAATTCTCGCCAAGGATGCAGGGACCCAAGTTCGCCAGATGTTCGCTGCA AGCTTCAAGCTCGGTCCTCGTTTTGAAAAAGATTTTCGTACCTTTTGGAGCTTACCGGAT AGCTGGACTCTGCCTGAAGAAGAAATCGGTGTGCCCACGTACGGTTCGCGCTATAGTAT GGACATTTCCGAGTTACCGGACGTGCACACCGTGCAATTTGTTGAAGAGTAAaggagatata ccatggcg
XAacx C-G1
ataaaggagatataccATGGCATATACCCGTTCGAAAATTGTTGACCTGGTTGACGGTAAAATC GATCCGGATACCTTACATCAGATGTTATCTACACCGAAAGATCCGGAACGCTTTGTGACG TATGTGGAAATTCTGCAAGAACGCATGCCGTGGGATGATAAAATCATTCTTCCCCTGGGT CCGAAGCTGTTTATCGTTCAGCAGAAAGTTAGCAAAAAATGGACAGTACGCTGTGAATGT GGCCATGATTTTTGCGATTGGAAAGACAATTGGAAACTCTCTGCACGCGTACACGTGCG TGACACCCCACAAAAGATGGAGGAAATTTATCCGCGTCTGATGGCTCCAACTCCGAGCT GGCAGGTAATTCGTGAATACTTTTGTCCCGAGTGTGGCACGCTCCATGACGTCGAAGCA CCGACTCCGTGGTACCCAGTAATCCACGATTTTAGCCCTGACATTGAGGGCTTTTATCAG GAATGGCTTGGTCTGCCAGTTCCGGAACGTGCGGATGCGTAAgtcgacctgcaggcatgcaagctt
AAacx A-G1
accaggagatataccATGGAAGCCCTGCAAGCGTCCCAAGTGCAGGTCATGGGCATTGATGC TGGAGGCACGATGACTGACACTTTTTTCGTCCGTGAAGATGGTCGTTTCGTTGTAGGCA AAGCGCAGAGCAACCCAGCGGATGAGTCTTTGGCGATCTTTAATAGCAGCCAGGATGCG CTGGCACATTGGGAACGTGATGTGGATGAAGTTTATCCGGAACTGGTTACCTGTGTGTA TTCCGGTACTGCCATGTTAAACCGTATCCTGATGCGCAAGGGTCTGGATGTGGGCTTAA TCTGTAACCGTGGATTCGAACAAATCCATAGCATGGGTCGTGCACTGCAGAGCTACTTA GGTTATGCGCTGGAAGATCGCATTCATTTGAATACACATCGTTATGACGAGCCTCTGGTT CCGGTTTCTCGTACGCGTGGTGTGGCTGAACGTACGGATGTACAGGGTAAAGTCGTGAT TCCGCTTCGCGAAGACGAAGTCCGTCAGGCCACCCGTGAGCTTGTAGAAGCAGGTTCT CAAGCAGTGGTGATTTGCCTGTTACAGAGTCATAAAAACGAAACCTCAGAACAACGTGCA CGCGATGTGGTCAAAGATGAATTGAAAAAACTGAAGGTAGACATTCCCGTTTTTGCTTCT GTGGACTATTACCCGAGTCGTAAAGAATCGCACCGTATGAACACGACAATTTTGGAAGC GTACGGAGCAGAACCTTCTCGCCAGACCCTGAAGAAG
23
AAacx A-G2
AAacx A-G3
AAacx B-G1
AAacx B-G2
AAacx B-G3
CAGAACCTTCTCGCCAGACCCTGAAGAAGGTGAGCGATCGCTTCAAGAAACATGGAGCC AAGTTTGATCTCCGTGTAATGGCTACGCACGGTGGTACGATCAGCTGGAAAGCCAAAGA GCTTGCACGTACCATCGTGAGCGGTCCGATCGGTGGCGTGATCGGTAGTAAATTACTTG GCGAATATCTGGGTGATGAGAACATTGCGTGCAGCGATATTGGAGGTACCAGCTTCGAT GTTGCTCTGATCACCAAAGGGTCGTTCGCGATCAAAAGCGATCCCGATATGGCACGCCT GGTACTGAGTCTGCCGTTGGTAGCAATGGACTCTGTTGGCGCTGGTGCAGGTTCGTTTG TGCGTCTGGATCCGTATAGTAAAAGCATTAAACTGGGACCGGATAGCGCAGGATATCGC GTGGGCACGTGTTGGCCAGAATCTGGGCTGGACACCGTATCCGTGAGTGACTGCCACG TGGTTCTGGGATACCTGAATCCGGATAATTTTCTGGGTGGAGCGATCAAATTAGATGTAC AGCGTGCACGCGACCATATTAAAGTCCAGATTGCCGATCCTCTGGGTCTTAGCGTCGAG GATGCTGCGAGCGGTGTTATTGAACTGCTGGATCTGACGCTCTCGGAGTATCTCCGTGC AAATATTTCAGCGAAAGGCTATAATCCAGCGGAATTTACCTGCTTCTCGTATGGTGGTGC AGGACCGGTGCATACTTACGGCTATACCGAAG ACCGGTGCATACTTACGGCTATACCGAAGGGGTGGGCTTCAAAGATGTCGTGGTGCCTG CTTGGGCAGCGGGTTTCTCTGCGTTTGGCTGTGCATGTGCGGATTTCGAATATCGCTAC GATAAATCCGTTGACCTGGGTGTCGCACAATTCGCGTCCGACGAAGTGAAAGCAGCAGC GTGCGTCAGCCTGCAAGCAGCCTGGGAAGAATTAGCGGTAAAAGTAATTGACGAGTTCA TTATCAACGGTTACAAACCTGAAGATGTATTGCTTATCCCAGGCTATAAGATGCAGTATAT GGGCCAACTGAACGATCTGGAGATTGTATCTCCGGTGTCAACGGCAGCCACGGCAGGT GACTGGAATCAGATTATCGAGGCGTTCGAGACCACCTATAGTCGCGTTTACGCTAATAG CGCTCGTAGCCCTGAACTGGGCTTCTCTATTACTGGTGCTATCCTTCGTGGTATGGTTGT GACCCAGAAACCAGTTCTGCCTGAAGATGCTGACTGCGGTCCGACCCCACCAAAGGAA GCATATCTGGGAACCCGTCCGTTCTATCGTCATAAACGCTGGGTCGAAGCCACTCTGTG GAAAATGGAATCCCTCAAAGCAGGTAACCACATTGTCGGTCCTGCGATTATCGAATCTGA TGCAACCACCTTTGTGGTTCCGGATGGTTTCGAGACTACGGTAGACAAGCATCGCCTGT TTCACCTGAAAGAAGTGAAATAAtctagaaggagatatac tctagaaggagatataccATGAATATGCTGACCAATAAAGAAGTTGGGTTCGCCGATCTGTTAA AAAATGGGCAGACACTGAAACAATTCCGTGATGGTATTATCGAACGTACCAAAGCCTCTG GCTGCTATAATGGTATCGAAAAGCTGGAATTTCGCGACTCGGATCCGATTGGCTATGAAA AACTGTTTTCGAAACTGCGTGGTGGTTTAGTGCACGCACGTGAGACTGCGAAGAAAATC GCAGCGTCTCCGATTGTGGAGCAGGAAGGGGAACTGTGTTTTACCTTGTACAACGCAGC AGGCGATTGCGTGCTGACCTCGACTGGCATCATTATCCATGTGGGTACCATGGGTGCAG CGATCAAATACATGATTGAGAACAATTGGGAAGGAAACCCTGGCATCAACCCACGTGAC ATGTTCACCACGAACGATTGTGCTATCGGCAATGTTCATCCGTGTGACATCGCTACCATC GTGCCGATTT CGTGTGACATCGCTACCATCGTGCCGATTTTTTGGGAAGGCAAGTTAATTGGATGGGTG GGCGGTGTCACGCATGTGATCGACACGGGTGCAGTTACACCAGGCAGCATGTCGACGG GTCAGACCCAACGTTTTGGCGATGGCTATATGATTACGTGTCGTAAAACGGGTGTTAAC GATGAACCGCTGCGTGACTGGCTGCATGAATCCCAGCGTAGCGTTCGTACTCCGAAATA TTGGATCCTGGACGAGAAAACCCGTATTGCAGGATGCCATATGATTCGTGAATTAGTGG AAGAAGTTATCCGTGCGGATGGCCTTGATGCTTATGAGAAGTTTGGCTTTGAGGTTATTG AGGAAGGTCGTCGTGGATTGATGTCTCGTGTTAAAGCTATGACGCTTCCAGGAAAATATC GCAAAGTTAGCTTTGTTGACGTTCCCTTTAAGCATGAGGACGTACAAGTGAGCAATGCAT TCGCCAAACTGGACAGCATCA CAATGCATTCGCCAAACTGGACAGCATCATGCACTCACCAGTTGAGATGACCATCAAAC CCGATGGTAAGTGGCGTTTAGACTTTGAAGGTGCAAGCCGCTGGGGATGGCACACCTTC AACGCTCACCAGGTGGCCTTCACGAGCGGTATTTGGGTCATGATGTCCCAGACGCTGGT TCCGACTCAACGTATCAACGATGGAGCCTACTTTGCCACGGAATTTAAACTGCCAAAGG GTACATGGTGTAATCCTGACGATCGTCGCACTGGGCATGCGTACGCATGGCACTTTTTA GTGTCAGGTTGGGCTGCCCTGTGGCGTGGCCTTAGTCAATCTTACTTCTCTCGTGGCTA TCTGGAGGAAGTGAACGCAGGTAATGCTAACACGTCGAATTGGCTTCAAGGTGGTGGCA TCAATCAGGATGGTGAGATTCATGCCGTAAACTCTTTTGAGGCGTCCTCGTGTGGAACG GGAGCCTGTGCGGTGAAAGATGGCCTTAATCACGCAGCAGCGATTTGGAATCCGGAAG GCGATATGGGTGACATCGAAATCTGGGAGATGGCTGAACCGCTCTTATATCTGGGTCGC AACGTTAAAGCGAACTCTGGAGGCTACGGGAAATATCGTGGTGGCTGTGGCTTTGAAAC CCTGCGCATGGTCTGGAATGCACAAGACTGGACCATGTTTTTCATGGGCAATGGTTATAT GAATAGTGATTG
24
AAacx B-G4
CATGGGCAATGGTTATATGAATAGTGATTGGGGTATGATGGGTGGCTACCCTTCAGCGA CGGGTTATCGTTTTGAAGCCCATAAAACGGGTCTGGAACGTCGTATTGCCAATGGGGAG AGCCTGCCGTTAGGTGGCGATATTGACCCAGCGGCCCCTACCTACGAACGTAATATCGA TGCGACTAGCGTGGTCAAACGTGATAAACAGTGCATGACCACTGAAGACTGCTACGAAA ATCACGACCTGTATCTGAATTATTTGCGTGGAGGTCCAGGTTTTGGCGATCCGATTGATC GTGAACCGAAGGCCATCGAAAACGATTTGAACCAGAAATTCTTATTACCTGAATACGCCG AGAAAGTATATGGTGCGGTTATTACCCAAGATGCGAAAGGTGTATTTACCGTGGACGCA GCAAAAACCGCAGCACGTCGTGCGGAAGTCCGCAAAGAACGTCTGGCTCGTGCTGTCC CAACTCACGAATGGATGAAACAGGAGCGTGAACGCATCTTGATCAAAGATGCAGCTGTC CAAGTACAACACATGTTCGCGACGTCGTTTGGCCTGTCTGAAAAATTCACCAAAGAATTT AAAGCGTTTTGGTCACTTCCGGATGATTGGCAGCTGCTGGAAAGCGAGCTTAATGTCCC GACCTACGGAAGCAAACATCGTATGGATCTCTCGCTGTTACCGGATGTGACCACGGTGG TCCAGGTAGAAGAAtaaaggagat
AAacx C-G1
tataccATGTCAACGTATACGAACGAACAGGTGGCTCATCTGGTTGAAGGTTCACTTGATT GGGAAACGACTTTTCGCATGCTGTCAATGCCGAAAGACAATTCTCGCTTCGAACAGTATC TGAAAGCCCTGCAAGCGAAAGTGAGCTTTCCAGACAAAATTGTGCTCCCGTTGGGTCCG CACTTGTACATTGTCCAGTCGGCAAAAACCAAACAATGGGTCACCATGTGTGACTGTGG GCACGTGTTTGGGGATTATCGCGAAAACTGGAAACTGAACGCAGCAATCTATGTTCGCG ATACCGAGGAGGCAATGACCGAAGTTTATCCGAAGCTGATGGCTCCGGATACCCAATGG CAGGTTTATCGCGAATATTATTGCCCAACCTGCGGTACTATGCATGACGTGGAAGCACC GACCCCTTGGTATCCGGTTATTCATGATTTTGAACCGGACATCGAAGCGTTTTACCAGGA ATGGGTAGGACTGCCTGTTCCCGAACGTGCAGCATAAgtcgacctgcaggcatgcaagcttggctg ttttg
Literature Cited.
(1) Lee, T. S., Krupa, R. A., Zhang, F., Hajimorad, M., Holtz, W. J., Prasad, N., Lee, S. K., and Keasling, J. D. (2011) BglBrick vectors and datasheets: A synthetic biology platform for gene expression. J. Biol. Eng. 5, 1-14.
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