生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 80-92.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0271
李仁瀚1,2(), 张乐乐1,2, 刘春立1,2,3, 刘秀霞1,2,3, 白仲虎1,2,3, 杨艳坤1,2,3(), 李业1,2,3()
收稿日期:
2023-03-23
出版日期:
2023-10-26
发布日期:
2023-11-28
通讯作者:
杨艳坤,男,博士,副教授,研究方向:分子生物学与合成生物学;E-mail: yangyankun@jiangnan.edu.cn;作者简介:
李仁瀚,男,研究方向:代谢工程与合成生物学;E-mail: 6200201078@stu.jiangnan.edu.cn
基金资助:
LI Ren-han1,2(), ZHANG Le-le1,2, LIU Chun-li1,2,3, LIU Xiu-xia1,2,3, BAI Zhong-hu1,2,3, YANG Yan-kun1,2,3(), LI Ye1,2,3()
Received:
2023-03-23
Published:
2023-10-26
Online:
2023-11-28
摘要:
结合生物传感器进行高通量筛选是鉴定L-色氨酸高产菌株的有力工具,但现有的L-色氨酸生物传感器普遍存在工作范围和动态范围都较低的缺点。在大肠杆菌中表达紫色杆菌素生物合成途径,测试不同来源的VioA酶,结合RBS工程,开发了一种新型酶偶联L-色氨酸生物传感器。测试发现来自Chromobacterium violaceum的VioA酶可使该传感器的检测上限扩大至10 g/L外源添加的L-色氨酸;将其RBS的初始翻译速率降低到约2 000时,传感器的动态范围扩大到55倍;通过肉眼可直观地区分L-色氨酸产量不同的大肠杆菌菌株。这种新型的L-色氨酸生物传感器可结合高通量筛选等手段,在鉴定L-色氨酸及其高附加值衍生物高产菌株等方面发挥巨大作用。
李仁瀚, 张乐乐, 刘春立, 刘秀霞, 白仲虎, 杨艳坤, 李业. 基于紫色杆菌素生物合成途径的L-色氨酸生物传感器的构建[J]. 生物技术通报, 2023, 39(10): 80-92.
LI Ren-han, ZHANG Le-le, LIU Chun-li, LIU Xiu-xia, BAI Zhong-hu, YANG Yan-kun, LI Ye. Development of an L-tryptophan Biosensor Based on the Violacein Biosynthesis Pathway[J]. Biotechnology Bulletin, 2023, 39(10): 80-92.
图1 紫色杆菌素生物合成途径 IPA:吲哚-3-丙酮酸;CPA:铬吡咯酸;VA:紫色杆菌酸;PVA:原紫色杆菌酸;PDVA:原脱氧紫色杆菌酸;DVA:脱氧紫色杆菌酸;Spont:自发反应
Fig. 1 Violacein biosynthetic pathway IPA: Indole-3-pyruvic acid; CPA: chromopyrrolic acid; VA: violaceinic acid; PVA: protoviolaceinic acid; PDVA: protoviolaceinic acid; DVA: deoxyviolaceinic acid; Spont: spontaneous reaction
质粒Plasmid | 描述Description | 来源Source |
---|---|---|
pVio | 在pRSFDuet载体的多克隆位点2内插入vioABCDE基因簇,卡那霉素抗性 pRSFDuet with vioABCDE gene cluster in MCS-2, KanR | [ |
pBL_401 | 删除了pVio中的vioD基因 pVio deleted vioD | 本研究构建This study |
pBL_403-407, 421-423 | 更换pBL_401中的RBSb区域 pBL_401 changed RBSb region | 本研究构建This study |
pBL_415 | 将pBL_401的Duganella sp. B2 vioA基因从多克隆位点2移到多克隆位点1,在多克隆位点前添加一个T7终止子 Duganella sp. B2 vioA of pBL_401 was moved to MCS-1 from MCS-2, and a T7 terminator was added before MCS-2 | 本研究构建This study |
pBL_416 | 将pBL_415的Duganella sp. B2 vioA替换为Janthinobacteriun violaceinigrum vioA Duganella sp. B2 vioA of pBL_415 was replaced by Janthinobacteriun violaceinigrum vioA | 本研究构建This study |
pBL_417 | 将pBL_415的Duganella sp. B2 vioA替换为Pseudoduganella violaceinigra vioA Duganella sp. B2 vioA of pBL_415 was replaced by Pseudoduganella violaceinigra vioA | 本研究构建This study |
pBL_418 | 将pBL_415的Duganella sp. B2 vioA替换为Myxococcus xanthus vioA Duganella sp. B2 vioA of pBL_415 was replaced by Myxococcus xanthus vioA | 本研究构建This study |
pBL_419 | 将pBL_415的Duganella sp. B2 vioA替换为Chromobacterium violaceinum vioA Duganella sp. B2 vioA of pBL_415 was replaced by Chromobacterium violaceinum vioA | 本研究构建This study |
pBL_420 | 将pBL_415的Duganella sp. B2 vioA替换为Duganella sp. HH105 vioA Duganella sp. B2 vioA of pBL_415 was replaced by Duganella sp. HH105 vioA | 本研究构建This study |
pBL_425 | 更换pBL_415中的RBSa区域 pBL_415 changed RBSa region | 本研究构建This study |
pBL_429 | 更换pBL_416中的RBSa区域 pBL_416 changed RBSa region | 本研究构建This study |
pBL_437-439 | 更换pBL_419中的RBSa区域 pBL_419 changed RBSa region | 本研究构建This study |
pED_RBS1-RBS4 | 携带trpEfbrD和不同RBS的pACYCDuet,氯霉素抗性 pACYCDuet with trpEfbrD and different RBSs, CmR | [ |
表1 本研究所用质粒
Table 1 Plasmids in this study
质粒Plasmid | 描述Description | 来源Source |
---|---|---|
pVio | 在pRSFDuet载体的多克隆位点2内插入vioABCDE基因簇,卡那霉素抗性 pRSFDuet with vioABCDE gene cluster in MCS-2, KanR | [ |
pBL_401 | 删除了pVio中的vioD基因 pVio deleted vioD | 本研究构建This study |
pBL_403-407, 421-423 | 更换pBL_401中的RBSb区域 pBL_401 changed RBSb region | 本研究构建This study |
pBL_415 | 将pBL_401的Duganella sp. B2 vioA基因从多克隆位点2移到多克隆位点1,在多克隆位点前添加一个T7终止子 Duganella sp. B2 vioA of pBL_401 was moved to MCS-1 from MCS-2, and a T7 terminator was added before MCS-2 | 本研究构建This study |
pBL_416 | 将pBL_415的Duganella sp. B2 vioA替换为Janthinobacteriun violaceinigrum vioA Duganella sp. B2 vioA of pBL_415 was replaced by Janthinobacteriun violaceinigrum vioA | 本研究构建This study |
pBL_417 | 将pBL_415的Duganella sp. B2 vioA替换为Pseudoduganella violaceinigra vioA Duganella sp. B2 vioA of pBL_415 was replaced by Pseudoduganella violaceinigra vioA | 本研究构建This study |
pBL_418 | 将pBL_415的Duganella sp. B2 vioA替换为Myxococcus xanthus vioA Duganella sp. B2 vioA of pBL_415 was replaced by Myxococcus xanthus vioA | 本研究构建This study |
pBL_419 | 将pBL_415的Duganella sp. B2 vioA替换为Chromobacterium violaceinum vioA Duganella sp. B2 vioA of pBL_415 was replaced by Chromobacterium violaceinum vioA | 本研究构建This study |
pBL_420 | 将pBL_415的Duganella sp. B2 vioA替换为Duganella sp. HH105 vioA Duganella sp. B2 vioA of pBL_415 was replaced by Duganella sp. HH105 vioA | 本研究构建This study |
pBL_425 | 更换pBL_415中的RBSa区域 pBL_415 changed RBSa region | 本研究构建This study |
pBL_429 | 更换pBL_416中的RBSa区域 pBL_416 changed RBSa region | 本研究构建This study |
pBL_437-439 | 更换pBL_419中的RBSa区域 pBL_419 changed RBSa region | 本研究构建This study |
pED_RBS1-RBS4 | 携带trpEfbrD和不同RBS的pACYCDuet,氯霉素抗性 pACYCDuet with trpEfbrD and different RBSs, CmR | [ |
菌株Strain | 描述Description | 来源 Source |
---|---|---|
E. coli JM109 | 用于构建质粒Construct plasmids | 实验室保存Lab stored |
E. coli BL21(DE3) | 用于生物传感器宿主菌The host of the biosensor | 实验室保存Lab stored |
E. coli B8 | 敲除trpR tnaA pheA的E. coli BL21(DE3)E. coli BL21(DE3)ΔtrpR ΔtnaA ΔpheA | [ |
EBL_400 | 携带pVio质粒的大肠杆菌BL21(DE3)E. coli BL21(DE3)with plasmid pVio | 本研究构建This study |
EBL_401-439 | 携带相应质粒的大肠杆菌BL21(DE3)E. coli BL21(DE3)with corresponding plasmids | 本研究构建This study |
表2 本研究所用菌株
Table 2 Strains in this study
菌株Strain | 描述Description | 来源 Source |
---|---|---|
E. coli JM109 | 用于构建质粒Construct plasmids | 实验室保存Lab stored |
E. coli BL21(DE3) | 用于生物传感器宿主菌The host of the biosensor | 实验室保存Lab stored |
E. coli B8 | 敲除trpR tnaA pheA的E. coli BL21(DE3)E. coli BL21(DE3)ΔtrpR ΔtnaA ΔpheA | [ |
EBL_400 | 携带pVio质粒的大肠杆菌BL21(DE3)E. coli BL21(DE3)with plasmid pVio | 本研究构建This study |
EBL_401-439 | 携带相应质粒的大肠杆菌BL21(DE3)E. coli BL21(DE3)with corresponding plasmids | 本研究构建This study |
引物Primer | 序列 Sequence(5'-3') | 用途Purpose |
---|---|---|
401_F | ACTTGGAAGGGTAAACTAAGCTGACCGCCAGCTTCG | 扩增pVio以构建pBL_401 Amplifying pVio for pBL_401 |
401_R | CTTAGTTTACCCTTCCAAGTTTGTACCAAACGT | |
403_F | GTATAAGAGAGAATTTTAATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_403 Amplifying pBL_401 for pBL_403 |
403_R | TTGTCATTAAAATTCTCTCTTATACTTAACTAATATACTAAGATGGG | |
404_F | GCAGGAAGTAAGTATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_404 Amplifying pBL_401 for pBL_404 |
404_R | TTGTCATACTTACTTCCTGCTTATACTTAACTAATATACTAAGATGGG | |
405_F | TAGTTAAGTATAAGCGGGGGCTCGAGATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_405 Amplifying pBL_401 for pBL_405 |
405_R | CCCCCGCTTATACTTAACTAATATACTAAGATGGG | |
406_F | TCGACACACATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_406 Amplifying pBL_401 for pBL_406 |
406_R | TAATTTGTCATGTGTGTCGAAACCCCTTATACTTAACTAATATACTAAGATGGG | |
407_F | AGAACCGAGAATTATCTCATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_407 Amplifying pBL_401 for pBL_407 |
407_R | ATGAGATAATTCTCGGTTCTTATACTTAACTAATATACTAAGATGGG | |
415V_F | ATAAGAAGGAGATATACATAATGAGCATTCTCGACTTTCC | 扩增pBL_401以构建pBL_415 Amplifying pBL_401 for pBL_415 |
415V_R | GTCATGGTATATCTCCTTATTAAAGTTAAACAAAATTATT | |
T7terA_F | GAGCGCGCATGAGGCAGCAGCCATCAC | 扩增pBL_401的T7终止子序列以构建pBL_415 Amplifying the T7 terminator sequence of pBL_401 for pBL_415 |
T7terA_R | CCGTTTAGAGGCCCCAAGGGGTTATGCTAGATACGATTACTTTCTGTTCGACT | |
T7terB_F | CCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGTGTACACGGCCGCAT | |
T7terB_R | TATGTATATCTCCTTCTTATACTTAACTAATATACTAAGA | |
415_VioA_F | TAAGGAGATATACCATGACAAATTATTCTGACATTTGCAT | 扩增pBL_401的Dug.B2_vioA序列以构建pBL_415 Amplifying Dug.B2_vioA sequence of pBL_401 for pBL_415 |
415_VioA_R | CTGCTGCCTCATGCGCGCTCAGTAAGA | |
416-420V_F | GGCAGCAGCCATCACCAT | 扩增pBL_415以作为pBL_416-420的载体 Amplifying pBL_415 as their vectors for pBL_416-420 |
416-420V_R | GGTATATCTCCTTATTAAAGTTAAACAAAATTATTTCTACAGGGG | |
416_VioA_F | TTAACTTTAATAAGGAGATATACCATGAGCACCTATAGCGATATTTGCATTG | 扩增Jan_vioA以构建pBL_416 Amplifying Jan_vioA for pBL_416 |
416_VioA_R | ATGGTGATGGCTGCTGCCTTACGCGCGTTCGGTGC | |
417_VioA_F | TTAACTTTAATAAGGAGATATACCATGACCAACTATAGCGATATTTGCATTATTGGC | 扩增Pse_vioA以构建pBL_417 Amplifying Pse_vioA for pBL_417 |
417_VioA_R | ATGGTGATGGCTGCTGCCTTACGCGCTTTCCGCCG | |
418_VioA_F | TTAACTTTAATAAGGAGATATACCATGACCCATCATAGCGATATTTGTATTGTTGG | 扩增Myx_vioA以构建pBL_418 Amplifying Myx_vioA for pBL_418 |
418_VioA_R | ATGGTGATGGCTGCTGCCTTACGGGGTGCGATACGCAAT | |
419_VioA_F | TTAACTTTAATAAGGAGATATACCATGAAACATAGCAGCGATATTTGCATTGT | 扩增Chr_vioA以构建pBL_419 Amplifying Chr_vioA for pBL_419 |
419_VioA_R | ATGGTGATGGCTGCTGCCTTACGCCGCAATGCGCTG | |
420_VioA_F | TTAACTTTAATAAGGAGATATACCATGGGCCGCCGCTTTC | 扩增Dug.HH105_vioA以构建pBL_420 Amplifying Dug.HH105_vioA for pBL_420 |
420_VioA_R | ATGGTGATGGCTGCTGCCTTACACGCGTTCCGCGGT | |
421_F | AGTATAAGGCATCGTAGAAGGGATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_421 Amplifying pBL_401 for pBL_421 |
421_R | CTTCTACGATGCCTTATACTTAACTAATATACTAAGATGGG | |
422_F | TATAAGACCTGGGACACAAATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_422 Amplifying pBL_401 for pBL_422 |
422_R | TTTGTGTCCCAGGTCTTATACTTAACTAATATACTAAGATGGG | |
423_F | CTAAACCCTTTTGCAAAATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_423 Amplifying pBL_401 for pBL_423 |
423_R | CATTTTGCAAAAGGGTTTAGCTTATACTTAACTAATATACTAAGATGG | |
425_F | CTGTAGAAATAATTTTGTTTAACTTTAATAAGGGGTCCAGAACATGACAAATTATTCTGACATTTGCATAGTT | 扩增pBL_415以构建pBL_425 Amplifying pBL_415 for pBL_425 |
429_F | CTGTAGAAATAATTTTGTTTAACTTTAATAGGTAGGGGGACATGAGCACCTATAGCGATATTTGC | 扩增pBL_416以构建pBL_429 Amplifying pBL_416 for pBL_429 |
437_F | CTGTAGAAATAATTTTGTTTAACTTTAATTCGGAGAGAAAACACATGAAACATAGCAGCGATATTTGCA | 扩增pBL_419以构建pBL_437 Amplifying pBL_419 for pBL_437 |
438_F | CTGTAGAAATAATTTTGTTTAACTTTAATAGGGTAAACACATGAAACATAGCAGCGATATTTGCA | 扩增pBL_419以构建pBL_438 Amplifying pBL_419 for pBL_438 |
439_F | CTGTAGAAATAATTTTGTTTAACTTTAATTACAGTATTGACATGAAACATAGCAGCGATATTTGCA | 扩增pBL_419以构建pBL_439 Amplifying pBL_419 for pBL_439 |
adj_RBSAll_R | ATTAAAGTTAAACAAAATTATTTCTACAGGGGAATT | 425_F, 429_F, 437_F, 438_F和439_F的反向引物 The reverse primer for 425_F, 429_F, 437_F, 438_F and 439_F |
表3 本研究所用引物
Table 3 Primers in this study
引物Primer | 序列 Sequence(5'-3') | 用途Purpose |
---|---|---|
401_F | ACTTGGAAGGGTAAACTAAGCTGACCGCCAGCTTCG | 扩增pVio以构建pBL_401 Amplifying pVio for pBL_401 |
401_R | CTTAGTTTACCCTTCCAAGTTTGTACCAAACGT | |
403_F | GTATAAGAGAGAATTTTAATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_403 Amplifying pBL_401 for pBL_403 |
403_R | TTGTCATTAAAATTCTCTCTTATACTTAACTAATATACTAAGATGGG | |
404_F | GCAGGAAGTAAGTATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_404 Amplifying pBL_401 for pBL_404 |
404_R | TTGTCATACTTACTTCCTGCTTATACTTAACTAATATACTAAGATGGG | |
405_F | TAGTTAAGTATAAGCGGGGGCTCGAGATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_405 Amplifying pBL_401 for pBL_405 |
405_R | CCCCCGCTTATACTTAACTAATATACTAAGATGGG | |
406_F | TCGACACACATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_406 Amplifying pBL_401 for pBL_406 |
406_R | TAATTTGTCATGTGTGTCGAAACCCCTTATACTTAACTAATATACTAAGATGGG | |
407_F | AGAACCGAGAATTATCTCATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_407 Amplifying pBL_401 for pBL_407 |
407_R | ATGAGATAATTCTCGGTTCTTATACTTAACTAATATACTAAGATGGG | |
415V_F | ATAAGAAGGAGATATACATAATGAGCATTCTCGACTTTCC | 扩增pBL_401以构建pBL_415 Amplifying pBL_401 for pBL_415 |
415V_R | GTCATGGTATATCTCCTTATTAAAGTTAAACAAAATTATT | |
T7terA_F | GAGCGCGCATGAGGCAGCAGCCATCAC | 扩增pBL_401的T7终止子序列以构建pBL_415 Amplifying the T7 terminator sequence of pBL_401 for pBL_415 |
T7terA_R | CCGTTTAGAGGCCCCAAGGGGTTATGCTAGATACGATTACTTTCTGTTCGACT | |
T7terB_F | CCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGTGTACACGGCCGCAT | |
T7terB_R | TATGTATATCTCCTTCTTATACTTAACTAATATACTAAGA | |
415_VioA_F | TAAGGAGATATACCATGACAAATTATTCTGACATTTGCAT | 扩增pBL_401的Dug.B2_vioA序列以构建pBL_415 Amplifying Dug.B2_vioA sequence of pBL_401 for pBL_415 |
415_VioA_R | CTGCTGCCTCATGCGCGCTCAGTAAGA | |
416-420V_F | GGCAGCAGCCATCACCAT | 扩增pBL_415以作为pBL_416-420的载体 Amplifying pBL_415 as their vectors for pBL_416-420 |
416-420V_R | GGTATATCTCCTTATTAAAGTTAAACAAAATTATTTCTACAGGGG | |
416_VioA_F | TTAACTTTAATAAGGAGATATACCATGAGCACCTATAGCGATATTTGCATTG | 扩增Jan_vioA以构建pBL_416 Amplifying Jan_vioA for pBL_416 |
416_VioA_R | ATGGTGATGGCTGCTGCCTTACGCGCGTTCGGTGC | |
417_VioA_F | TTAACTTTAATAAGGAGATATACCATGACCAACTATAGCGATATTTGCATTATTGGC | 扩增Pse_vioA以构建pBL_417 Amplifying Pse_vioA for pBL_417 |
417_VioA_R | ATGGTGATGGCTGCTGCCTTACGCGCTTTCCGCCG | |
418_VioA_F | TTAACTTTAATAAGGAGATATACCATGACCCATCATAGCGATATTTGTATTGTTGG | 扩增Myx_vioA以构建pBL_418 Amplifying Myx_vioA for pBL_418 |
418_VioA_R | ATGGTGATGGCTGCTGCCTTACGGGGTGCGATACGCAAT | |
419_VioA_F | TTAACTTTAATAAGGAGATATACCATGAAACATAGCAGCGATATTTGCATTGT | 扩增Chr_vioA以构建pBL_419 Amplifying Chr_vioA for pBL_419 |
419_VioA_R | ATGGTGATGGCTGCTGCCTTACGCCGCAATGCGCTG | |
420_VioA_F | TTAACTTTAATAAGGAGATATACCATGGGCCGCCGCTTTC | 扩增Dug.HH105_vioA以构建pBL_420 Amplifying Dug.HH105_vioA for pBL_420 |
420_VioA_R | ATGGTGATGGCTGCTGCCTTACACGCGTTCCGCGGT | |
421_F | AGTATAAGGCATCGTAGAAGGGATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_421 Amplifying pBL_401 for pBL_421 |
421_R | CTTCTACGATGCCTTATACTTAACTAATATACTAAGATGGG | |
422_F | TATAAGACCTGGGACACAAATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_422 Amplifying pBL_401 for pBL_422 |
422_R | TTTGTGTCCCAGGTCTTATACTTAACTAATATACTAAGATGGG | |
423_F | CTAAACCCTTTTGCAAAATGACAAATTATTCTGACATTTG | 扩增pBL_401以构建pBL_423 Amplifying pBL_401 for pBL_423 |
423_R | CATTTTGCAAAAGGGTTTAGCTTATACTTAACTAATATACTAAGATGG | |
425_F | CTGTAGAAATAATTTTGTTTAACTTTAATAAGGGGTCCAGAACATGACAAATTATTCTGACATTTGCATAGTT | 扩增pBL_415以构建pBL_425 Amplifying pBL_415 for pBL_425 |
429_F | CTGTAGAAATAATTTTGTTTAACTTTAATAGGTAGGGGGACATGAGCACCTATAGCGATATTTGC | 扩增pBL_416以构建pBL_429 Amplifying pBL_416 for pBL_429 |
437_F | CTGTAGAAATAATTTTGTTTAACTTTAATTCGGAGAGAAAACACATGAAACATAGCAGCGATATTTGCA | 扩增pBL_419以构建pBL_437 Amplifying pBL_419 for pBL_437 |
438_F | CTGTAGAAATAATTTTGTTTAACTTTAATAGGGTAAACACATGAAACATAGCAGCGATATTTGCA | 扩增pBL_419以构建pBL_438 Amplifying pBL_419 for pBL_438 |
439_F | CTGTAGAAATAATTTTGTTTAACTTTAATTACAGTATTGACATGAAACATAGCAGCGATATTTGCA | 扩增pBL_419以构建pBL_439 Amplifying pBL_419 for pBL_439 |
adj_RBSAll_R | ATTAAAGTTAAACAAAATTATTTCTACAGGGGAATT | 425_F, 429_F, 437_F, 438_F和439_F的反向引物 The reverse primer for 425_F, 429_F, 437_F, 438_F and 439_F |
质粒Plasmids | RBSa/RBS | 翻译起始速率TIR | RBSb | 翻译起始速率TIR |
---|---|---|---|---|
pBL_401 | AAGGAGATATACC | / | AAGGAGATATACATA | 11 602.81 |
pBL_403 | AAGGAGATATACC | / | AGAGAATTTTA | 23 837.83 |
pBL_404 | AAGGAGATATACC | / | CAGGAAGTAAGT | 17 039.41 |
pBL_405 | AAGGAGATATACC | / | CGGGGGCTCGAG | 7 953.16 |
pBL_406 | AAGGAGATATACC | / | GGGTTTCGACACAC | 4 941.17 |
pBL_407 | AAGGAGATATACC | / | AACCGAGAATTATCTC | 1 875.68 |
pBL_415 | AAGGAGATATACC | 40 994.72 | AAGGAGATATACATA | / |
pBL_416 | AAGGAGATATACC | 15 231.17 | AAGGAGATATACATA | / |
pBL_417 | AAGGAGATATACC | 51 571.35 | AAGGAGATATACATA | / |
pBL_418 | AAGGAGATATACC | 28 729.01 | AAGGAGATATACATA | / |
pBL_419 | AAGGAGATATACC | 94 682.27 | AAGGAGATATACATA | / |
pBL_420 | AAGGAGATATACC | 18 153.47 | AAGGAGATATACATA | / |
pBL_421 | AAGGAGATATA | / | GCATCGTAGAAGGG | 998.41 |
pBL_422 | AAGGAGATATA | / | ACCTGGGACACAA | 510.31 |
pBL_423 | AAGGAGATATA | / | CTAAACCCTTTTGCAAA | 99.95 |
pBL_425 | AAGGGGTCCAGAAC | 10 027.16 | AAGGAGATATACATA | / |
pBL_429 | AGGTAGGGGGAC | 5 184.17 | AAGGAGATATACATA | / |
pBL_437 | TCGGAGAGAAAACAC | 10 446.34 | AAGGAGATATACATA | / |
pBL_438 | AGGGTAAACAC | 4 924.30 | AAGGAGATATACATA | / |
pBL_439 | TACAGTATTGAC | 2 004.59 | AAGGAGATATACATA | / |
pED-RBS1 | AAAAAAAGTTTTG | 995.63 | / | / |
pED-RBS2 | GAAAAAACTCAGC | 393.13 | / | / |
pED-RBS3 | AAGGAGACCAGC | 12 922.15 | / | / |
pED-RBS4 | AAAAGGAAAATAA | 7 438.00 | / | / |
表4 本研究设计的RBS序列及其TIR
Table 4 RBS sequence and its TIR designed in this study
质粒Plasmids | RBSa/RBS | 翻译起始速率TIR | RBSb | 翻译起始速率TIR |
---|---|---|---|---|
pBL_401 | AAGGAGATATACC | / | AAGGAGATATACATA | 11 602.81 |
pBL_403 | AAGGAGATATACC | / | AGAGAATTTTA | 23 837.83 |
pBL_404 | AAGGAGATATACC | / | CAGGAAGTAAGT | 17 039.41 |
pBL_405 | AAGGAGATATACC | / | CGGGGGCTCGAG | 7 953.16 |
pBL_406 | AAGGAGATATACC | / | GGGTTTCGACACAC | 4 941.17 |
pBL_407 | AAGGAGATATACC | / | AACCGAGAATTATCTC | 1 875.68 |
pBL_415 | AAGGAGATATACC | 40 994.72 | AAGGAGATATACATA | / |
pBL_416 | AAGGAGATATACC | 15 231.17 | AAGGAGATATACATA | / |
pBL_417 | AAGGAGATATACC | 51 571.35 | AAGGAGATATACATA | / |
pBL_418 | AAGGAGATATACC | 28 729.01 | AAGGAGATATACATA | / |
pBL_419 | AAGGAGATATACC | 94 682.27 | AAGGAGATATACATA | / |
pBL_420 | AAGGAGATATACC | 18 153.47 | AAGGAGATATACATA | / |
pBL_421 | AAGGAGATATA | / | GCATCGTAGAAGGG | 998.41 |
pBL_422 | AAGGAGATATA | / | ACCTGGGACACAA | 510.31 |
pBL_423 | AAGGAGATATA | / | CTAAACCCTTTTGCAAA | 99.95 |
pBL_425 | AAGGGGTCCAGAAC | 10 027.16 | AAGGAGATATACATA | / |
pBL_429 | AGGTAGGGGGAC | 5 184.17 | AAGGAGATATACATA | / |
pBL_437 | TCGGAGAGAAAACAC | 10 446.34 | AAGGAGATATACATA | / |
pBL_438 | AGGGTAAACAC | 4 924.30 | AAGGAGATATACATA | / |
pBL_439 | TACAGTATTGAC | 2 004.59 | AAGGAGATATACATA | / |
pED-RBS1 | AAAAAAAGTTTTG | 995.63 | / | / |
pED-RBS2 | GAAAAAACTCAGC | 393.13 | / | / |
pED-RBS3 | AAGGAGACCAGC | 12 922.15 | / | / |
pED-RBS4 | AAAAGGAAAATAA | 7 438.00 | / | / |
图4 EBL_401的脱氧紫色杆菌素生产曲线(A)、菌株生长曲线(B)和葡萄糖消耗曲线(C)
Fig. 4 Deoxyviolacein production curve(A), strain gro-wth curve(B), and glucose consumption curve(C)of EBL_401
图5 EBL_401, 403-407, 421-423的脱氧紫色杆菌素生产(A)、菌株生长(B)及生物传感器pBL_406的测试(C)
Fig. 5 Deoxyviolacein production(A)and the strain growth(B)of EBL_401, 403-407, 421-423 and the test of biosensor pBL_406(C)
比对蛋白Comparative protein | 相似度Identity/% | |
---|---|---|
Dug.B2_VioA | Jan_VioA | 62.5 |
Pse_VioA | 62.0 | |
Myx_VioA | 57.9 | |
Chr_VioA | 54.7 | |
Dug.HH105_VioA | 59.5 | |
Jan_VioA | Pse_VioA | 82.5 |
Myx_VioA | 74.3 | |
Chr_VioA | 66.4 | |
Dug.HH105_VioA | 81.4 | |
Pse_VioA | Myx_VioA | 73.1 |
Chr_VioA | 65.8 | |
Dug.HH105_VioA | 80.4 | |
Myx_VioA | Chr_VioA | 67.1 |
Dug.HH105_VioA | 73.8 | |
Chr_VioA | Dug.HH105_VioA | 64.9 |
表5 5种不同来源VioA的序列相似性比对
Table 5 Sequence comparison of 5 VioAs from different sources
比对蛋白Comparative protein | 相似度Identity/% | |
---|---|---|
Dug.B2_VioA | Jan_VioA | 62.5 |
Pse_VioA | 62.0 | |
Myx_VioA | 57.9 | |
Chr_VioA | 54.7 | |
Dug.HH105_VioA | 59.5 | |
Jan_VioA | Pse_VioA | 82.5 |
Myx_VioA | 74.3 | |
Chr_VioA | 66.4 | |
Dug.HH105_VioA | 81.4 | |
Pse_VioA | Myx_VioA | 73.1 |
Chr_VioA | 65.8 | |
Dug.HH105_VioA | 80.4 | |
Myx_VioA | Chr_VioA | 67.1 |
Dug.HH105_VioA | 73.8 | |
Chr_VioA | Dug.HH105_VioA | 64.9 |
图7 不同来源VioA(A)、调整RBSa(B)及生物传感器pBL_438、pBL_439的测试(C,D)
Fig. 7 Testing of different sources VioA(A), adjusted RBSa(B), and biosensors pBL_438 and pBL_439(C, D)
图8 生物传感器pBL_439在L-色氨酸产量不同的大肠杆菌中的测试A:生物传感器pBL_439响应值与L-色氨酸产量之间的相关性测试;B:肉眼观测照片及pED系列质粒示意图
Fig. 8 Testing of the biosensor pBL_439 in different E. coli strains with varying L-Trp production A: Correlation test between biosensor pBL_439 response values and L-Trp production. B: Observational photos by naked eye and schematic diagram of pED series plasmids
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