• 研究论文 •
大肠杆菌中终止子对下游转录单元基因表达的影响
任家卫1,2, 张金鹏1,2, 徐国强1,2, 张晓梅3, 许正宏4, 张晓娟1,2
- 1.江南大学，生物工程学院工业生物技术教育部重点实验室，江苏 无锡 214122
2.江南大学，粮食发酵与食品生物制造国家工程研究中心，江苏 无锡 214122
3.江南大学，生命科学与健康工程学院，江苏 无锡 214122
4.四川大学 轻工科学与工程学院，四川 成都 610065
-
收稿日期:2024-06-17修回日期:2024-08-22出版日期:2024-08-23 -
通讯作者:张晓娟 -
作者简介:任家卫 （1999—），男，硕士。研究方向为终止子调控元件开发研究。 E-mail：297887816@qq.com张晓娟 （1982—），女，教授。研究方向为酿造食品微生物开发与应用、新型工业菌株的基因表达调控元件开发研究。 E-mail：zhangxj@jiangnan.edu.cn -
基金资助:国家自然科学基金(32171421)
Effect of terminators in Escherichia coli on gene expression in downstream transcription unit
Jiawei REN1,2, Jinpeng ZHANG1,2, Guoqiang XU1,2, Xiaomei ZHANG3, Zhenghong XU4, Xiaojuan ZHANG1,2
- 1.Jiangnan University，Key Laboratory of Industrial Biotechnology，Ministry of Education，，Wuxi 214122，China
2.Jiangnan University，National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing，Wuxi 214122，China
3.Jiangsu Engineering Research Center for Bioactive Products Processing Technology，Wuxi 214122，China
4.Sichuan University，College of Light Industry Science and Engineering，Chengdu 610065，China
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Received:2024-06-17Revised:2024-08-22Online:2024-08-23 -
Contact:Xiaojuan ZHANG
摘要：
在基因转录过程中，RNA聚合酶通过识别启动子序列启动转录过程，而当其识别到位于3'-UTR的终止子序列后，转录复合物解离，转录过程终止。因此，转录单元内的启动子和终止子分别发挥启动和终止转录的作用。然而，对于下游的转录单元，终止子除了终止转录本通读这一直接作用以外，其与RNA聚合酶之间的解离可能会影响后续转录单元中启动子与RNA聚合酶的结合，从而间接改变下游转录单元的表达。这种跨转录单元的终止子和启动子之间的互作关系目前尚缺少研究，因此，明确终止子对下游转录单元的转录反应强度的影响对于精准调控基因表达，开发高效终止子具有重要的意义。本研究通过one-pot技术将9种终止子、5种间隔序列和9种启动子进行组合构建了一个包含405种不同组合元件（终止子-间隔序列-启动子）的组装文库，基于FlowSeq技术对文库中所有组合进行测序和荧光强度分析，进而建立组合序列-下游基因表达之间的相关性。结果表明弱终止子、短间隔以及强启动子的组合更有利于提高下游基因的表达。通过对转录本的定量分析发现弱终止子不仅提高下游的渗漏转录（提高21-70倍），同时也促进了下游启动子重新招募RNA聚合酶进行重启转录（提高2-3倍）。本研究解析了终止子对下游转录单元基因表达的调控效果和机制，为利用终止子构建基因回路提供设计依据。
中图分类号:
引用本文
任家卫, 张金鹏, 徐国强, 张晓梅, 许正宏, 张晓娟. 大肠杆菌中终止子对下游转录单元基因表达的影响[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-046.
Jiawei REN, Jinpeng ZHANG, Guoqiang XU, Xiaomei ZHANG, Zhenghong XU, Xiaojuan ZHANG. Effect of terminators in Escherichia coli on gene expression in downstream transcription unit[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-046.
表1 本研究所涉及的菌株和质粒信息
Tab. 1 Strains and plasmids used in this study
| Name | Description | Source |
|---|---|---|
| Escherichia coli JM109 | EUROSCARF | |
| Escherichia coli BL21 | EUROSCARF | |
| PTK | Dual Fluorescent Probe Plasmid，Kanr, T7 promoter, EGFP, mRFP1, T7 terminator | this study |
| PTK-pot | PTK derivatives carrying one-pot assembly libraries | this study |
| PTK-pheA-13-e | PTK derivatives carrying the terminator pheA, the spacer sequence sp-13, and the promoter dap-e | this study |
| PTK-pheA-13-e12 | PTK derivatives carrying the terminator pheA, the spacer sequence sp-13, and the promoter dap-e12 | this study |
| PTK-pheA-40-e | PTK derivatives carrying the terminator pheA, the spacer sequence sp-40, and the promoter dap-e | this study |
| PTK-pheA-40-e12 | PTK derivatives carrying the terminator pheA, the spacer sequence sp-40, and the promoter dap-e12 | this study |
| PTK-recA-13-e | PTK derivatives carrying the terminator recA, the spacer sequence sp-13 and the promoter dap-e | this study |
| PTK-recA-13-e12 | PTK derivatives carrying the terminator recA, the spacer sequence sp-13 and the promoter dap-e12 | this study |
| PTK-recA-40-e | PTK derivatives carrying the terminator recA, the spacer sequence sp-40 and the promoter dap-e | this study |
| PTK-recA-40-e12 | PTK derivatives carrying the terminator recA, the spacer sequence sp-40 and the promoter dap-e12 | this study |
表1 本研究所涉及的菌株和质粒信息
Tab. 1 Strains and plasmids used in this study
| Name | Description | Source |
|---|---|---|
| Escherichia coli JM109 | EUROSCARF | |
| Escherichia coli BL21 | EUROSCARF | |
| PTK | Dual Fluorescent Probe Plasmid，Kanr, T7 promoter, EGFP, mRFP1, T7 terminator | this study |
| PTK-pot | PTK derivatives carrying one-pot assembly libraries | this study |
| PTK-pheA-13-e | PTK derivatives carrying the terminator pheA, the spacer sequence sp-13, and the promoter dap-e | this study |
| PTK-pheA-13-e12 | PTK derivatives carrying the terminator pheA, the spacer sequence sp-13, and the promoter dap-e12 | this study |
| PTK-pheA-40-e | PTK derivatives carrying the terminator pheA, the spacer sequence sp-40, and the promoter dap-e | this study |
| PTK-pheA-40-e12 | PTK derivatives carrying the terminator pheA, the spacer sequence sp-40, and the promoter dap-e12 | this study |
| PTK-recA-13-e | PTK derivatives carrying the terminator recA, the spacer sequence sp-13 and the promoter dap-e | this study |
| PTK-recA-13-e12 | PTK derivatives carrying the terminator recA, the spacer sequence sp-13 and the promoter dap-e12 | this study |
| PTK-recA-40-e | PTK derivatives carrying the terminator recA, the spacer sequence sp-40 and the promoter dap-e | this study |
| PTK-recA-40-e12 | PTK derivatives carrying the terminator recA, the spacer sequence sp-40 and the promoter dap-e12 | this study |
表2 本实验所用的引物
Tab. 2 Primers used in this study
| Primer | Description |
|---|---|
| pheA-F | TAGCAACAATAAGGCCTCCCAAATCGGGGGGCCTTTTTTATTGAT |
| pheA-R | TTAGATCAATAAAAAAGGCCCCCCGATTTGGGAGGCCTTATTGTT |
| thrL-F | TAGCTCAAAAAAGCCCGCACCTGACAGTGCGGGCTTTTTTTTTACT |
| thrL-R | TTAGAGTAAAAAAAAAGCCCGCACTGTCAGGTGCGGGCTTTTTTGA |
| rpsO-F | TAGCCAGAAAAGGGGGCCTGAGTGGCCCCTTTTTTCAAGCT |
| rpsO-R | TTAGAGCTTGAAAAAAGGGGCCACTCAGGCCCCCTTTTCTG |
| arcA-F | TAGCAATAAAAACGGCGCTAAAAAGCGCCGTTTTTTTTGACG |
| arcA-R | TTAGCGTCAAAAAAAACGGCGCTTTTTAGCGCCGTTTTTATT |
| fhuE-F | TAGCGTAAAAAAGGCAGCCATCTGGCTGCCTTAGTCTCCCCA |
| fhuE-R | TTAGTGGGGAGACTAAGGCAGCCAGATGGCTGCCTTTTTTAC |
| hisI-F | TAGCCCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGT |
| hisI-R | TTAGACGCGAAGTAATCTTTTCGGTTTTAAAGAAAAAGGGCAGGG |
| recA-F | TAGCAAGCAAAAGGGCCGCAGATGCGACCCTTGTGTATCAAC |
| recA-R | TTAGGTTGATACACAAGGGTCGCATCTGCGGCCCTTTTGCTT |
| T7-MOD-F | TAGCAAACAGATAGGCCCTCttcgGAGGGCCtatctgttTTTTTTT |
| T7-MOD-R | TTAGAAAAAAAaacagataGGCCCTCcgaaGAGGGCCTATCTGTTT |
| SP-TE-F | TAGCCCAATGTTTACTCATATCCAGTCACAGAAACTGAACTATC |
| SP-TE-R | TTAGGATAGTTCAGTTTCTGTGACTGGATATGAGTAAACATTGG |
| SPACE-5-F | CTAACTATC |
| SPACE-5-R | TTATGATAG |
| SPACE-13-F | CTAAAAACTGAACTATC |
| SPACE-13-R | TTATGATAGTTCAGTTT |
| SPACE-20-F | CTAAGTCACAGAAACTGAACTATC |
| SPACE-20-R | TTATGATAGTTCAGTTTCTGTGAC |
| SPACE-28-F | CTAACATATCCAGTCACAGAAACTGAACTATC |
| SPACE-28-R | TTATGATAGTTCAGTTTCTGTGACTGGATATG |
| SPACE-35-F | CTAAGTTTACTCATATCCAGTCACAGAAACTGAACTATC |
| SPACE-35-R | TTATGATAGTTCAGTTTCTGTGACTGGATATGAGTAAAC |
| SPACE-40-F | CTAACCAATGTTTACTCATATCCAGTCACAGAAACTGAACTATC |
| SPACE-40-R | TTATGATAGTTCAGTTTCTGTGACTGGATATGAGTAAACATTGG |
| dap-A16-F | ATAATTGTTTAACCCCCAAATGAGGGAAGAAGGTATAATTGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dap-A16-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCAATTATACCTTCTTCCCTCATTTGGGGGTTAAACAA |
| dap-e-F | ATAATTGTTTAGCCACCAAATGAGGGAAAGAGGCACAATGGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dap-e-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCCATTGTGCCTCTTTCCCTCATTTGGTGGCTAAACAA |
| dapA-e10-F | ATAATTGTTTTGACACCAAATGAGGGAATGTGGTATAATTGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dapA-e10-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCAATTATACCACATTCCCTCATTTGGTGTCAAAACAA |
| dapA-e11-F | ATAATTGTTTTGACACCAAATGAGGGAATGTGCTATAATGGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dapA-e11-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCCATTATAGCACATTCCCTCATTTGGTGTCAAAACAA |
| dapA-e12-F | ATAATTGTTTTGACACCAAATGAGGGAATGTGGTAGAGTGGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dapA-e12-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCCACTCTACCACATTCCCTCATTTGGTGTCAAAACAA |
| dapA-e10-35-F | ATAATTGTTTAACCCCCAAATGAGGGAATGTGGTATAATTGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dapA-e10-35-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCAATTATACCACATTCCCTCATTTGGGGGTTAAACAA |
| J1-F | ATAATTGACAATTTTCTTAAATTGTGTTACAATGGGTTTCGCTCAAGGCGCAAGGAGCACACACA |
| J1-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAAACCCATTGTAACACAATTTAAGAAAATTGTCAA |
| J2-F | ATAATTGACATTTTTTTAGTTTTGAGTTACAATGGTTGTCGCTCAAGGCGCAAGGAGCACACACA |
| J2-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGACAACCATTGTAACTCAAAACTAAAAAAATGTCAA |
| SP-PRO-F | ATAAGTTTACTCATATCCAGTCACAGAAACTGAACTATCTCGCTCAAGGCGCAAGGAGCACACACA |
| SP-TRO-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGATAGTTCAGTTTCTGTGACTGGATATGAGTAAAC |
| T7-CG-F | AATTCCGGCCGCGGGGCCCGCTTCGGCGGGCCCCGCGGCCGTTTTTTTAAACTGAACTATCG |
| T7-CG-R | GATCCGATAGTTCAGTTTAAAAAAACGGCCGCGGGGCCCGCCGAAGCGGGCCCCGCGGCCGG |
| WEAK-F | AATTCAGCCCCTCAGTATAGGGGCGTATTTTCTCAAACTGAACTATCG |
| WEAK-R | GATCCGATAGTTCAGTTTGAGAAAATACGCCCCTATACTGAGGGGCTG |
表2 本实验所用的引物
Tab. 2 Primers used in this study
| Primer | Description |
|---|---|
| pheA-F | TAGCAACAATAAGGCCTCCCAAATCGGGGGGCCTTTTTTATTGAT |
| pheA-R | TTAGATCAATAAAAAAGGCCCCCCGATTTGGGAGGCCTTATTGTT |
| thrL-F | TAGCTCAAAAAAGCCCGCACCTGACAGTGCGGGCTTTTTTTTTACT |
| thrL-R | TTAGAGTAAAAAAAAAGCCCGCACTGTCAGGTGCGGGCTTTTTTGA |
| rpsO-F | TAGCCAGAAAAGGGGGCCTGAGTGGCCCCTTTTTTCAAGCT |
| rpsO-R | TTAGAGCTTGAAAAAAGGGGCCACTCAGGCCCCCTTTTCTG |
| arcA-F | TAGCAATAAAAACGGCGCTAAAAAGCGCCGTTTTTTTTGACG |
| arcA-R | TTAGCGTCAAAAAAAACGGCGCTTTTTAGCGCCGTTTTTATT |
| fhuE-F | TAGCGTAAAAAAGGCAGCCATCTGGCTGCCTTAGTCTCCCCA |
| fhuE-R | TTAGTGGGGAGACTAAGGCAGCCAGATGGCTGCCTTTTTTAC |
| hisI-F | TAGCCCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGT |
| hisI-R | TTAGACGCGAAGTAATCTTTTCGGTTTTAAAGAAAAAGGGCAGGG |
| recA-F | TAGCAAGCAAAAGGGCCGCAGATGCGACCCTTGTGTATCAAC |
| recA-R | TTAGGTTGATACACAAGGGTCGCATCTGCGGCCCTTTTGCTT |
| T7-MOD-F | TAGCAAACAGATAGGCCCTCttcgGAGGGCCtatctgttTTTTTTT |
| T7-MOD-R | TTAGAAAAAAAaacagataGGCCCTCcgaaGAGGGCCTATCTGTTT |
| SP-TE-F | TAGCCCAATGTTTACTCATATCCAGTCACAGAAACTGAACTATC |
| SP-TE-R | TTAGGATAGTTCAGTTTCTGTGACTGGATATGAGTAAACATTGG |
| SPACE-5-F | CTAACTATC |
| SPACE-5-R | TTATGATAG |
| SPACE-13-F | CTAAAAACTGAACTATC |
| SPACE-13-R | TTATGATAGTTCAGTTT |
| SPACE-20-F | CTAAGTCACAGAAACTGAACTATC |
| SPACE-20-R | TTATGATAGTTCAGTTTCTGTGAC |
| SPACE-28-F | CTAACATATCCAGTCACAGAAACTGAACTATC |
| SPACE-28-R | TTATGATAGTTCAGTTTCTGTGACTGGATATG |
| SPACE-35-F | CTAAGTTTACTCATATCCAGTCACAGAAACTGAACTATC |
| SPACE-35-R | TTATGATAGTTCAGTTTCTGTGACTGGATATGAGTAAAC |
| SPACE-40-F | CTAACCAATGTTTACTCATATCCAGTCACAGAAACTGAACTATC |
| SPACE-40-R | TTATGATAGTTCAGTTTCTGTGACTGGATATGAGTAAACATTGG |
| dap-A16-F | ATAATTGTTTAACCCCCAAATGAGGGAAGAAGGTATAATTGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dap-A16-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCAATTATACCTTCTTCCCTCATTTGGGGGTTAAACAA |
| dap-e-F | ATAATTGTTTAGCCACCAAATGAGGGAAAGAGGCACAATGGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dap-e-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCCATTGTGCCTCTTTCCCTCATTTGGTGGCTAAACAA |
| dapA-e10-F | ATAATTGTTTTGACACCAAATGAGGGAATGTGGTATAATTGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dapA-e10-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCAATTATACCACATTCCCTCATTTGGTGTCAAAACAA |
| dapA-e11-F | ATAATTGTTTTGACACCAAATGAGGGAATGTGCTATAATGGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dapA-e11-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCCATTATAGCACATTCCCTCATTTGGTGTCAAAACAA |
| dapA-e12-F | ATAATTGTTTTGACACCAAATGAGGGAATGTGGTAGAGTGGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dapA-e12-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCCACTCTACCACATTCCCTCATTTGGTGTCAAAACAA |
| dapA-e10-35-F | ATAATTGTTTAACCCCCAAATGAGGGAATGTGGTATAATTGAACTCTCGCTCAAGGCGCAAGGAGCACACACA |
| dapA-e10-35-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGAGTTCAATTATACCACATTCCCTCATTTGGGGGTTAAACAA |
| J1-F | ATAATTGACAATTTTCTTAAATTGTGTTACAATGGGTTTCGCTCAAGGCGCAAGGAGCACACACA |
| J1-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAAACCCATTGTAACACAATTTAAGAAAATTGTCAA |
| J2-F | ATAATTGACATTTTTTTAGTTTTGAGTTACAATGGTTGTCGCTCAAGGCGCAAGGAGCACACACA |
| J2-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGACAACCATTGTAACTCAAAACTAAAAAAATGTCAA |
| SP-PRO-F | ATAAGTTTACTCATATCCAGTCACAGAAACTGAACTATCTCGCTCAAGGCGCAAGGAGCACACACA |
| SP-TRO-R | TCATTGTGTGTGCTCCTTGCGCCTTGAGCGAGATAGTTCAGTTTCTGTGACTGGATATGAGTAAAC |
| T7-CG-F | AATTCCGGCCGCGGGGCCCGCTTCGGCGGGCCCCGCGGCCGTTTTTTTAAACTGAACTATCG |
| T7-CG-R | GATCCGATAGTTCAGTTTAAAAAAACGGCCGCGGGGCCCGCCGAAGCGGGCCCCGCGGCCGG |
| WEAK-F | AATTCAGCCCCTCAGTATAGGGGCGTATTTTCTCAAACTGAACTATCG |
| WEAK-R | GATCCGATAGTTCAGTTTGAGAAAATACGCCCCTATACTGAGGGGCTG |
图1 不同强度终止子和启动子组合调控效果质粒中上游和下游转录单元结构示意图（a）以及9种突变质粒中下游转录单元红色荧光转录水平（b）和表达水平 （c）
Fig. 1 Analysis of the regulatory effects of combinations of terminators and promoters of different strengths(Schematic structure of upstream and downstream transcriptional units in the plasmid (a) and red fluorescent transcript levels (b) and expression levels (c) of downstream transcriptional units in the nine mutant plasmids)
图1 不同强度终止子和启动子组合调控效果质粒中上游和下游转录单元结构示意图（a）以及9种突变质粒中下游转录单元红色荧光转录水平（b）和表达水平 （c）
Fig. 1 Analysis of the regulatory effects of combinations of terminators and promoters of different strengths(Schematic structure of upstream and downstream transcriptional units in the plasmid (a) and red fluorescent transcript levels (b) and expression levels (c) of downstream transcriptional units in the nine mutant plasmids)
表3 文库中包含的组件
Tab. 3 Parts included in the library
| Assembly | Part Name | Activity | Sequence（5'→3'） |
|---|---|---|---|
| Terminator | pheA | Strong ↓ weak | AATCATAAGGCCTGACAAATCGGGGGGGATTTTTTCTTGA |
| thrL | TCCAAAAAGCCCCGACCTGACAGTGACGGCTTTTTTATTAC | ||
| T7-MOD | AAACAGATAGGCCCTCTTCGGAGGGCCTATCTGTTTTTTTTT | ||
| arcA | AATATAAACGGCGCTAAAACGCGCGGTTTTTTTTGCC | ||
| rpsO | CAGCAAAGGGTGCCTGAGTGCTCCCTTTTTGCAAGC | ||
| fhuE | GTAAAAAAGGCAGCCATCTGGCTGCCTTAGTCTCCCCA | ||
| hisI | CCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGT | ||
| recA | AAGCAAAAGGGCCGCAGATGCGACCCTTGTGTATCAAC | ||
| SP-TE | CCAATGTTTACTCATATCCAGTCACAGAAACTGAACTATC | ||
| Spacer | SP-13 | Short ↓ Long | AAACTGAACTATC |
| SP-20 | GTCACAGAAACTGAACTATC | ||
| SP-28 | CATATCCAGTCACAGAAACTGAACTATC | ||
| SP-35 | GTTTACTCATATCCAGTCACAGAAACTGAACTATC | ||
| SP-40 | CCAATGTTTACTCATATCCAGTCACAGAAACTGAACTATC | ||
| Promoter | dapA-e12 | Strong ↓ weak | TTGTTTTGACACCAAATGAGGGAATGTGGTAGAGTGGAACTC |
| dapA-e11 | TTGTTTTGACACCAAATGAGGGAATGTGCTATAATGGAACTC | ||
| dapA-e10 | TTGTTTTGACACCAAATGAGGGAATGTGGTATAATTGAACTC | ||
| dap-A16 | TAGGTTTTTTGCGGGGTTGTTTAACCCCCAAATGAGGGAAGAAGGTATAATTGAACTC | ||
| J2 | TTGACATTTTTTTAGTTTTGAGTTACAATGGTTG | ||
| dapA-e10-35 | TTGTTTAACCCCCAAATGAGGGAATGTGGTATAATTGAACTC | ||
| J1 | TTGACAATTTTCTTAAATTGTGTTACAATGGGTT | ||
| dap-e | TTGTTTAGCCACCAAATGAGGGAAAGAGGCACAATGGAACTC | ||
| SP-PRO | GTTTACTCATATCCAGTCACAGAAACTGAAC |
表3 文库中包含的组件
Tab. 3 Parts included in the library
| Assembly | Part Name | Activity | Sequence（5'→3'） |
|---|---|---|---|
| Terminator | pheA | Strong ↓ weak | AATCATAAGGCCTGACAAATCGGGGGGGATTTTTTCTTGA |
| thrL | TCCAAAAAGCCCCGACCTGACAGTGACGGCTTTTTTATTAC | ||
| T7-MOD | AAACAGATAGGCCCTCTTCGGAGGGCCTATCTGTTTTTTTTT | ||
| arcA | AATATAAACGGCGCTAAAACGCGCGGTTTTTTTTGCC | ||
| rpsO | CAGCAAAGGGTGCCTGAGTGCTCCCTTTTTGCAAGC | ||
| fhuE | GTAAAAAAGGCAGCCATCTGGCTGCCTTAGTCTCCCCA | ||
| hisI | CCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGT | ||
| recA | AAGCAAAAGGGCCGCAGATGCGACCCTTGTGTATCAAC | ||
| SP-TE | CCAATGTTTACTCATATCCAGTCACAGAAACTGAACTATC | ||
| Spacer | SP-13 | Short ↓ Long | AAACTGAACTATC |
| SP-20 | GTCACAGAAACTGAACTATC | ||
| SP-28 | CATATCCAGTCACAGAAACTGAACTATC | ||
| SP-35 | GTTTACTCATATCCAGTCACAGAAACTGAACTATC | ||
| SP-40 | CCAATGTTTACTCATATCCAGTCACAGAAACTGAACTATC | ||
| Promoter | dapA-e12 | Strong ↓ weak | TTGTTTTGACACCAAATGAGGGAATGTGGTAGAGTGGAACTC |
| dapA-e11 | TTGTTTTGACACCAAATGAGGGAATGTGCTATAATGGAACTC | ||
| dapA-e10 | TTGTTTTGACACCAAATGAGGGAATGTGGTATAATTGAACTC | ||
| dap-A16 | TAGGTTTTTTGCGGGGTTGTTTAACCCCCAAATGAGGGAAGAAGGTATAATTGAACTC | ||
| J2 | TTGACATTTTTTTAGTTTTGAGTTACAATGGTTG | ||
| dapA-e10-35 | TTGTTTAACCCCCAAATGAGGGAATGTGGTATAATTGAACTC | ||
| J1 | TTGACAATTTTCTTAAATTGTGTTACAATGGGTT | ||
| dap-e | TTGTTTAGCCACCAAATGAGGGAAAGAGGCACAATGGAACTC | ||
| SP-PRO | GTTTACTCATATCCAGTCACAGAAACTGAAC |
图2 组合调控元件（终止子-间隔-启动子）调控的下游基因表达强度（a） 根据下游红色荧光表达水平对包含组合元件的细胞进行流式筛选；（b） 根据下游相对荧光加权强度对组合元件进行高表达、中表达、低表达分组
Fig. 2 Downstream gene expression regμLated by assemblies of terminator-spacer-promoter(a) Screening of combinatorial elements based on downstream red fluorescence expression levels by Flow cytometry; (b) Grouping of assemblies of terminator-spacer-promoter into High, Medium, and Low groups based on downstream relative fluorescence intensity of mRFP1
图2 组合调控元件（终止子-间隔-启动子）调控的下游基因表达强度（a） 根据下游红色荧光表达水平对包含组合元件的细胞进行流式筛选；（b） 根据下游相对荧光加权强度对组合元件进行高表达、中表达、低表达分组
Fig. 2 Downstream gene expression regμLated by assemblies of terminator-spacer-promoter(a) Screening of combinatorial elements based on downstream red fluorescence expression levels by Flow cytometry; (b) Grouping of assemblies of terminator-spacer-promoter into High, Medium, and Low groups based on downstream relative fluorescence intensity of mRFP1
图3 组件中的各元件在高、中和低表达组的出现频率（a）高、中和低表达组中不同元件出现频率的叠加；（b）高、中和低表达组中不同元件出现频率的三元图
Fig. 3 Frequency of each part in the assemblies at the "High", "Medium" and "Low" groups(a) Stacked histograms of the frequency of occurrence of different components in the "High", "Medium" and "Low" groups; (b) Ternary results of the frequency of occurrence of different components in the "High", "Medium" and "Low"groups
图3 组件中的各元件在高、中和低表达组的出现频率（a）高、中和低表达组中不同元件出现频率的叠加；（b）高、中和低表达组中不同元件出现频率的三元图
Fig. 3 Frequency of each part in the assemblies at the "High", "Medium" and "Low" groups(a) Stacked histograms of the frequency of occurrence of different components in the "High", "Medium" and "Low" groups; (b) Ternary results of the frequency of occurrence of different components in the "High", "Medium" and "Low"groups
图4 典型组合元件调控的下游基因mRNA水平（a）和红色荧光蛋白表达水平（b）（强弱启动子分别为dap-e11 和 dap-e， 强弱终止子分别为pheA和recA，长短间隔序列分别为sp-40和sp-13）
Fig. 4 mRNA and red fluorescent protein levels of downstream transcription unit regulated by typical assemblies selected from one-pot assembly library(The strong and weak promoters were dap-e11 and dap-e, the strong and weak terminators were pheA and recA, and the long and short interval sequences were sp-40 and sp-13, respectively)
图4 典型组合元件调控的下游基因mRNA水平（a）和红色荧光蛋白表达水平（b）（强弱启动子分别为dap-e11 和 dap-e， 强弱终止子分别为pheA和recA，长短间隔序列分别为sp-40和sp-13）
Fig. 4 mRNA and red fluorescent protein levels of downstream transcription unit regulated by typical assemblies selected from one-pot assembly library(The strong and weak promoters were dap-e11 and dap-e, the strong and weak terminators were pheA and recA, and the long and short interval sequences were sp-40 and sp-13, respectively)
图5 不同强度启动子和终止子组合对下游转录单元mRNA水平和荧光蛋白的影响（a） 固定间隔序列的情况下不同终止子和启动子组合对基因表达的影响；（b） 固定启动子的情况下不同间隔序列和终止子组合对基因表达的影响；；（c） 固定终止子的情况下不同启动子和启动子组合对基因表达的影响；强弱启动子分别为dap-e11 和 dap-e， 强弱终止子分别为pheA和recA，长短间隔序列分别为sp-40和sp-13
Fig.5 Influence of the combination of promoters and terminators on the mRNA level and fluorescence protein(a) The effect of different terminator and promoter combinations on gene expression with fixed interval sequences; (b) The effects of different spacer sequences and terminator combinations on gene expression with fixed promoters; (c) The effects of different promoters and combinations of promoters on gene expression with fixed terminators; The strong and weak promoters were dap-e11 and dap-e, the strong and weak terminators were pheA and recA, and the long and short interval sequences were sp-40 and sp-13, respectively
图5 不同强度启动子和终止子组合对下游转录单元mRNA水平和荧光蛋白的影响（a） 固定间隔序列的情况下不同终止子和启动子组合对基因表达的影响；（b） 固定启动子的情况下不同间隔序列和终止子组合对基因表达的影响；；（c） 固定终止子的情况下不同启动子和启动子组合对基因表达的影响；强弱启动子分别为dap-e11 和 dap-e， 强弱终止子分别为pheA和recA，长短间隔序列分别为sp-40和sp-13
Fig.5 Influence of the combination of promoters and terminators on the mRNA level and fluorescence protein(a) The effect of different terminator and promoter combinations on gene expression with fixed interval sequences; (b) The effects of different spacer sequences and terminator combinations on gene expression with fixed promoters; (c) The effects of different promoters and combinations of promoters on gene expression with fixed terminators; The strong and weak promoters were dap-e11 and dap-e, the strong and weak terminators were pheA and recA, and the long and short interval sequences were sp-40 and sp-13, respectively
图6 不同组合元件调控下的下游整体mRNA、渗漏转录mRNA以及重启mRNA水平分析（a） 通过不同引物的qPCR获得渗漏转录mRNA和重启转录mRNA示意图；（b） 不同组合元件调控下的下游mRNA水平；（c） 不同组合元件调控下的渗漏转录mRNA水平；（d） 渗漏转录mRNA在下游mRNA中的占比；（e） 不同组合元件调控下的重启转录的mRNA水平；强弱启动子分别为dap-e11 和 dap-e， 强弱终止子分别为pheA和recA，长短间隔序列分别为sp-40和sp-13
Fig.6 Schematic representation of downstream mRNA, leakage transcription mRNA, and re-promotered mRNA levels under the regulation of different combinatorial elements.(a) Schematic representation of leakage transcription mRNA and re-promotered mRNA; (b) Downstream mRNA levels under the regμLation of different combinatorial elements; (c) Leakage transcription mRNA levels under the regμLation of different combinatorial elements; (d) Percentage of Leakage transcription mRNA in downstream mRNA; (e) Re-promotered mRNA levels under the regulation of different combinatorial elements; The strong and weak promoters were dap-e11 and dap-e, the strong and weak terminators were pheA and recA, and the long and short interval sequences were sp-40 and sp-13, respectively
图6 不同组合元件调控下的下游整体mRNA、渗漏转录mRNA以及重启mRNA水平分析（a） 通过不同引物的qPCR获得渗漏转录mRNA和重启转录mRNA示意图；（b） 不同组合元件调控下的下游mRNA水平；（c） 不同组合元件调控下的渗漏转录mRNA水平；（d） 渗漏转录mRNA在下游mRNA中的占比；（e） 不同组合元件调控下的重启转录的mRNA水平；强弱启动子分别为dap-e11 和 dap-e， 强弱终止子分别为pheA和recA，长短间隔序列分别为sp-40和sp-13
Fig.6 Schematic representation of downstream mRNA, leakage transcription mRNA, and re-promotered mRNA levels under the regulation of different combinatorial elements.(a) Schematic representation of leakage transcription mRNA and re-promotered mRNA; (b) Downstream mRNA levels under the regμLation of different combinatorial elements; (c) Leakage transcription mRNA levels under the regμLation of different combinatorial elements; (d) Percentage of Leakage transcription mRNA in downstream mRNA; (e) Re-promotered mRNA levels under the regulation of different combinatorial elements; The strong and weak promoters were dap-e11 and dap-e, the strong and weak terminators were pheA and recA, and the long and short interval sequences were sp-40 and sp-13, respectively
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