Oligonucleotide library assisted sequence mining reveals promoter sequences with distinct temporal expression dynamics for applications in Curvibacter sp. AEP1-3.

IF 2.6 Q2 BIOCHEMICAL RESEARCH METHODS
Synthetic biology (Oxford, England) Pub Date : 2025-05-21 eCollection Date: 2025-01-01 DOI:10.1093/synbio/ysaf001
Maurice Mager, Lukas Becker, Nina Schulten, Sebastian Fraune, Ilka M Axmann
{"title":"Oligonucleotide library assisted sequence mining reveals promoter sequences with distinct temporal expression dynamics for applications in <i>Curvibacter</i> sp. AEP1-3.","authors":"Maurice Mager, Lukas Becker, Nina Schulten, Sebastian Fraune, Ilka M Axmann","doi":"10.1093/synbio/ysaf001","DOIUrl":null,"url":null,"abstract":"<p><p>The <i>β-proteobacterial</i> species <i>Curvibacter</i> sp. AEP1-3 is a model organism for the study of symbiotic interactions as it is the most abundant colonizer of <i>Hydra vulgaris</i>. Yet, genetic tools for <i>Curvibacter</i> are still in their infancy; few promoters have been characterized so far. Here, we employ an oligonucleotide-based strategy to develop novel expression systems <i>Curvibacter</i>. Potential promoters were systematically mined from the genome <i>in silico</i>. The sequences were cloned as a mixed library into a mCherry reporter vector and positive candidates were selected by Flow Cytometry to be further analysed through plate reader measurements. From 500 candidate sequences, 25 were identified as active promoters of varying expression strength levels. Plate reader measurements revealed unique activity profiles for these sequences across growth phases. The expression levels of these promoters ranged over two orders of magnitudes and showed distinct temporal expression dynamics over the growth phases: while three sequences showed higher expression levels in the exponential phase, we found 12 sequences saturating expression during stationary phase and 10 that showed little discrimination between growth phases. From our library, promoters of the genes <i>dnaK, rpsL</i> and an acyl-homoserine-lactone (AHL) synthase stood out as the most interesting candidates fit for a variety of applications. We identified enriched transcription factor binding motifs among the sorted 33 sequences and genes encoding for homologs of these transcription factors in close proximity to the identified motifs. In this work, we show the value of employing comprehensive high-throughput strategies to establish expression systems for novel model organisms.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"10 1","pages":"ysaf001"},"PeriodicalIF":2.6000,"publicationDate":"2025-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12094071/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Synthetic biology (Oxford, England)","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1093/synbio/ysaf001","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/1/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

Abstract

The β-proteobacterial species Curvibacter sp. AEP1-3 is a model organism for the study of symbiotic interactions as it is the most abundant colonizer of Hydra vulgaris. Yet, genetic tools for Curvibacter are still in their infancy; few promoters have been characterized so far. Here, we employ an oligonucleotide-based strategy to develop novel expression systems Curvibacter. Potential promoters were systematically mined from the genome in silico. The sequences were cloned as a mixed library into a mCherry reporter vector and positive candidates were selected by Flow Cytometry to be further analysed through plate reader measurements. From 500 candidate sequences, 25 were identified as active promoters of varying expression strength levels. Plate reader measurements revealed unique activity profiles for these sequences across growth phases. The expression levels of these promoters ranged over two orders of magnitudes and showed distinct temporal expression dynamics over the growth phases: while three sequences showed higher expression levels in the exponential phase, we found 12 sequences saturating expression during stationary phase and 10 that showed little discrimination between growth phases. From our library, promoters of the genes dnaK, rpsL and an acyl-homoserine-lactone (AHL) synthase stood out as the most interesting candidates fit for a variety of applications. We identified enriched transcription factor binding motifs among the sorted 33 sequences and genes encoding for homologs of these transcription factors in close proximity to the identified motifs. In this work, we show the value of employing comprehensive high-throughput strategies to establish expression systems for novel model organisms.

寡核苷酸库辅助序列挖掘揭示了具有不同时间表达动态的启动子序列在Curvibacter sp. AEP1-3中的应用。
β-变形菌Curvibacter sp. AEP1-3是水螅(Hydra vulgaris)最丰富的定殖菌,是研究共生相互作用的模式生物。然而,弯曲杆菌的遗传工具仍处于起步阶段;到目前为止,很少有启动子被描述出来。在这里,我们采用基于寡核苷酸的策略来开发新的表达系统曲线杆菌。通过计算机系统地从基因组中挖掘潜在的启动子。将这些序列作为混合文库克隆到mCherry报告载体中,并通过流式细胞术选择阳性候选序列,通过平板阅读器测量进一步分析。从500个候选序列中,鉴定出25个不同表达强度水平的活性启动子。平板阅读器测量揭示了这些序列在生长阶段的独特活动概况。这些启动子的表达量在两个数量级以上,并且在生长阶段表现出明显的时间表达动态:其中3个序列在指数期表现出较高的表达水平,我们发现12个序列在稳定期表达饱和,10个序列在生长阶段之间表现出不明显的差异。从我们的文库中,基因dnaK, rpsL和酰基-同丝氨酸-内酯(AHL)合成酶的启动子脱颖而出,成为适合各种应用的最有趣的候选基因。我们在分类的33个序列中发现了丰富的转录因子结合基序,以及这些转录因子的同源编码基因在鉴定基序附近。在这项工作中,我们展示了采用综合高通量策略建立新型模式生物表达系统的价值。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
自引率
0.00%
发文量
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信