Construction and model-based analysis of a promoter library for E. coli: an indispensable tool for metabolic engineering.
Construction and model-based analysis of a promoter library for E. coli: an indispensable tool for metabolic engineering.
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DOI：
10.1186/1472-6750-7-34
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发表时间：
2007-06-18
影响因子：
3.5
通讯作者：
Vandamme EJ
中科院分区：
文献类型：
--
作者：
De Mey M;Maertens J;Lequeux GJ;Soetaert WK;Vandamme EJ
目前，代谢工程研究的重点正在从基因的大量过表达和失活转向基于模型的基因表达微调。在这种情况下，大肠杆菌的合成启动子库的建设作为一个有用的工具微调基因表达进行了讨论。编码大肠杆菌共有序列的简并寡核苷酸序列。设计并合成了由随机序列间隔区分隔的大肠杆菌启动子。该序列长57 bp，包含24个保守核苷酸、13个半保守核苷酸（W、R和D）和20个随机核苷酸。将该DNA片段的混合物克隆到启动子探测载体（pVIK 165）中。将连接混合物转化到感受态E. coliMA8，通过测定相对荧光单位筛选GFP活性，发现部分克隆产生高荧光强度，另一些克隆产生弱荧光强度。克隆覆盖了从21.79 RFU/OD 600 ml到7606.83 RFU/OD 600 ml的启动子活性范围。对57个启动子进行测序并用于启动子分析。目前的结果最终表明，将启动子强度与-10盒和/或-35盒中的异常以及间隔区的长度联系起来的假设通常是无效的。然而，通过应用偏最小二乘回归，建立并验证了描述启动子强度的模型。对于大肠杆菌，启动子强度不能与-10盒和/或-35盒中的异常以及间隔区的长度相关联。将启动子序列与其强度相关联的概率方法也具有一些缺点。然而，通过应用偏最小二乘回归，发现启动子序列和启动子强度之间具有良好的相关性。该PLS模型可以为合理设计合适的启动子以微调基因表达提供有用的工具。
Nowadays, the focus in metabolic engineering research is shifting from massive overexpression and inactivation of genes towards the model-based fine tuning of gene expression. In this context, the construction of a library of synthetic promoters of Escherichia coli as a useful tool for fine tuning gene expression is discussed here. A degenerated oligonucleotide sequence that encodes consensus sequences for E. coli promoters separated by spacers of random sequences has been designed and synthesized. This 57 bp long sequence contains 24 conserved, 13 semi-conserved (W, R and D) and 20 random nucleotides. This mixture of DNA fragments was cloned into a promoter probing vector (pVIK165). The ligation mixtures were transformed into competent E. coli MA8 and the resulting clones were screened for GFP activity by measuring the relative fluorescence units; some clones produced high fluorescence intensity, others weak fluorescence intensity. The clones cover a range of promoter activities from 21.79 RFU/OD600 ml to 7606.83 RFU/OD600 ml. 57 promoters were sequenced and used for promoter analysis. The present results conclusively show that the postulates, which link promoter strength to anomalies in the -10 box and/or -35 box, and to the length of the spacer, are not generally valid. However, by applying Partial Least Squares regression, a model describing the promoter strength was built and validated. For Escherichia coli, the promoter strength can not been linked to anomalies in the -10 box and/or -35 box, and to the length of the spacer. Also a probabilistic approach to relate the promoter sequence to its strength has some drawbacks. However, by applying Partial Least Squares regression, a good correlation was found between promoter sequence and promoter strength. This PLS model can be a useful tool to rationally design a suitable promoter in order to fine tune gene expression.
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DOI：
10.1016/s1387-2656(08)70049-8
发表时间：
1995-01-01
期刊：
Biotechnology annual review
影响因子：
--
作者：
Goldstein, M A;Doi, R H
通讯作者：
Doi, R H
影响因子：
4.4
作者：
Jensen, Kyle;Alper, Hal;Stephanopoulos, Gregory
通讯作者：
Stephanopoulos, Gregory
DOI：
10.1016/s0169-7439(02)00051-5
发表时间：
2002-10-28
影响因子：
3.9
作者：
Li, BB;Morris, J;Martin, EB
通讯作者：
Martin, EB
影响因子：
3.2
作者：
Mashego, Mlawule R.;van Gulik, Walter M.;Heijnen, Joseph J.
通讯作者：
Heijnen, Joseph J.
影响因子：
14.9
作者：
HARLEY, CB;REYNOLDS, RP
通讯作者：
REYNOLDS, RP