Analyzing and enhancing mRNA translational efficiency in an Escherichia coli in vitro expression system

Voges, Dieter; Watzele, Manfred; Nemetz, Cordula; Wizemann, S.; Buchberger, Bernd

doi:10.1016/j.bbrc.2004.04.064

Cited by 67 publications

(70 citation statements)

References 41 publications

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“…As secondary structure elements in the region upstream of the ribosomal binding site can interfere with the efficiency of translation (see refs. [29][30] ), it is very likely that a similar effect due to the highly stable structure of the T8 terminator is the reason for the observed reduced reporter gene expression in this construct. The last non-functional riboswitch RS8D4bp is also not fulfilling both criteria.…”

Section: Design Criteria For Synthetic Terminators In the Riboswitch mentioning

confidence: 92%

Design criteria for synthetic riboswitches acting on transcription

et al. 2015

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Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell.

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Section: Design Criteria For Synthetic Terminators In the Riboswitch mentioning

confidence: 92%

Design criteria for synthetic riboswitches acting on transcription

et al. 2015

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“…Previous investigations demonstrated that the usage of codon following the initiation codon could affect gene expression at translation level in E. coli [24][25][26][27]. In this sturdy, only a small amount of transcriptional products were detected by qRT-PCR without any leading sequence adjacent to the 5'end of LMW-GS genes, which indicated that transcriptional suppression should be responsible for LMW-GS gene failing to express in E. coli.…”

Section: Discussionmentioning

confidence: 67%

High-level expression of LMW-GS and α-gliadin genes promoted by the expressed tag sequence of 5′ end in Escherichia coli

Wang

Gao

et al. 2015

Protein Expression and Purification

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractWheat storage protein genes, especially low molecular weight glutenin subunit (LMW-GS) and gliadin genes are difficult to be expressed in E. coli, mainly due to the presence of highly repetitive sequences. In order to establish a high efficiency expression system for these genes, five different expression plasmids combining with 2 9 genes, viz. 6 LMW-GS and 3 α-gliadin genes isolated from common wheat and related species, were studied for heterologous expression in E. coli. In this study, when an expressed tag sequence encoding signal peptide, His-S or GST-tag was fused to the 5'end of LMW-GS or gliadin gene as the leading sequence, all recombination genes could be stably expressed at a high level. On the contrast, as expected, the inserted genes encoding mature protein failed without an expressed tag sequence. This result indicated that using expressed tag sequences as leading sequences could promote LMW-GS and gliadin genes to be well expressed in E. coli. Further transcriptional analysis by quantitative real-time PCR (qRT-PCR) showed transcription levels of recombination genes (e.g. GST-Glutenin, His-S-Glutenin and SP*-His-Glutenin) were 4-fold to 33-fold higher than those of the LMW-GS genes, which suggested these expressed tag sequences might play an important role in stimulating transcription. The possible molecular mechanism under this phenomenon was discussed.

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“…4). The fact that the E. coli-optimized operon was functionally expressed, while the native R. palustris pathway was not expressed in E. coli, is partially supported by a study linking E. coli translation initiation efficiency in vitro to lower GC content (Voges et al, 2004). The authors tested 756 constructs which differed in a 39 bp insert downstream of the start codon, fused to GFP.…”

Section: Discussionmentioning

confidence: 99%

Transfer of the high-GC cyclohexane carboxylate degradation pathway from Rhodopseudomonas palustris to Escherichia coli for production of biotin

Bernstein¹,

Bülter²,

Liao³

2008

Metabolic Engineering

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This work demonstrates the transfer of the five gene cyclohexane carboxylate (CHC) degradation pathway from the high GC alphaproteobacterium Rhodopseudomonas palustris to Escherichia coli, a gammaproteobacterium. The degradation product of this pathway is pimeloyl-CoA, a key metabolite in E. coli's biotin biosynthetic pathway. This pathway is useful for biotin overproduction in E. coli, however, the expression of GC-rich genes is troublesome in this host. When the native R. palustris CHC degradation pathway is transferred to a ΔbioH pimeloyl-CoA auxotroph of E. coli, it is unable to complement growth in the presence of CHC. To overcome this expression problem we redesigned the operon with decreased GC content and removed stretches of high GC intergenic DNA which comprise the 5' untranslated region of each gene, replacing these features with shorter low GC sequences. We show this synthetic construct enables growth of the ΔbioH strain in the presence of CHC. When the synthetic degradation pathway is overexpressed in conjunction with the downstream genes for biotin biosynthesis, we measured significant accumulation of biotin in the growth medium, showing that the pathway transfer is successfully integrated with the host metabolism.

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Analyzing and enhancing mRNA translational efficiency in an Escherichia coli in vitro expression system

Cited by 67 publications

References 41 publications

Design criteria for synthetic riboswitches acting on transcription

Design criteria for synthetic riboswitches acting on transcription

High-level expression of LMW-GS and α-gliadin genes promoted by the expressed tag sequence of 5′ end in Escherichia coli

Transfer of the high-GC cyclohexane carboxylate degradation pathway from Rhodopseudomonas palustris to Escherichia coli for production of biotin

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