Shine-Dalgarno sequence enhances the efficiency of lacZ repression by artificial anti-lac antisense RNAs in Escherichia coli

Stefan, Alessandra; Schwarz, Flavio; Bressanin, Daniela; Hochkoeppler, Alejandro

doi:10.1016/j.jbiosc.2010.05.012

Cited by 13 publications

(12 citation statements)

References 22 publications

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“…The position of annealing region within mRNA is another significant factor. In many cases, long asRNAs are designed to cover RBS and the start codon region of a target mRNA (Stefan et al, 2010). This is because translation initiation is the rate-limiting step and thus prevention of ribosomes from binding to RBS of target mRNAs is a decisive factor determining the efficiency of asRNAs.…”

Section: Metabolic Engineering Of E Colimentioning

confidence: 99%

Rapid and high-throughput construction of microbial cell-factories with regulatory noncoding RNAs

Chaudhary

Lee

2015

Biotechnology Advances

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Section: Metabolic Engineering Of E Colimentioning

confidence: 99%

Rapid and high-throughput construction of microbial cell-factories with regulatory noncoding RNAs

Chaudhary

Lee

2015

Biotechnology Advances

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“…For target sequences, the ribosome-binding site (RBS) and start codon regions (typically 70 to 160 bp) were always chosen, because these regions are thought to be the most sensitive to silencing (21).…”

Section: Genes Chosen and Construction Of Ptasrna Expression Vectorsmentioning

confidence: 99%

“…Probably, these PTasRNAs prevent ribosomes from recognizing the RBS and thus inhibit translation without mRNA degradation. The precise silencing mechanisms used by artificial asRNAs and native small regulatory RNAs are still controversial (21).…”

Section: Genes Chosen and Construction Of Ptasrna Expression Vectorsmentioning

confidence: 99%

A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

Nakashima

Ohno

Yoshikawa

et al. 2014

Appl Environ Microbiol

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We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination. R ecent advances in biotechnology have allowed genome-wide studies of metabolic pathways (1). In Escherichia coli, a singlegene disruption library of all nonessential genes (the Keio collection) has been constructed and distributed all over the world (2, 3). In addition, predicting metabolic flux change after gene disruption is becoming possible with in silico simulation (4, 5). Information obtained by these studies is used for understanding the metabolic pathway as a system and for rationally designing the metabolic pathway to produce valuable compounds. However, the Keio collection is not applicable to all E. coli strains and does not include growth essential genes.We previously reported the construction of vectors that express antisense RNAs (asRNAs) to silence the function of any gene in E. coli (6, 7). The vectors are isopropyl-␤-D-thiogalactoside (IPTG)-inducible, allowing for the conditional silencing of target genes. Moreover, the asRNAs have novel structures called pairedterminus (PT) asRNAs, consisting of 38-bp inverted repeats that create paired double-stranded RNA termini for any antisense sequence. The PTasRNAs have much higher silencing efficacies than ordinary asRNAs because of their improved stability, which increases the abundance of asRNAs in cells. The advantages of the gene silencing approach over the gene disruption approach include their inducibility, portability, and high throughput.The final goal of our research is the construction of PTasRNA expression vectors for all E. coli genes (ca. 4,000 to 5,000) in order to understand the whole cell as a system. As a first step, we focused on central carbon metabolism, since it is a good example of a miniaturized cellular system. Similarly, in silico simulation study initially focused on 14 central carbon-metabolism genes and 4 additional genes for the metabolic precursors needed for cell growth (the first core model) (8), whereas the current simulati...

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“…In many cases, asRNAs are designed to hybridize to the ribosome-binding site (RBS) and the start codon region of the target mRNAs (Figure 5) [98]. This is because translation initiation is the limiting step in the translational process; thus, preventing the ribosome from binding to the RBS site of target mRNAs is most critical for its efficacy.…”

Section: Gene Silencing Using Asrnasmentioning

confidence: 99%

Bacterial Cellular Engineering by Genome Editing and Gene Silencing

Nakashima

Miyazaki

2014

IJMS

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Genome editing is an important technology for bacterial cellular engineering, which is commonly conducted by homologous recombination-based procedures, including gene knockout (disruption), knock-in (insertion), and allelic exchange. In addition, some new recombination-independent approaches have emerged that utilize catalytic RNAs, artificial nucleases, nucleic acid analogs, and peptide nucleic acids. Apart from these methods, which directly modify the genomic structure, an alternative approach is to conditionally modify the gene expression profile at the posttranscriptional level without altering the genomes. This is performed by expressing antisense RNAs to knock down (silence) target mRNAs in vivo. This review describes the features and recent advances on methods used in genomic engineering and silencing technologies that are advantageously used for bacterial cellular engineering.

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Shine-Dalgarno sequence enhances the efficiency of lacZ repression by artificial anti-lac antisense RNAs in Escherichia coli

Cited by 13 publications

References 22 publications

Rapid and high-throughput construction of microbial cell-factories with regulatory noncoding RNAs

Rapid and high-throughput construction of microbial cell-factories with regulatory noncoding RNAs

A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

Bacterial Cellular Engineering by Genome Editing and Gene Silencing

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