Evaluation of GFP reporter utility for analysis of transcriptional slippage during gene expression.

Abstract:

BACKGROUND:Epimutations arising from transcriptional slippage seem to have more important role in regulating gene expression than earlier though. Since the level and the fidelity of transcription primarily determine the overall efficiency of gene expression, all factors contributing to their decrease should be identified and optimized. RESULTS:To examine the influence of A/T homopolymeric sequences on introduction of erroneous nucleotides by slippage mechanism green fluorescence protein (GFP) reporter was chosen. The in- or out-of-frame gfp gene was fused to upstream fragment with variable number of adenine or thymine stretches resulting in several hybrid GFP proteins with diverse amino acids at N-terminus. Here, by using T7 phage expression system we showed that the intensity of GFP fluorescence mainly depends on the number of the retained natural amino acids. While the lack of serine (S2) residue results in negligible effects, the lack of serine and lysine (S2K3) contributed to a significant reduction in fluorescence by 2.7-fold for polyA-based in-frame controls and twofold for polyTs. What is more, N-terminal tails amino acid composition was rather of secondary importance, since the whole-cell fluorescence differed in a range of 9-18% between corresponding polyA- and polyT-based constructs. CONCLUSIONS:Here we present experimental evidence for utility of GFP reporter for accurate estimation of A/T homopolymeric sequence contribution in transcriptional slippage induction. We showed that the intensity of GFP hybrid fluorescence mainly depends on the number of retained natural amino acids, thus fluorescence raw data need to be referred to appropriate positive control. Moreover, only in case of GFP hybrids with relatively short N-terminal tags the fluorescence level solely reflects production yield, what further indicates the impact of an individual slippage sequence. Our results demonstrate that in contrast to the E. coli enzyme, T7 RNA polymerase exhibits extremely high propensity to slippage even on runs as short as 3 adenine or 4 thymine residues.

journal_name

Microb Cell Fact

journal_title

Microbial cell factories

authors

Wons E,Koscielniak D,Szadkowska M,Sektas M

doi

10.1186/s12934-018-0999-3

subject

Has Abstract

pub_date

2018-09-21 00:00:00

pages

150

issue

1

issn

1475-2859

pii

10.1186/s12934-018-0999-3

journal_volume

17

pub_type

杂志文章
  • Comparison of two codon optimization strategies to enhance recombinant protein production in Escherichia coli.

    abstract:BACKGROUND:Variations in codon usage between species are one of the major causes affecting recombinant protein expression levels, with a significant impact on the economy of industrial enzyme production processes. The use of codon-optimized genes may overcome this problem. However, designing a gene for optimal expressi...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/1475-2859-10-15

    authors: Menzella HG

    更新日期:2011-03-03 00:00:00

  • Surface components and metabolites of probiotics for regulation of intestinal epithelial barrier.

    abstract::The gut microbiota can significantly affect the function of the intestinal barrier. Some intestinal probiotics (such as Lactobacillus, Bifidobacteria, a few Escherichia coli strains, and a new generation of probiotics including Bacteroides thetaiotaomicron and Akkermansia muciniphila) can maintain intestinal epithelia...

    journal_title:Microbial cell factories

    pub_type: 杂志文章,评审

    doi:10.1186/s12934-020-1289-4

    authors: Liu Q,Yu Z,Tian F,Zhao J,Zhang H,Zhai Q,Chen W

    更新日期:2020-02-05 00:00:00

  • A new-generation of Bacillus subtilis cell factory for further elevated scyllo-inositol production.

    abstract:BACKGROUND:A stereoisomer of inositol, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer's disease. However, this compound is relatively rare, whereas another stereoisomer of inositol, myo-inositol (MI) is abundant in nature. Bacillus subtilis 168 has the ability to metabolize inosi...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-017-0682-0

    authors: Tanaka K,Natsume A,Ishikawa S,Takenaka S,Yoshida KI

    更新日期:2017-04-21 00:00:00

  • Combination of uniform design with artificial neural network coupling genetic algorithm: an effective way to obtain high yield of biomass and algicidal compound of a novel HABs control actinomycete.

    abstract::Controlling harmful algae blooms (HABs) using microbial algicides is cheap, efficient and environmental-friendly. However, obtaining high yield of algicidal microbes to meet the need of field test is still a big challenge since qualitative and quantitative analysis of algicidal compounds is difficult. In this study, w...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/1475-2859-13-75

    authors: Cai G,Zheng W,Yang X,Zhang B,Zheng T

    更新日期:2014-05-24 00:00:00

  • Bacterial magnetosomes as an efficient gene delivery platform for cancer theranostics.

    abstract:BACKGROUND:Gene therapy has gained an increasing interest in its anti-tumor efficiency. However, numerous efforts are required to promote them to clinics. In this study, a novel and efficient delivery platform based on bacterial magnetosomes (BMs) were developed, and the efficiency of BMs in delivering small interferin...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-017-0830-6

    authors: Dai Q,Long R,Wang S,Kankala RK,Wang J,Jiang W,Liu Y

    更新日期:2017-11-28 00:00:00

  • Genetic manipulation of longevity-related genes as a tool to regulate yeast life span and metabolite production during winemaking.

    abstract:BACKGROUND:Yeast viability and vitality are essential for different industrial processes where the yeast Saccharomyces cerevisiae is used as a biotechnological tool. Therefore, the decline of yeast biological functions during aging may compromise their successful biotechnological use. Life span is controlled by a varie...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/1475-2859-12-1

    authors: Orozco H,Matallana E,Aranda A

    更新日期:2013-01-02 00:00:00

  • Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

    abstract:BACKGROUND:Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-016-0532-5

    authors: Vogt CM,Schraner EM,Aguilar C,Eichwald C

    更新日期:2016-08-11 00:00:00

  • The comprehensive profile of fermentation products during in situ CO2 recycling by Rubisco-based engineered Escherichia coli.

    abstract:BACKGROUND:In our previous study, the feasibility of Rubisco-based engineered E. coli (that contains heterologous phosphoribulokinase (PrkA) and Rubisco) for in situ CO2 recycling during the fermentation of pentoses or hexoses was demonstrated. Nevertheless, it is perplexing to see that only roughly 70 % of the carbon ...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-016-0530-7

    authors: Yang CH,Liu EJ,Chen YL,Ou-Yang FY,Li SY

    更新日期:2016-08-02 00:00:00

  • Enzymes: An integrated view of structure, dynamics and function.

    abstract:: Microbes utilize enzymes to perform a variety of functions. Enzymes are biocatalysts working as highly efficient machines at the molecular level. In the past, enzymes have been viewed as static entities and their function has been explained on the basis of direct structural interactions between the enzyme and the sub...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/1475-2859-5-2

    authors: Agarwal PK

    更新日期:2006-01-12 00:00:00

  • Expressing a cytosolic pyruvate dehydrogenase complex to increase free fatty acid production in Saccharomyces cerevisiae.

    abstract:BACKGROUND:Saccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels. Previous studies found that both precursor supply and fatty acid metabolism deregulation are essential for enhanced fatty acid synthesis. A bacterial pyruvate dehydrogenase (PDH) complex ex...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-020-01493-z

    authors: Zhang Y,Su M,Qin N,Nielsen J,Liu Z

    更新日期:2020-12-10 00:00:00

  • Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine.

    abstract:BACKGROUND:The rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and cho...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/1475-2859-6-30

    authors: Báez-Viveros JL,Flores N,Juárez K,Castillo-España P,Bolivar F,Gosset G

    更新日期:2007-09-19 00:00:00

  • Whole cell biosynthesis of a functional oligosaccharide, 2'-fucosyllactose, using engineered Escherichia coli.

    abstract:BACKGROUND:2'-Fucosyllactose (2-FL) is a functional oligosaccharide present in human milk which protects against the infection of enteric pathogens. Because 2-FL can be synthesized through the enzymatic fucosylation of lactose with guanosine 5'-diphosphate (GDP)-l-fucose by α-1,2-fucosyltransferase (FucT2), an 2-FL pro...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/1475-2859-11-48

    authors: Lee WH,Pathanibul P,Quarterman J,Jo JH,Han NS,Miller MJ,Jin YS,Seo JH

    更新日期:2012-04-30 00:00:00

  • Genetic engineering of Pseudomonas chlororaphis GP72 for the enhanced production of 2-Hydroxyphenazine.

    abstract:BACKGROUND:The biocontrol strain Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere synthesizes three antifungal phenazine compounds, 2-Hydroxyphenazine (2-OH-PHZ), 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) and phenazine-1-carboxylic acid (PCA). PCA has been a commercialized antifungal pest...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-016-0529-0

    authors: Liu K,Hu H,Wang W,Zhang X

    更新日期:2016-07-28 00:00:00

  • Metabolic engineering of Escherichia coli for efficient production of L-alanyl-L-glutamine.

    abstract:BACKGROUND:L-Alanyl-L-glutamine (AQ) is a functional dipeptide with high water solubility, good thermal stability and high bioavailability. It is widely used in clinical treatment, post-operative rehabilitation, sports health care and other fields. AQ is mainly produced via chemical synthesis which is complicated, time...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-020-01369-2

    authors: Zhu J,Yang W,Wang B,Liu Q,Zhong X,Gao Q,Liu J,Huang J,Lin B,Tao Y

    更新日期:2020-06-11 00:00:00

  • Production of human blood group B antigen epitope conjugated protein in Escherichia coli and utilization of the adsorption blood group B antibody.

    abstract:BACKGROUND:In the process of ABO-incompatible (ABOi) organ transplantation, removal of anti-A and/or B antibodies from blood plasma is a promising method to overcome hyperacute rejection and allograft loss caused by the immune response between anti-A and/or B antibodies and the A and/or B antigens in the recipient. Alt...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-016-0538-z

    authors: Shang W,Zhai Y,Ma Z,Yang G,Ding Y,Han D,Li J,Zhang H,Liu J,Wang PG,Liu XW,Chen M

    更新日期:2016-08-11 00:00:00

  • Comparison of quenching and extraction methodologies for metabolome analysis of Lactobacillus plantarum.

    abstract:BACKGROUND:A reliable quenching and metabolite extraction method has been developed for Lactobacillus plantarum. The energy charge value was used as a critical indicator for fixation of metabolism. RESULTS:Four different aqueous quenching solutions, all containing 60% of methanol, were compared for their efficiency. O...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/1475-2859-6-27

    authors: Faijes M,Mars AE,Smid EJ

    更新日期:2007-08-20 00:00:00

  • Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus.

    abstract:BACKGROUND:Targeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein. These signal sequences usually contain an N-terminal basic amino acid followed by a stretch containing hydrophobic residues, although no consensus signa...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-015-0203-y

    authors: Yarimizu T,Nakamura M,Hoshida H,Akada R

    更新日期:2015-02-14 00:00:00

  • Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity.

    abstract:BACKGROUND:Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-018-0895-x

    authors: Kidibule PE,Santos-Moriano P,Jiménez-Ortega E,Ramírez-Escudero M,Limón MC,Remacha M,Plou FJ,Sanz-Aparicio J,Fernández-Lobato M

    更新日期:2018-03-22 00:00:00

  • An artificial neural network for membrane-bound catechol-O-methyltransferase biosynthesis with Pichia pastoris methanol-induced cultures.

    abstract:BACKGROUND:Membrane proteins are important drug targets in many human diseases and gathering structural information regarding these proteins encourages the pharmaceutical industry to develop new molecules using structure-based drug design studies. Specifically, membrane-bound catechol-O-methyltransferase (MBCOMT) is an...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-015-0304-7

    authors: Pedro AQ,Martins LM,Dias JM,Bonifácio MJ,Queiroz JA,Passarinha LA

    更新日期:2015-08-07 00:00:00

  • Engineering building blocks for self-assembling protein nanoparticles.

    abstract::Like natural viruses, manmade protein cages for drug delivery are to be ideally formed by repetitive subunits with self-assembling properties, mimicking viral functions and molecular organization. Naturally formed nanostructures (such as viruses, flagella or simpler protein oligomers) can be engineered to acquire spec...

    journal_title:Microbial cell factories

    pub_type: 社论

    doi:10.1186/1475-2859-9-101

    authors: Vázquez E,Villaverde A

    更新日期:2010-12-30 00:00:00

  • Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone.

    abstract:BACKGROUND:β-Ionone is a fragrant terpenoid that generates a pleasant floral scent and is used in diverse applications as a cosmetic and flavoring ingredient. A growing consumer desire for natural products has increased the market demand for natural β-ionone. To date, chemical extraction from plants remains the main ap...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-018-0984-x

    authors: Czajka JJ,Nathenson JA,Benites VT,Baidoo EEK,Cheng Q,Wang Y,Tang YJ

    更新日期:2018-09-01 00:00:00

  • Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel 34S labeling procedure.

    abstract:BACKGROUND:The budding yeast Pichia pastoris is widely used for protein production. To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. Especially the balance between intracellular protein for...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/1475-2859-10-47

    authors: Pfeffer M,Maurer M,Köllensperger G,Hann S,Graf AB,Mattanovich D

    更新日期:2011-06-26 00:00:00

  • Homologous high-throughput expression and purification of highly conserved E coli proteins.

    abstract:BACKGROUND:Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD). Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intest...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/1475-2859-6-18

    authors: Ergin A,Büssow K,Sieper J,Thiel A,Duchmann R,Adam T

    更新日期:2007-06-06 00:00:00

  • Systematic pathway engineering of Corynebacterium glutamicum S9114 for L-ornithine production.

    abstract:BACKGROUND:L-Ornithine is a non-protein amino acid with extensive applications in medicine and the food industry. Currently, L-ornithine production is based on microbial fermentation, and few microbes are used for producing L-ornithine owing to unsatisfactory production titer. RESULTS:In this study, Corynebacterium gl...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-017-0776-8

    authors: Zhang B,Yu M,Zhou Y,Li Y,Ye BC

    更新日期:2017-09-22 00:00:00

  • Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production.

    abstract:BACKGROUND:In the biopharmaceutical industry, Escherichia coli (E. coli) strains are among the most frequently used bacterial hosts for producing recombinant proteins because they allow a simple process set-up and they are Food and Drug Administration (FDA)-approved for human applications. Widespread use of E. coli in ...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/1475-2859-12-58

    authors: Marisch K,Bayer K,Cserjan-Puschmann M,Luchner M,Striedner G

    更新日期:2013-06-11 00:00:00

  • Lactic acid fermentation as a tool to enhance the functional features of Echinacea spp.

    abstract:BACKGROUND:Extracts and products (roots and/or aerial parts) from Echinacea ssp. represent a profitable market sector for herbal medicines thanks to different functional features. Alkamides and polyacetylenes, phenols like caffeic acid and its derivatives, polysaccharides and glycoproteins are the main bioactive compou...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/1475-2859-12-44

    authors: Rizzello CG,Coda R,Macías DS,Pinto D,Marzani B,Filannino P,Giuliani G,Paradiso VM,Di Cagno R,Gobbetti M

    更新日期:2013-05-04 00:00:00

  • A novel bi-directional promoter system allows tunable recombinant protein production in Pichia pastoris.

    abstract:BACKGROUND:The methylotrophic yeast Pichia pastoris is a well-studied host organism for recombinant protein production, which is usually regulated either by a constitutive promoter (e.g. promoter of glyceraldehyde-3-phosphate dehydrogenase; PGAP) or an inducible promoter (e.g. promoter of alcohol oxidase 1; PAOX1). Bot...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-017-0768-8

    authors: Rajamanickam V,Metzger K,Schmid C,Spadiut O

    更新日期:2017-09-13 00:00:00

  • Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli.

    abstract:BACKGROUND:Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (D...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-020-01321-4

    authors: Ding T,Huang C,Liang Z,Ma X,Wang N,Huo YX

    更新日期:2020-03-10 00:00:00

  • Isolation and characterization of high exopolysaccharide-producing Weissella confusa VP30 from young children's feces.

    abstract:BACKGROUND:Lactic acid bacteria (LAB) are known to have a significant ability to colonize the human intestinal tract and adhere to the surface of intestinal epithelial cells. Among the various lactic acid bacteria, exopolysaccharide (EPS) producing strains are known to provide a variety of health benefits for their hos...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-019-1158-1

    authors: Jin H,Jeong Y,Yoo SH,Johnston TV,Ku S,Ji GE

    更新日期:2019-06-13 00:00:00

  • Characterization of a promiscuous cadmium and arsenic resistance mechanism in Thermus thermophilus HB27 and potential application of a novel bioreporter system.

    abstract:BACKGROUND:The characterization of the molecular determinants of metal resistance has potential biotechnological application in biosensing and bioremediation. In this context, the bacterium Thermus thermophilus HB27 is a metal tolerant thermophile containing a set of genes involved in arsenic resistance which, differen...

    journal_title:Microbial cell factories

    pub_type: 杂志文章

    doi:10.1186/s12934-018-0918-7

    authors: Antonucci I,Gallo G,Limauro D,Contursi P,Ribeiro AL,Blesa A,Berenguer J,Bartolucci S,Fiorentino G

    更新日期:2018-05-18 00:00:00