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  1. Article: Translation initiation from sequence variants of the bacteriophage T7 g10RBS in Escherichia coli and Agrobacterium fabrum

    Benedict, Alex B. / Chamberlain, Joshua D. / Calvopina, Diana G. / Griffitts, Joel S.

    Molecular biology reports. 2022 Jan., v. 49, no. 1

    2022  

    Abstract: BACKGROUND: The bacteriophage T7 gene 10 ribosome binding site (g10RBS) has long been used for robust expression of recombinant proteins in Escherichia coli. This RBS consists of a Shine–Dalgarno (SD) sequence augmented by an upstream translational “ ... ...

    Abstract BACKGROUND: The bacteriophage T7 gene 10 ribosome binding site (g10RBS) has long been used for robust expression of recombinant proteins in Escherichia coli. This RBS consists of a Shine–Dalgarno (SD) sequence augmented by an upstream translational “enhancer” (Enh) element, supporting protein production at many times the level seen with simple synthetic SD-containing sequences. The objective of this study was to dissect the g10RBS to identify simpler derivatives that exhibit much of the original translation efficiency. METHODS AND RESULTS: Twenty derivatives of g10RBS were tested using multiple promoter/reporter gene contexts. We have identified one derivative (which we call “CON_G”) that maintains 100% activity in E. coli and is 33% shorter. Further minimization of CON_G results in variants that lose only modest amounts of activity. Certain nucleotide substitutions in the spacer region between the SD sequence and initiation codon show strong decreases in translation. When testing these 20 derivatives in the alphaproteobacterium Agrobacterium fabrum, most supported strong reporter protein expression that was not dependent on the Enh. CONCLUSIONS: The g10RBS derivatives tested in this study display a range of observed activity, including a minimized version (CON_G) that retains 100% activity in E. coli while being 33% shorter. This high activity is evident in two different promoter/reporter sequence contexts. The array of RBS sequences presented here may be useful to researchers in need of fine-tuned expression of recombinant proteins of interest.
    Keywords Agrobacterium ; Escherichia coli ; bacteriophages ; molecular biology ; protein synthesis ; reporter genes ; ribosomes ; start codon
    Language English
    Dates of publication 2022-01
    Size p. 833-838.
    Publishing place Springer Netherlands
    Document type Article
    ZDB-ID 186544-4
    ISSN 1573-4978 ; 0301-4851
    ISSN (online) 1573-4978
    ISSN 0301-4851
    DOI 10.1007/s11033-021-06891-z
    Database NAL-Catalogue (AGRICOLA)

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  2. Article ; Online: Translation initiation from sequence variants of the bacteriophage T7 g10RBS in Escherichia coli and Agrobacterium fabrum.

    Benedict, Alex B / Chamberlain, Joshua D / Calvopina, Diana G / Griffitts, Joel S

    Molecular biology reports

    2021  Volume 49, Issue 1, Page(s) 833–838

    Abstract: Background: The bacteriophage T7 gene 10 ribosome binding site (g10RBS) has long been used for robust expression of recombinant proteins in Escherichia coli. This RBS consists of a Shine-Dalgarno (SD) sequence augmented by an upstream translational " ... ...

    Abstract Background: The bacteriophage T7 gene 10 ribosome binding site (g10RBS) has long been used for robust expression of recombinant proteins in Escherichia coli. This RBS consists of a Shine-Dalgarno (SD) sequence augmented by an upstream translational "enhancer" (Enh) element, supporting protein production at many times the level seen with simple synthetic SD-containing sequences. The objective of this study was to dissect the g10RBS to identify simpler derivatives that exhibit much of the original translation efficiency.
    Methods and results: Twenty derivatives of g10RBS were tested using multiple promoter/reporter gene contexts. We have identified one derivative (which we call "CON_G") that maintains 100% activity in E. coli and is 33% shorter. Further minimization of CON_G results in variants that lose only modest amounts of activity. Certain nucleotide substitutions in the spacer region between the SD sequence and initiation codon show strong decreases in translation. When testing these 20 derivatives in the alphaproteobacterium Agrobacterium fabrum, most supported strong reporter protein expression that was not dependent on the Enh.
    Conclusions: The g10RBS derivatives tested in this study display a range of observed activity, including a minimized version (CON_G) that retains 100% activity in E. coli while being 33% shorter. This high activity is evident in two different promoter/reporter sequence contexts. The array of RBS sequences presented here may be useful to researchers in need of fine-tuned expression of recombinant proteins of interest.
    MeSH term(s) Agrobacterium/genetics ; Agrobacterium/metabolism ; Agrobacterium/virology ; Bacteriophage T7/genetics ; Binding Sites ; Codon, Initiator/genetics ; Enhancer Elements, Genetic/genetics ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Escherichia coli/virology ; Genes, Reporter ; Genetic Engineering/methods ; Plasmids ; Promoter Regions, Genetic/genetics ; Protein Biosynthesis/genetics ; Recombinant Proteins/metabolism ; Ribosomes/metabolism
    Chemical Substances Codon, Initiator ; Recombinant Proteins
    Language English
    Publishing date 2021-11-07
    Publishing country Netherlands
    Document type Journal Article
    ZDB-ID 186544-4
    ISSN 1573-4978 ; 0301-4851
    ISSN (online) 1573-4978
    ISSN 0301-4851
    DOI 10.1007/s11033-021-06891-z
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Convergent evolution of bacterial ceramide synthesis.

    Stankeviciute, Gabriele / Tang, Peijun / Ashley, Ben / Chamberlain, Joshua D / Hansen, Matthew E B / Coleman, Aimiyah / D'Emilia, Rachel / Fu, Larina / Mohan, Eric C / Nguyen, Hung / Guan, Ziqiang / Campopiano, Dominic J / Klein, Eric A

    Nature chemical biology

    2021  Volume 18, Issue 3, Page(s) 305–312

    Abstract: The bacterial domain produces numerous types of sphingolipids with various physiological functions. In the human microbiome, commensal and pathogenic bacteria use these lipids to modulate the host inflammatory system. Despite their growing importance, ... ...

    Abstract The bacterial domain produces numerous types of sphingolipids with various physiological functions. In the human microbiome, commensal and pathogenic bacteria use these lipids to modulate the host inflammatory system. Despite their growing importance, their biosynthetic pathway remains undefined since several key eukaryotic ceramide synthesis enzymes have no bacterial homolog. Here we used genomic and biochemical approaches to identify six proteins comprising the complete pathway for bacterial ceramide synthesis. Bioinformatic analyses revealed the widespread potential for bacterial ceramide synthesis leading to our discovery of a Gram-positive species that produces ceramides. Biochemical evidence demonstrated that the bacterial pathway operates in a different order from that in eukaryotes. Furthermore, phylogenetic analyses support the hypothesis that the bacterial and eukaryotic ceramide pathways evolved independently.
    MeSH term(s) Bacteria/genetics ; Bacteria/metabolism ; Biosynthetic Pathways ; Ceramides/chemistry ; Ceramides/metabolism ; Humans ; Phylogeny ; Sphingolipids/chemistry ; Sphingolipids/metabolism
    Chemical Substances Ceramides ; Sphingolipids
    Language English
    Publishing date 2021-12-30
    Publishing country United States
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2202962-X
    ISSN 1552-4469 ; 1552-4450
    ISSN (online) 1552-4469
    ISSN 1552-4450
    DOI 10.1038/s41589-021-00948-7
    Database MEDical Literature Analysis and Retrieval System OnLINE

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