Development of designer <i>S</i>. <i>meliloti</i> strains and multi-functional vectors. Stephanie L. Brumwell Michael R. MacLeod Tony Huang Ryan R. Cochrane Rebecca S. Meaney Maryam Zamani Ola Matysiakiewicz Kaitlyn N. Dan Preetam Janakirama David R. Edgell Trevor C. Charles Turlough M. Finan Bogumil J. Karas 10.1371/journal.pone.0206781.g001 https://plos.figshare.com/articles/figure/Development_of_designer_i_S_i_i_meliloti_i_strains_and_multi-functional_vectors_/8284355 <p>(a) Schematic of the designer <i>S</i>. <i>meliloti</i> ΔpSymA Δ<i>hsdR</i> strains including the genome reduction and deletion of the restriction-system. Several versions of the designer strains were developed including strains with the pTA-Mob conjugative plasmid, and/or compatible genome engineering vectors (pAGE or pBGE) installed. (b) Vector map of pAGE/pBGE MHS vector with both standard and interchangeable components. Standard components include the BAC and YAC backbone (BAC/YAC), an origin of transfer (<i>oriT</i>), and the selectable marker for <i>P</i>. <i>tricornutum</i> nourseothricin N-acetyl transferase (Ntc). Interchangeable components include selectable markers for <i>S</i>. <i>meliloti</i>: tetracycline (Tet), kanamycin/neomycin (Neo), and spectinomycin (Spec); and origins of replication for <i>S</i>. <i>meliloti</i>: the pSymA origin (<i>repA2B2C2</i>), and pSymB origin (<i>repA1B1C1</i>). Three vectors (pAGE1.0, pAGE2.0, and pAGE3.0) were constructed utilizing the <i>repA2B2C2</i> origin with Spec, Tet or Neo selection, respectively. Three vectors (pBGE1.0, pBGE2.0, and pBGE3.0) were constructed utilizing the <i>repA1B1C1</i> origin with Spec, Tet or Neo selection, respectively. (c) Following conjugation of three pAGE plasmids from <i>E</i>. <i>coli</i> ECGE101 conjugative strain to <i>S</i>. <i>meliloti</i> RmP4122 <i>∆</i>pSymA <i>∆hsdR</i> Nm<sup>s</sup>, an antibiotic selection test for each plasmid was performed. The pAGE1.0, pAGE2.0, and pAGE3.0 vectors in <i>S</i>. <i>meliloti</i> contain antibiotic resistance markers for Spec, Tet, and Neo, respectively. <i>S</i>. <i>meliloti</i> without a plasmid (control) was plated alongside <i>S</i>. <i>meliloti</i> harbouring pAGE1.0, pAGE2.0, or pAGE3.0 on LBmc agar, and LBmc agar supplemented with either spectinomycin (200 μg mL<sup>-1</sup>), tetracycline (10 μg mL<sup>-1</sup>), or neomycin (100 μg mL<sup>-1</sup>).</p> 2019-06-17 17:26:37 coli hsdR restriction-system drought resistance inter-kingdom DNA transfer chemical product polyethylene glycol transformation method Saccharomyces cerevisiae biology applications multi-functional shuttle vectors 2.1 x 10 3 CFU μ g MHS vectors inter-kingdom DNA transfer Storage Designer Sinorhizobium meliloti strains genome strains DNA fragments multi-functional vectors DNA transformation novel host DNA efficiency biosynthetic pathways nitrogen-fixing plant 1.4 x 10 5 host organisms biology research Sinorhizobium meliloti eukaryotic microalgae Phaeodactylum tricornutum