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