Plasmids were constructed using golden-gate assembly (47 (link), 48 (link)), HiFi assembly (New England Biolabs), and yeast recombination (49 (link)), as described in SI Appendix, Supplementary Materials and Methods . All inserts were confirmed by Sanger sequencing. Plasmids were chemically transformed into E. coli strains (50 (link)) and mobilized into rhizobia via biparental conjugation with E. coli ST18 (51 (link)) or via triparental conjugation with a helper E. coli strain carrying plasmid pRK2013. Plasmids with R6K replicons were maintained at low copy number in E. coli EC100D Pir+ where possible.
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Constructing Plasmids via Multiple Assembly Methods
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Corresponding Organization :
Other organizations : University of Oxford, University of Cambridge, HUN-REN Szegedi Biológiai Kutatóközpont, Hungarian Academy of Sciences, Sainsbury Laboratory, North Dakota State University, Massachusetts Institute of Technology, John Innes Centre
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Variable analysis
independent variables
- Golden-gate assembly
- HiFi assembly
- Yeast recombination
- Plasmid construction
- Plasmid confirmation by Sanger sequencing
- Plasmid transformation into E. coli strains
- Plasmid mobilization into rhizobia via biparental or triparental conjugation
- E. coli strain ST18 for biparental conjugation
- Helper E. coli strain carrying plasmid pRK2013 for triparental conjugation
- Maintenance of plasmids with R6K replicons in E. coli EC100D Pir+ at low copy number
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