design: To generate a suitably diverse library of plasmids for A. tumefaciens mediated transformation of T. atroviride, we transformed the pooled barcoded plasmids into A. tumefaciens EHA 105 using a method modified from {Mersereau, 1990 #185}. To generate electrocompetent A. tumefaciens, we inoculated A. tumefaciens into 5 ml LB and incubated at 30oC with shaking at 250 rpm for 16 h. We then backdiluted the A. tumefaciens culture into 5 ml LB and incubated at 30oC with shaking at 250 rpm for at least 2 doublings until the cultures were at OD 600 1. We then backdiluted a second time into 100 ml LB flasks such that they would be at OD 600 1 after an overnight incubation at 30oC with shaking at 250 rpm. A. tumefaciens cultures at OD 600 1 were chilled on ice with slow swirling for 45 m. These cultures were then decanted into ice-cold 50 ml conical tubes and spun at 4000 rpm for 15 m at 4oC. The cell pellet was then washed once in 50 ml ice-cold ddH2O, once in 25 ml ice-cold ddH2O, and once in 2 ml ice-cold 10% glycerol before resuspension in 150 ul of ice-cold 10% glycerol. Aliquots of 55 ul of cell suspension were then flash frozen in liquid nitrogen.We transformed 153 aliquots of frozen cell suspensions. We thawed the frozen electrocompetent A. tumefaciens cells on ice and mixed the 55 ul cell suspension with 2 ug of barcoded plasmid DNA in ddH2O. We then incubated the cell/DNA mixtures on ice for 2 m before transferring the cell/DNA mixtures to chilled 2 mm gap 96-well electroporation plates (BTX) and electroporating using a 12.5 kV/cm field strength exponential decay wave (electroporation settings: 2500 V, 25 uF, 400 ohms) on a Gemini X2 high throughput electroporation system (BTX). We then immediately transferred the electroporated cells to 1 ml of 30oC LB and gently suspended the cells prior to incubating the culture for 2 h at 30oC with shaking at 250 rpm. Transformed A. tumefaciens cells were selected with kanamycin. We pooled all transformants, sequenced the barcodes, and estimated the diversity of the barcoded vector pool as described above. We estimate the barcoded plasmid pool housed in A. tumefaciens contains ~140 million uniquely barcoded plasmids.