Plasmid library construction and high-throughput sequencing

In the first step, the positions 1 and 2 transgenes were barcoded through amplification, with the termini of the position 1 amplicons harboring identical sequences to the termini of the position 2 amplicons, permitting Gibson assembly of the fragments (Supplementary Figure S1a, top). A total of 40 ng of plasmid DNA was mixed with 0.333 μM of forward primer, 0.333 μM of reverse primer and 2× Kapa Hifi added as half the volume (Supplementary Table S2). The mixtures were cycled 12 times in a thermocycler. A total of 1 μl of DPNI enzyme (20 units) was added to each tube, and incubated at 37°C for 2 h. A total of 15 μl of product was mixed with 3 μl of (6× gel loading dye), and run on a 0.8% agarose gel in Tris-borate-EDTA buffer. Bands were cut, position 1 bands and position 2 bands were pooled separately, and extracted using the Qiagen Gel Extraction kit. Position 1 and position 2 DNA were eluted in 10 μl nuclease-free water. A total of 5 μl of each eluate were mixed together with 10 μl 2× Gibson mix and incubated at 50°C for 30 min. After incubation, the product was eluted in 5 μl, and 4 μl of library was electroporated into 10-beta cells (NEB) using 0.1 μm cuvettes (Bio-Rad) according to manufacturer instructions. Approximately 2.16 million colony forming units of transformants were grown in 200 ml Luria Broth overnight, and plasmid DNA was extracted with a Qiagen Midiprep Kit (Qiagen). In the second step, polymerase chain reaction (PCR) and Gibson assembly was used to insert an IRES-Puromcyin-2A-mCherry cassette in the reverse orientation 5′ of the Bxb1 attB site, so the library could be selected in the absence of Dox induction (Supplementary Figure S1a, bottom). A total of 320 ng of library DNA was mixed with 2× Kapa Hifi Readymix and a final concentration of 0.333 μM KAM2311 and 0.333 μM KAM2312, with 30 μl distributed into eight replicate tubes. A total of 160 ng of rIP2M_attB_GFP plasmid was mixed with 2× Kapa Hifi and a final concentration of 0.333 μM KAM2313 and 0.333 μM KAM2314, with 30 μl distributed into four replicate tubes. Both sets of tubes were amplified over 18 cycles. A total of 1 μl (20 units) of DPNI enzyme was added to each tube, and incubated at 37°C for 2 h. After incubation, the rIP2M_attB_GFP amplicon was gel extracted from a 0.8% agarose gel and eluted in 10 μl, whereas the library amplicon was cleaned and concentrated (Zymo), and eluted in 5 μl. A total of 4 μl of each were mixed together with 8 μl of 2× Gibson mix and incubated at 50°C for 30 min. The product was clean-and-concentrated, eluted in 5 μl, and electroporated in 10-beta cells using 0.1 μm cuvettes (Bio-Rad) according to manufacturer's instructions. Approximately 0.5 million colony forming units were amplified in 200 ml Luria Broth overnight, and extracted using the Qiagen Midiprep kit. Sanger sequencing of eight colonies revealed six to be intended library members. This library was called attB-TSPCR.

The barcode region within the 10 ng of plasmid DNA was amplified with 14 cycles of PCR in a 50 μl NEB Q5 polymerase reaction (New England Biolabs) using a Bio Rad mini opticon and Sybr Green I. Cycling was stopped at 14 cycles during low to mid log phase amplification. The amplicons were mixed equally based on their final relative fluorescence units as measured by the mini opticon and run on a 1% TBE-agarose gel. The product band was excised and extracted using the Freeze and Squeeze Kit according to the manufacturer's instructions (Bio-Rad). Gel-purified amplicons were quantified by qPCR using the Illumina Library Quantification Kit (Kapa Biosystems). Then, indexed amplicons were pooled and sequenced on a Nextseq (NextSeq 75 High V2 kit, Illumina, for primers see Supplementary Data Set 1).

The library contained ∼400 000 unique plasmid barcodes, which we were able to quantitate with reasonable accuracy across technical replicate amplification and sequencing reactions (Pearson's r: 0.9; Supplementary Figure S1b). When plasmid identifiers were combined for each of the 216 observed combinations out of the 224 possible (Supplementary Table S3), we observed precise quantitation across the technical replicates (Pearson's r: 1; Supplementary Figure S1b). Notably, there was a strong correlation between the frequency of each transgene identifier combination within the plasmid library with the number of times that combination was associated with a unique plasmid identifier combination (Supplementary Figure S1d; Pearson's r: 0.99), showing there was little selection of each plasmid combination during growth in bacteria.