High-Throughput DNA Library Assembly Services

Client requested the construction of a four-gene metabolic pathway with four constitutive promoters in a low-copy number vector. Fifty variants for each pathway gene were identified and designed through database blasting and literature search. These gene variants were randomly inserted into each of the coding DNA sequence (CDS) positions in the pathway, making the theoretical library size 6,250,000.

Library was constructed as a pool by putting all fragments into the reaction. Then, 2,000 colonies were randomly picked and tested for positive inserts. More than 80% positive insertion rate was achieved. In addition, all plasmids with inserts were mini-prepped with high-throughput platform, followed by the transformation of plasmids into yeast cells for performance testing and screening.

48 randomly-picked positive clones were analyzed by sequencing. Results demonstrated 100% diversity and little assembly bias.

After screening all positive clones for their performance, the top 96 best performing clones were sequenced. The enrichments of some variants were higher than others in the good-performing clones. These good-performing enzyme variants can be further combinatorically tested in the next round of optimization.