Figure 4

Progressive minimization of P. tricornutum fragment 26–3 using yeast homologous recombination. A. Diagram of the deletion strategy. To generate reduced versions of fragment 26–3, we inserted yeast deletion cassettes (which had 40-bp homology at the 5’ end (F) common for all deletions, 40-bp homology at the 3’ end specific for each fragment (RX where X represents homology sites 1–9), and a URA3 marker). These cassettes were PCR amplified, and yeast transformation was performed using the lithium acetate method (Step 1). Homologous recombination between the deletion cassettes and fragment 26–3 would produce desired fragments (Step 2). URA3 (yellow box), Yeast vector (orange box), P. tricornutum DNA representing fragment 26–3 (blue box and blue dotted line). B. After creation of the deletion in yeast, the plasmids were transferred to E. coli and purified. Supercoiled plasmids were separated by gel electrophoresis in the following order: BAC-Tracker ladder (lane 1), unmodified fragment 26–3 (lane 2), deletion of fragment 26–3 into 100 kb (lane 3), 90 kb (lane 4), 80 kb (lane 5), 70 kb (lane 6), 60 kb (lane 7), 50 kb (lane 8), 40 kb (lane 9), 30 kb (lane 10), 20 kb (lane 11).