Fig. 5

Deletion of increasing amount of DNA and simultaneous insertion of complex DNA. a Schematic of the assembly approach followed for the presented experiments. A pUC19 plasmid was cut with KpnI, and a RFP cassette was introduced to the cut vector using Gibson assembly. The different inserts were produced using PCR, each utilizing different primers with different homologous harms to delete increasing amounts of nucleotides around the KpnI site upon Gibson Deletion reaction. The RFP cassettes were designed to have 20 nucleotides of homology with the plasmid (represented by the red and yellow lines and boxes). The RFP primers were designed to delete 0, 10 (5 on each sides), 20 (10 on each sides), 30 (15 on each sides), 40 (20 on each sides), 50 (25 on each sides), 60 (30 on each sides), and 200 (100 on each sides) nucleotides around the KpnI site. b The Gibson reactions were transformed and plated on carbenicillin plates (1/10 or 9/10 of the transformation mixture). Colonies expressing DNA that underwent likely successful assembly, appear red due to the expression of the inserted RFP. An incorrect assembly would result in a white colony. c The number of red versus white colonies were counted and recorded. d Number of correct clones after Sanger sequencing. Gibson Deletion reactions were performed with a 50 °C incubation for 30 min or with a pre-incubation at 37 °C for 30 min before a 50 °C incubation for additional 30 min