Figure 2. Improvement in minicircle quality and quantity by the new manufacturing system.

(a) Strain BW27783 produced minicircle (MC) with enhanced purity. Minicircles were produced according to the protocol described previously⁶. p2øC3.hFIX, minicircle producer plasmid (PP); BglII+EcoN1, two restriction enzymes used to restrict MC before electrophoresis; L-arab(%), percent of L-arabinose in the minicircle induction reaction; PB, plasmid backbone circle. (b) Strategy for inactivation of endA. pKanR.endA, the plasmid used to generate the Pme1-restricted targeting DNA fragment; KanR, kanamycin-resistance gene; attB and attP, the bacterial and phage attachment sites of bacteriophage øC31 integrase; boxed endA, PCR-generated 329- and 754-bp end A fragments; pBAD.Red, a plasmid expressing the bacteriophage Lambda homology recombination complex (Red) under the control of araC.BAD (BAD); p2øC31, a complementing plasmid encoding two copies of BAD.øC31 gene, one copy of BAD.I-SceI gene and one I-SceI site; BWΔendA, a strain derived from BW27783 with the endA interrupted. (c) Minicircle DNA integrity before and after disruption of the endA gene. Before disrupting endA, we observed repeatedly large variations in the degree of plasmid degradation as demonstrated in Figures 2a&c. Because the Endonuclease A is a membrane-bound enzyme, it was possible that its membrane release and activation varied during plasmid preparation. 32°C and 37°C, the incubation temperature; hr., hours; all reactions contained 1% L-arabinose. (d) Quality of the minicircle determined by gel analyses. Minicircle was made according to the simplified protocol outlined in Figure 1b and Supplementary Figure 9, DNAs were restricted before electrophoresis; (e) Yield of minicircle producer plasmids and minicircle vector DNAs. The yield was derived from triplicate 400-ml overnight cultures; PP, Minicircle producer plasmid; MC, minicircle; Wilcoxon rank sum test comparing the yield of minicircle and its minicircle producer plasmid: (I), p<0.05; (II), p>0.05. We used the following formula to convert the yield from mg/L to mol/L: mol/L=[yield (mg/L)×10E-3 gram/L]/[size (kb)×1000 ×330×2 gram/mol], where 330 is the average molecular weight of dNTP. The minicircle producer plasmid, pMC.RSV-hAAT is schematically illustrated in Supplementary Figure 4d, while the pMC.CMV.LGNSO was described in details in the Constructs section of the Online Methods.