Figure 3

Comparison of the influence of detergent, autoinduction, post-assembly purification and competency of used bacteria on DNA assembling efficiency. (a) In four of the five PPY lysis conditions induction of Redα had moderate effects. Five different detergents were tested on PPY-cells grown with either lactose (Redα un-induced) or arabinose (Redα induced). All assemblies were column-purified before transformation into NEB 5-alpha. Bars indicate standard error of three independent replicates of an assembly reaction in all following graphs. PPY recombinogenic capacity was assessed in three-fragment assemblies. (b) Column-purification of the three-fragment-assembly reactions led to markedly increased number of recombinant colonies for all tested detergents. In the case of CHAPS and SB-12, unpurified samples resulted in no colonies. The OTG derived PPY-extract resulted in the highest number of recombinant colonies without purification. All assemblies were arabinose-induced. (c) Chemical competency has profound influence on recombination efficiency. OTG prepared PPY-lysate was used in a three-fragment-assembly and transformed into commercial NEB 5-alpha competent E. coli (1 × 10⁹ cfu/µg pUC DNA) or the same strain prepared by the Inoue-method²¹ (2.3 × 10⁶ cfu/µg DNA). (d) For convenient readout of the potency of the PPY-extracts PCR-fragments used for the three- and four-way assembly reactions consisted of a blue chromoprotein coding ORF, a kanamycin resistance gene an origin-of-replication (on one fragment for the three-fragment assembly) and a bacterial basal-promoter-fragment. Only successful recombinants could produce blue colonies on kanamycin plates. The PCR-fragments to be assembled had overlapping bases that summed up to about 15 bp overlapping ends.