Figure 3.

Minicircle DNA production test and long range PCR analysis of HB101Cre clones. (A) Minicircle DNA production test. Cultures of the prospective HB101Cre strain harbouring minicircle producer plasmid pFIXluc were grown overnight in the LB medium supplemented with 0.2% D-glucose under Cm selection. The cells from each bacterial clone were washed in the minimal M9 medium, split into 2 tubes containing the M9 medium supplemented with 0.2% D-glucose (control) and 0.2% L-arabinose, cultured for 18 hrs and used for closed circular DNA extraction. The resultant DNA was studied by electrophoresis in a 0.7% agarose gel. Analysis for 6 clones is shown. (B) Long range PCR analysis to show successful introduction of the araC-Cre expression cassette into E. coli HB101. Primers LACZ-FA and LACZ2 were used to amplify the 3094 bp DNA fragment with the araC-Cre expression cassette. In all PCR analysis experiments bacteria were added directly to the PCR reaction mixtures and electrophoresis was performed in a 0.7% agarose gel. (C) Long range PCR analysis to show successful insertion of the araC-Cre cassette into the chromosomal lacZ gene. The araC-Cre segment replaced 886 bp of the lacZ gene. Primers LACZ-START and LACZ-END are homologous to the start and the end of the β-galactosidase coding sequence respectively. These primers were used to amplify the entire 5219 bp lacZ::(araC-Cre) fragment of the generated strain HB101Cre (the lacZ gene is 3075 bp).