Figure 3

Replica plating of randomly chosen colonies from transformed amber suppressor strain LE392 with the ligation mixtures of p24MGFPm with GCSF, PTH, SAK, MAP respectively (Please refer to methods section for details). The transformants were plated on inducer (13 mM L+Arabinose) containing plates that indicate both glowing and non glowing colonies upon UV exposure. Colony PCR of randomly chosen two glowing and two non-glowing colonies from each plate were carried out for GFP and for genes of interest. Panel A, C, E and G show the GFP fluorescence from recombinants of p24MGFPm-GCSF, p24MGFPm-PTH, p24MGFPm-SAK, p24MGFPm-MAP from LB amp plates containing inducer (13 mM arabinose). Panel B: GCSF-GFP fusion: Lanes 1 & 2 are the PCR products from glowing colonies nos. 1 & 3 respectively and lanes 3 & 4 are the same from two non-glowing colonies nos. 2 & 5. Lanes 5 & 6 are positive and negative controls, respectively. Panel D: PTH-GFP fusion: Lanes 1 & 2 is the PCR products from glowing colonies nos. 1 & 2 respectively and lanes 3 & 4 are the same from two non-glowing colonies nos. 7 & 10. Lanes 5 & 6 is positive and negative controls, respectively. Panel F: SAK-GFP fusion: Lanes 1 & 2 are the PCR products from glowing colonies nos. 1 & 3 respectively, and lanes 3 & 4 are the same from two non-glowing colonies nos. 2 & 4. Lanes 5 & 6 are positive and negative controls, respectively. Panel H: MAP-GFP fusion: Lanes 1 & 2 are the PCR products from glowing colonies nos. 1 & 2 respectively, and lanes 3 & 4 are the same from two non-glowing colonies nos. 3 & 6. Lanes 5 & 6 are negative and positive controls, respectively. In all the cases M stands for molecular weight marker (Lambda DNA EcoRI/HindIII digest); Upper panels are gene specific PCR and Lower panels indicate GFP specific PCR. Arrows indicate corresponding PCR product. Panel I: Representative diagram showing the lay out of the clones in the replica plate.