Figure 2
From: Control of total GFP expression by alterations to the 3′ region nucleotide sequence
Comparison of fluorescence (A) and RT-PCR (B) data for various MK 6 -GFP clones with different orders of 5×AAA and 1×AAG in the K 6 . To obtain the corresponding clones, we constructed MK6-GFP-Stop(TAA)-# clones with varying sequences of 5×AAA and 1×AAG codons within the K6 sequence, as described in the Methods. The determination of total protein fluorescence was conducted as in Figure 1. A semi-quantitative RT-PCR analysis of 1-h IPTG-induced cultures of the MK6-GFP clones was conducted at 30°C after 30 cycles, as described in the Methods. To check for saturation of the PCR products before 30 cycles, an RT-PCR analysis was conducted after 20 cycles, but the general thin and unsaturated density band patterns of the 1-h induced cultures were similar amongst the clones (data not shown). The upper and lower bands correspond to the gfp and 16S rRNA genes, respectively. We used an Invitrogen 1 kb plus ladder as the DNA size marker. All cultures, mean values, and error bars are as in Figure 1. Lanes: 1, MK(AAA)6-GFP-LE-6H-Stop(TGA), undeleted control; 2, MK(AAA)6-GFP-Stop(TAA)-#, deleted control; 3, MK(AAG)1(AAA)5-GFP-Stop(TAA)-#; 4, MK(AAA)1(AAG)1(AAA)4-GFP-Stop(TAA)-#; 5, MK(AAA)2(AAG)1(AAA)3-GFP-Stop(TAA)-#; 6, MK(AAA)3 (AAG)1(AAA)2-GFP-Stop(TAA)-#; 7, MK(AAA)4(AAG)1(AAA)1-GFP-Stop(TAA)-#; and 8, MK(AAA)5(AAG)1-GFP-Stop(TAA)-#.