K1725040 represses expression driven by K1725000 (PhlF repressible promoter) as shown in Figure 1. K1725042 is K1725040 driven by the lacI regulated promoter K1725080. Our control was K1725083 (the Tet repressor C0040 also driven by K1725080) and K1725082 (the TetR repressible promoter R0040 driving expression of I13500).



Figure 1. Represser constructs with pSB1C3 backbone; promoter driving GFP constructs with pSB3K3 backbone. Repressor protein expression induced with 100μM IPTG. Replicates of constructs and controls of three dilutions from one colony, under the same conditions. Mean and standard deviation of replicates were calculated to give value and error bars.


We also tested if K1725040 is orthogonal (does the repressor protein specifically repress K1725000) K1725042 is expected to repress GFP expression from K1725001 so these cells should not fluoresce, however, it is not expected to repress GFP expression from K1725021, or K1725082 so these cells should fluoresce green. As shown in Figure 2, K1725042 represses GFP expression from K1725001, but not from K1725082 or K1725021, as expected.


Figure 2 Characterising Repressors. Repressor constructs in pSB1C3 backbone; promoter driving GFP constructs in pSB3K3 backbone. Cells were grown overnight in 100μM IPTG, to induce expression of the repressor proteins. Three replicates of the sample were diluted and tested under the same conditions for each sample. Mean and standard deviation of replicates were calculated to give value and error bars.


In addition to showing that K1725042 was capable of repressing K1725001, quantification of repression of GFP expression was calculated. Figure 3 shows that K1725042 represses K1725001 GFP expression by 83-fold.



Figure 3 Fold Repression. Repressor protein expression induced with 100μM IPTG. Values and error bars from experiments described above.


To further characterise K1725042, the concentration of IPTG used to induce repressor expression was reduced to investigate the range of regulation of GFP expression. Figure 4 shows that K1725083 has a wider range of regulation, whereas K1725042 shows no significant difference between 100μM and 10μM IPTG, implying that K1725042 (PhlF) can repress to equivalent to 0 with less repressor protein expressed, than K1725083 (TetR).



Figure 4. Repressor constructs with pSB1C3 backbone; promoter driving GFP constructs with pSB3K3 backbone. Cells were grown overnight in 100μM, 30 μM, 10 μM, 3 μM, and 0 μM IPTG, to induce expression of the repressor proteins. Three replicates of the sample were diluted and tested under the same conditions for each sample. Mean and standard deviation of replicates were calculated to give value and error bars.