Part:BBa_K1725040

phlF encoding PhlF repressor from Pseudomonas protegens Pf-5

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Figure 1 shows the PhlF repressible promoter driving GFP expression (K1725001) first without, then with the repressor expressed (K1725042).

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.