Difference between revisions of "Part:BBa K1725000"

Revision as of 22:56, 18 September 2015

PhlF repressible promoter

Sequence and Features

This promoter was characterised by using it to drive expression of GFP with two different Ribosome Binding Sites, BBa_B0032 in E5501 and BBa_B0034 in I13500.

GFP fluorescence of K1725001 (K1725000.I13500), K1725002 (K1725000.E5501), K1725021 (SrpR repressible promoter.I13500), K1725022 (SrpR repressible promoter.E5501), K1725082 (TetR repressible promoter.I13500), and E5504 (TetR repressible promoter.E5501) with plasmid backbone pSB3K3 was measured to compare the relative strengths of promoters K1725000 and K1725020 (SrpR repressible promoter) to a promoter already well documented in the registry, R0040 (TetR repressible promoter). Figure 1 the fluorescence scan image and a graph of approximate molecules of GFP per cell. These results indicated that K1725000 is a significantly stronger promoter than R0040 or K1725020.

Figure 1. All constructs with pSB3K3 plasmid backbone, in DH5α cells. Replicates of constructs and controls from three colonies, under the same conditions. Mean and standard deviation of replicates were calculated to give value and error bars.

K1725000 driven expression is repressed by K1725040 (phlF encoding PhlF repressor) as shown in Figure 2. 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 2. 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.