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Team:Sheffield/FadR
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| + | <h2>FadR Characterisation</h2> | ||
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<p>We investigated FadR expression using a Tecan Plate Reader. LB broth containing varying concentrations of oleic acid were innoculated with DH5a <i>E. coli</i> cells expressing fadR (BBa_K861060), fadR-RFP (BBa_K861060), or consituative RFP (BBa_J23110). These were loaded into a clear flat bottomed black walled 96 well plate, some wells contained no cells and were used as blanks. Cells were grown in the Tecan plate reader overnight at 37oC with shaking for a period of 16 hours. OD600 measurements were taken every 30 minutes. In addition RFP readings were also taken with 565nm excitation and 605nm emission wavelengths. </p> | <p>We investigated FadR expression using a Tecan Plate Reader. LB broth containing varying concentrations of oleic acid were innoculated with DH5a <i>E. coli</i> cells expressing fadR (BBa_K861060), fadR-RFP (BBa_K861060), or consituative RFP (BBa_J23110). These were loaded into a clear flat bottomed black walled 96 well plate, some wells contained no cells and were used as blanks. Cells were grown in the Tecan plate reader overnight at 37oC with shaking for a period of 16 hours. OD600 measurements were taken every 30 minutes. In addition RFP readings were also taken with 565nm excitation and 605nm emission wavelengths. </p> | ||
Latest revision as of 03:49, 18 October 2014
Results
FadR Characterisation
We investigated FadR expression using a Tecan Plate Reader. LB broth containing varying concentrations of oleic acid were innoculated with DH5a E. coli cells expressing fadR (BBa_K861060), fadR-RFP (BBa_K861060), or consituative RFP (BBa_J23110). These were loaded into a clear flat bottomed black walled 96 well plate, some wells contained no cells and were used as blanks. Cells were grown in the Tecan plate reader overnight at 37oC with shaking for a period of 16 hours. OD600 measurements were taken every 30 minutes. In addition RFP readings were also taken with 565nm excitation and 605nm emission wavelengths.
Values were normalised by OD and are shown as a mean of four repeats.
In the graphs above, a trend can be seen for all concentrations of oleic acid, that fluorescence produced from constitutively expressed RFP increases over time. In comparison there was no observable trend in fadR-RFP expressing cells, showing no increase in fluorescence in any oleic acid concentration. In addition there was no difference in the amount of fluorescence observed between the fadR-RFP and the fadR only and empty cells.
The FadR promoter and BBa_J23110 constitutive promoter upstream of RFP are the same in strength and so this disregards the observed difference in expression under the two promoters being one based on strength. Further investigation was carried out using fluorescence microscopy to measure RFP fluorescence in response to different oleic acid concentrations and a similar result was observed.
Based on this information and the results shown above, we suggest that the FadR promoter does not result in expression of RFP downstream in the presence of oleic acid.
