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So what next? Well I now had confirmation of my suspicions about those three CRE elements and which genes they might control. The next step was to determine whether expression of those genes was actually tied to competence-inducing stimuli. To do this, I used a relatively new technique called Quantitative Real-time PCR. In essence, this technique would allow me to examine the expression of genes from the CRE elements as cells became competence by monitoring the change in quanitity of the mRNA from those genes. Expression profile of tfoX, rec-2 and comA during competence development. Q-PCR was employed to examine transcription of known competence genes during development of competence in MIV media. Values represent the fold change in expression compared to levels in the control (pre-treated) sample as determined by the 2-ΔΔCt method. Error bars indicate the range in fold change as determined by this method. tfoX is represented by black bars, rec-2 by gray bars and comA by white bars. CRE sites are present upstream of rec-2 and comA and both have been shown to be dependent on tfoX for expression. The transformation frequency at each timepoint is shown by the closed circles. Expression profiles of genes contiguous with CRE0364 during competence development. Q-PCR was employed to examine transcription of HI0364 (black), HI0366 (gray), and HI0368 (white) during competence development in MIV media. Values represent the fold change in expression compared to levels in the control (pre-treated) sample as determined by the 2-ΔΔCt method. Error bars indicate the range in fold change as determined by this method. The transformation frequency at each timepoint is shown by the closed circles. Expression profiles of genes contiguous with CRE0937 during competence development. Q-PCR was employed to examine transcription of HI0937 (black), HI0938 (gray), HI0939 (hatched gray), and HI0942 (white) during competence development in MIV media. Values represent the fold change in expression compared to levels in the control (pre-treated) sample as determined by the 2-ΔΔCt method. Error bars indicate the range in fold change as determined by this method. The transformation frequency at each timepoint is shown by the closed circles. Expression profiles of genes contiguous with CRE1181 during competence development. Q-PCR was employed to examine transcription of HI1181 (white) and HI1182 (gray) during competence development in MIV media. Values represent the fold change in expression compared to levels in the control (pre-treated) sample as determined by the 2-ΔΔCt method. Error bars indicate the range in fold change as determined by this method. The transformation frequency at each timepoint is shown by the closed circles. These data basically proved that I was correct in my hypothesis that these three operons were the true targets of CRE0364, CRE0937 and CRE1181. So, could any of those genes help me fill in that model I previously discussed?
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