OK, so the induction of CRP-S genes experiments established clearly that the ppdA gene's CRP-S promoter isn't being induced by the growth conditions I tried. And that changes in beta-gal activity don't necessarily result from changes in promoter activity. What to do now?
To make progress in these experiments, I need better tools for manipulating genes. I want to put the ppdD::lacZ fusion into the chromosome (it's on a plasmid now) and I want to test the effect of knocking out various genes, and I want to try a 'wild' E. coli strain. All of these require recombining desired genes into the chromosome, so I need to get the recombineering technique working for me. One of the post-docs was working on this, so I just need to take up where she left off.
I also need to get back to the tests I was setting up of components of the laser-tweezers project. I was just about ready to test attaching DNA to beads (have the beads with the bound streptavidin, have the biotinylated nucleotides to put on the ends of the DNA, have the DNA prep). Separately, I need to work with the new post-doc on an antibody-based method to attach H. influenzae cells to other beads, so they can easily be pushed around under the microscope.
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Why do not you do your experiments on solid surface? That is, instead of testing these competence genes in liquid culture, you can test their activities on solid LB-Agar surface. You might get a group of different data. The gene expression pattern on solid surface should be different from that in liquid culture. The problem is how to assay protein activity on plates. I am also seriously thinking about this question.
ReplyDeleteI'm using growth in well mixed liquid because this exposes all cells to the same conditions. Cells growing on agar experience very heterogeneous conditions. This increases the likelihood that SOME cells will encounter conditions that induce competence genes, but makes it harder to detect any such induction and to investigate its cause.
ReplyDeletep.s. It's nice to see another open-science blog about competence!
I competely agree with you that it is more difficult to investigate transformation on plates and that investigation in liquid culture is desired and preferred. But the recent evidences forced us to face the difficulty if we want to know natural transformation in E. coli. Firstly, three independent groups(including our group) found that transformation of E. coli occurrs on the surface of solid medium (J Biomed Sci 2002; 9:246-252)(FEMS Microbiol Lett 2004; 236:61-64)(FEMS Microbiol Lett 2006; 265:249-255). All of these reports reported that their attemps to transform E. coli naturally in their transformation systems are not successful. Secondly, at least three independent groups found that none or very weak expression of type IV pili in liquid culture ( J Bacteriol 2000; 182:848-54; J Bacteriol 2001; 183:6288-93). These evidences might imply that competence genes might be dorminant in liquid culture but could be turned on when they were exposed to solid surfaces. Another conception biofilm might be able to partially explain this (FEMS Microbiol Lett 2004; 236:61-64), but genetic approaches combining with other techniques are required to reveal the detailed picture. Although some of our later investigations did not support the conclusions made by previous investigations, all of our evidences support the idea that transformation of E. coli occurs only on plates. So we might have to face the challenge.
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