Ok, I should forget about the traditional pathway and try to find other ways. We should return back to the initial question --how does DNA pass throught the first membrane? There are two pilQ homologue in E. coli, hofQ and gspD. HofQ has been reported to be required for DNA as a nutrient, however, it is not the channel for our transformation. I will test the second candidate gspD and the primers should be on the way. If it is true, this might tell us the story of the criptic regulon gsp. If not, we will be blind again.
I discussed with my mentor about exploring real chromosomal transformation. He warned me that innumerous failures were on this direction for years before. We do not know whether sxy gene, which is required for natural transformation in H. influenzae and V. cholerae, express in E. coli and if so under which condition it can be turned on. In addition, in V. cholerae, except for sxy, HapR is required for the V. cholerae transformation. We do not know whether similar or additional regulators are required for turnning on competent genes in E. coli. We have evidences showing that rpoS is required for our transformation. HapR is under the regulation of rpoS in V. cholerae. However, hapR homologue was not found in E. coli genome.
没有评论:
发表评论