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Design to detect transcription factor specificity

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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Design to detect transcription factor specificity

Postby sam2400 » Thu Jun 23, 2011 1:29 pm

Hi I am creating an experimental design to identify the target of a theoretical bacterial transcription factor of unknown specificity, but known sequence. Basically I need to know which genes it regulates. My thoughts so far were to

a) Run a blast search for the nucleotide sequence of the TF which would shed some light on its possible function by returning homologues/similar sequence from other bacteria.

b) Disrupt the gene and clone into a plasmid (with detectable marker), and homologous sequence for recombination, transform plasmid into the original strain and select for recombinant bacteria. Isolate and grow on cell culture representative of the bacteria's naturally colonised tissue. Compare to a non-recombinant control strain to detect functional changes alluding to which genes are controlled by the unknown transcription factor.

Does anybody have any thoughts on whether this design would be suitable to detect the genes which the transcription factor operates on. I have managed to confuse myself a little and am not sure whether I have got the wrong idea or not.

Thanks
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Postby JackBean » Thu Jun 23, 2011 1:51 pm

a) probably won't work. The TFs are similar in global, but the local sequence, which binds DNA may be completely different.

b) yeah, either knok-in or knock-out your gene and see the difference on chip.

c) Chip-on-CHIP

d) get library of bacteria DNA and marked TF, put it together and see, which DNA part will be bound (like if you were doing hybridization with probe)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby sam2400 » Mon Jun 27, 2011 3:54 am

Thanks, thats brilliant. I was looking into the hybridisation with the tagged TF which seems like it would work. Also with the chip will it be appropriate for bacteria? I just got a little confused as bacteria do not have chromatin?

Thanks
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