Hi Experts,
Recently, I performed a mutagenesis experiment and correctly generated a mutated binding site on the promoter region of my reporter gene assay for my TF of interest. I verified the mutation and it is correct. I have an issue interpreting the data though.
My questions:
1. Is it possible to have a 100-fold decrease in promoter activity by changing just one TF binding site?
2. Isn't there are also other TF sites that could be important for basal expression? I'm now wondering if that binding site is so "critical" even for basal expression of the gene.
Has anyone ever experienced this phenomenon? Please let me know your thoughts. Thanks in advance!
Hi Edward,
I am trying to do what you successfully did in the above experiment. What would you suggest is the right strategy to go about doing mutagenesis. Should I simple replace the binding seq with something inert i.e. multiple Cs. Or is there a more refined way to go about doing this?
Hi Ila,
I designed at least two site-mutagenesis to investigate the role of a cis-regulator element. Changing those nucleotides depends on what you want to achieve, for me, I just destroyed the binding capacity my mutating at least 2 nucleotides into their non-conserved counterpart. Are you trying to achieve the same thing as my experiment? I hope this helps. Ila Mishra
Hi Edward, thanks for your response.
Yes, I want to destroy the binding capacity.
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