Hello researchers,
I am currently performing Dual-Luciferase assays in S. cerevisiae to study the response of a promoter to Mn2+. As a control, I integrated the expression cassette containing the (constitutive) promoter of the triose phosphate isomerase (pTPI), the Firefly luciferase and the CYC1 terminator into the his3delta locus.
We surprisingly observed a decrease in the signal of the Firefly after exposure to Mn2+. I thought that the "constitutive" nature of pTPI would be sufficient to give us a constant signal despite manganese presence. We are investigating the potential interaction of Mn2+ with Firefly, but it does not seem to be responsible.
Is it possible that the promoter of HIS3 gene still present in the genome right before pTPI could impact its expression pattern?
Should have I added a terminator before the pTPI promoter in my cassette?
Thanks in advance
Yes, the expression of a gene integrated into the yeast genome can be influenced by upstream promoters. In molecular biology, the term "promoter" refers to a specific DNA sequence that regulates the initiation of transcription, which is the process by which a gene's DNA sequence is copied into RNA. Promoters are typically found upstream of the gene they regulate.
In yeast and other eukaryotic organisms, including humans, the expression of a gene is controlled by the presence of specific promoter sequences and associated regulatory elements. These promoter regions contain binding sites for transcription factors, which are proteins that can either enhance or repress gene expression. When transcription factors bind to the promoter region, they can either facilitate or inhibit the binding of RNA polymerase, the enzyme responsible for transcribing the gene into RNA.
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