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Making T7 Promoter Variants

To produce these T7 promoter variants, we use a two-step PCR process. The first PCR, which we call "gene-specific" PCR, is a typical PCR that adds a ribosome-binding site (rbs) in front of either RFP or the lysis operon and adds a terminator downstream.

With this PCR product now serving as the DNA template, we start a second PCR which we call the "extension" PCR. It extends the product by adding the T7 promoter, with or without a lac operator downstream. In these illustrations, we have substituted the lysis operon for the RFP gene but the same process is done for RFP.

In the last stage, we run another PCR with a set of primers that will add Gibson overhangs for the Gibson assembly that will add this promoter construct into the desired plasmid.