Team:Virginia Commonwealth/Design

Promoter design

Promoters classes

• Constitutive promoters
  • There are several types of constitutive promoters that we can focus on. These promoters are separate in regard to their respective sigma binding domains. Constitutive promoters that interact with the sigma 70 domain of RNAP are most common most prevalent being as how sigma 70 RNAP is most common at normal cellular growth conditions. The alternatives would be sigma 54 and sigma 32 binding factors, these types of polymerases are more active when the cell is under certain strain, such as heat shock of nitrogen starvation.
  • There has already been considerable work done with sigma 70 RNAP including an established promoter library of varying promoter strengths available on the parts registry. We will develop and implement a method to fully characterize and standardize these promoters' activity and upload it to the registry.

• Promoter UP Elements
  • Work has been done to identify a consensus UP element on some promoters which interacts with an alpha subunit on RNAP. This UP element has been shown to significantly increase the recruiting power of promoters. We are working on creating this UP element as a modular BioBrick part and characterizing its activity using the promoter characterization method that we develop. We will also manipulate this element using a bottom-up design approach to get UP elements of varying degree's of strength. Thus increasing the level of control we have over gene expression.

• T7 Promoters
  • T7 phage promoters have long been utilized as a way to create orthogonal gene expression. There is very little variation in the strength of T7 promoters available via the BioBrick registry. Our goal is to identify a consensus T7 promoter sequence and use our bottom-up design approach to create a library of T7 promoters which we will characterize and upload to the registry.

Design strategies

Promoter design will be approached from the bottom up. First, a consensus promoter sequence will be studied and accepted by researching the work done by others on various promoters. This consensus will be our starting point as we identify which nucleotide and nucleotide sequences are most important to RNAP binding affinity. We will use direct synthesis to construct promoter sequences that we will test.