Many synthetic biology applications require that a given gene be expressed in either a constitutive (always on) or binary (on-off) fashion, and to meet these needs a number of constitutive and inducible systems have been characterized. However, for other applications, it would be desirable to express a transgene at one finite, specified level while in the basal state, and then to increase expression upon induction some upstream pathway or biosensor. However, to date, no such technology exists. Because our project requires such a dual-state expression system, we therefore developed a new technology we term a "dual-state promoter,” which can provide specialized transcriptional control over a gene of interest. In its construction, we placed an inducible promoter upstream a constitutive promoter and separated the two by a nonsense spacer region. We hope our experimentation with this novel transcription regulatory construct will lay the groundwork for future iGEM teams to design alternative dual-state promoters.

A dual-state promoter provides two modes of transcription: the constant basal state, and the induced elevated state.

The utility of a dual-state promoter

There are several advantages to dual-state promoter. For example, when our system is used in conjunction with the correct gene, it will be useful not only to combat drops in pH due to secretion of lactic acid by harmful oral bacteria like S. mutans. It will also be useful in instances where a pH drop is related to consuming a particularly acidic food, or when it is related to vomiting.

Additionally, a dual-state promoter is able to achieve the same effect as duplicated genes regulated by two separate promoters in less space. This means we are able to place an entire suite of genes on a plasmid without running into constraints in length. Most importantly, as stated before, some circumstances require a constant, basal level of expression until an upstream pathway or biosensor inducts an elevated response. Since our dual-state promoter is modular, it can be used in any system where such transcriptional control is useful.

For example, as it relates to oral health, a constant, tunable, basal level of expression of genes which promote oral health may be enough to combat slight fluctuations in pH in the oral microbiome. However, after a large meal or a particularly sugary snack, this constant, basal level of expression may not be insufficient in maintaining healthy pH levels, at which point the dual-state promoter should induce transcription of these orally-beneficial genes at elevated levels. Nonetheless, a basal level of expression is important both to conserve resources for the bacteria, and to make sure an elevated response is not induced unnecessarily. Depending on the gene regulated by the dual-state promoter, an unnecessary, exaggerated response could actually be harmful. Both the basal state and the elevated state are necessary levels of expression when addressing the issue of oral health.

Design of a pH-inducible dual-state promoter

The NU-tralize team identified the need for a pH-inducible promoter in a system designed to combat tooth decay. When the pH of the mouth drops below 5.5, demineralization of the tooth enamel and dentin occurs, over time leading to cavity formation. By using a pH-inducible promoter that is sensitive to pH levels at or near pH 5.5, the dual-state promoter can respond with elevated levels of transcription of a gene of interest only when necessary. Such a situation may occur when basal levels of expression, as defined by the constitutive promoter, are not enough to combat a particularly large drop in pH in the oral environment. Our team focused on the promoter regions of two acid-inducible genes native to E. coli: the asr and gadA genes. For more information on why we targeted these pH-inducible promoter regions, please see: pH Detection.

Inclusion of a spacer region

Including a nonsense spacer region accounts for the simple geometric fact that the length of the RNA polymerase is slightly longer than the promoter. The RNA polymerase? could block the DNA immediately upstream of the promoter. We hypothesize that if two promoters are connected in series, the probability of having two polymerases binding to the the two promoters at the same time is either very low or impossible. To test this, we included a construct in which we did not include a nonsense spacer. If our hypothesis is true, the construct should behave more as a superposition of the two promoters rather than two promoters. If the two promoters in our dual state promoter are acting independently, we should have a significant increase in translational products upon induction. This spacer-less construct will act as a control to verify our hypothesis.

Choosing a spacer length was based on the size of RNA polymerase. No literature has looked closely at the biophysics of RNA polymerase initiation from nearby sites, so the team predicted the length scale of the polymerase would be most important in affecting how the two promoters interact. We found a polymerase from pdb.org (the protein data bank website) and visualized it on VMD (Visual Molecular Dynamics) to determine the length of the polymerase. The length roughly corresponds to 64 base-pair (bp). For the second spacer, we decided to double the length, making a 128 base-pair spacer.