Northwestern iGEM

Abstract

The pH of the mouth drops several times per day after mealtimes, which results in severe tooth decay over time if not addressed. One way to combat this problem is to engineer the microbiome of the mouth to respond dynamically to changes in pH. To enable this, here we present the development of a novel dual-state promoter for the detection of and response to pH fluctuations in the mouth has been started. This novel transcription regulation element will be capable of having both constitutive and inducible activity. This is achieved by linking a constitutive promoter downstream a pH-inducible promoter. We plan to characterize our dual-state promoter by first linking single promoters upstream green fluorescent protein (GFP) and assess the transcriptional activity via fluorescence assays. This will be followed by assays on the dual-state promoter. The hypothesis is that the pH-inducible promoters will have minimal activity at a neutral pH, with an intense activity as the pH nears and crossed the cavity formation threshold. Furthermore, it is hypothesized that the constitutive promoters will have a constant activity, independent of pH. The dual-state promoter construct is hypothesized to have the combined effect of both of the promoters, with a constant basal expression as well as a pH-inducible response. This technology will have future applications in any system requiring pH detection and response.

Figure 1: The Stephan Curve4, depicting pH fluctuation in the mouth over 24 hours.

The ultimate goal of this research is to create a novel biological system that ensures that the demineralization threshold is never crossed in the oral biome. In order to do this, one of the native bacteria, Veillonella parvula, will be engineered to: (1) detect and respond to pH drops due to lactic acid and (2) catabolize lactic acid in order to negate a drop in pH. Since V. parvula naturally contains lactic acid catabolic pathways, the detection and response aspect of this system will be the scope of this project. This technology has the potential to dramatically change the oral health industry, but it also has applications to any biological system where pH monitoring would be necessary, including water sampling, food processing, and many other industries. The newly expanding field of synthetic biology enables this problem to be addressed in ways never possible before. The standardization inherent to the synthetic biology field means that genetic manipulation of a model system can in theory be easily translated to other species with the same genetic framework. Since the genome of Escherichia coli is well characterized, it will be used as the model system. The goal of this research is to create a system that ensures that oral pH is never low enough to cause enamel demineralization, so construction of a simple promoter will not suffice, as the system needs to respond to the dynamic pH environment found on the surface of the enamel. Instead, it is desired for gene expression to be directly proportional to the concentration of lactic acid. In order to address this particular problem, we must fundamentally create a new transcription regulation system: a dual-state promoter. A dual-state promoter is a promoter in which two “states” are possible. One obvious example would be similar to an electric switch – the potential states being on and off. For our system, the promoter must be more complex. In order to create the promoter, a two-part approach will be utilized. First, a constantly active, constitutive, promoter will be used to continually activate the given response genes at a background, or basal, level. This will cause gene expression when lactic acid concentration is low. However when the lactic acid concentration increases, such as after meals when S. mutans catabolized sucrose, an inducible promoter is also desired. This promoter will be pH-induced and will respond to large increases in lactic acid concentration. As a result, the desired promoter will function as both constitutive and inducible. The promoter will be constitutive at a basal level and then induce the gene(s) of interest at a higher level in response to a pH that nears and potentially drops below 5.5. Therefore the two states for the promoter will be low activity and high activity, and the state the promoter is in will be dependent upon pH. This will be done by placing a pH-dependent promoter upstream of a constitutive promoter to allow dual control of the downstream gene expression (Figure 2).

put image of dual state

Figure 2: A schematic of the dual-state promoter that will be developed in this research.

The asr and gadA promoters5 will both be options for the pH-induced promoter in Figure 2. The asr promoter will be taken from the acid-shock response (asr) gene in Escherichia coli. The gadA promoter will be taken from the first of the glutamic acid decarboxylase (gad) genes (known as gadA). Two known constitutive promoters, Tac and Lpp6,7, will be tested in combination with these pH-induced promoters to find the optimum construct. Further optimization will be explored by varying the length of the spacer region between the two promoters. The spacing between the two promoters is important in the event that two RNA polymerases bind to the both promoters at the same time. Optimal spacing will decrease any potential steric hindrance between the two enzymes while also minimizing the distance said enzymes must travel along the DNA prior to encountering the start codon and beginning the elongations step of transcription. Modeling using Pymol has shown that a RNA polymerase is roughly equivalent in length to 64 base pairs of DNA when the DNA is in the helix formation. A total of 12 combinations will be used to create the library of dual-state promoters. Our hypothesis is that the Asr promoter combined with a 64 base pair spacer and the Tac promoter will have the greatest ability to ensure that oral pH does not drop below 5.5 for two reasons: (1) the Asr promoter has been seen to express at a slightly higher level than gadA at low pH5, and (2) the 64 bp spacer should minimize both steric hindrance and the distance RNA polymerase must travel in order to begin elongation of mRNA. Green fluorescent protein (GFP) will be used to measure the effect of the promoter combinations tested at varying pH levels in order to show both basal and inductive gene expression since it is a simple qualitative assay for measuring the level of transcription. The developed promoter will have numerous applications for use in pH environments to increase the regulation of pH related genes.