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

put image of dual state

Insert IMAGE 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.

e low copy plasmid pSB4A5 will be used for the different promoter constructs. The asr and gadA promoters were extracted from the E. coli genome via colony polymerase chain reaction (PCR). The constitutive promoters TacI and Lpp were amplified from pDAK1 and pDAK2 donated by the Jewett Lab6,7. The restriction enzyme cut sites EcoR1, Pst1, Spe1, and Xba1 were used in ligation to create the different constructs (Figure 3). When multiple parts were connected a mixed site was formed between Spe1 and Xba1, which cannot be cut, by any of the restriction enzymes. This leads to the benefit of not having a restriction site in the middle of the construct, and furthermore these standard restriction enzymes can always be used with the final dual-state promoter constructs.