Team:PEA Exeter NH/Abstract Raw

Three integral concepts underlie the design of our parts: upstream regulation, fluorescence and pigmentation. Upstream regulation is the idea that the expression (quantity of transcribed protein material) of a gene primarily depends on the region directly upstream of the coding region of the gene, which is known as the binding site for RNA Polymerase. In addition, expression of a gene can be regulated by other DNA sequences, called signature sequences. Signature sequences are binding sites for proteins called transcription factors. These transcription factors modify the affinity of the binding of RNA Polymerase to the promoter region, thus modifying the rate of transcription. If the binding affinity increases, then the transcription factor is an activator for that specific gene. Likewise, if RNA polymerase is less likely to bind, then the transcription factor is a repressor for that specific gene.

Fluorescent proteins are those that emit visible wavelengths of light upon exposure to lower wavelengths. The chemical mechanism for fluorescence comes from the nature of quantum mechanics, which states, in short, that electrons may only occupy discrete energy shells associated with discrete amounts of potential energy. The further away an electron is from its neighboring atoms, the more potential energy it has. However, electrons far away from nuclei of the atoms will be attracted by the Coulomb force, causing a discrete decrease in potential energy. Due to the conservation of energy, this energy is then emitted as a photon. Fluorescence occurs when a step of the return of the electron to the ground state, where it has the lowest possible potential energy, causes an emission of visible light. The emitted light is necessarily of a lower energy level and thus higher wavelength than the light triggering fluorescence due to the conservation of energy; heat (high wavelength light) releasing steps along with one or many visible light releasing steps combine to convert the energy from the absorbed light back to light energy. Biologically, a fluorescent protein is typically used as smaller fluorescent particles such as metals cannot be expressed via genes. The green fluorescent protein (GFP), expressed naturally in the jellyfish Aequorea Victoria, is the first example of a fluorescent protein successfully expressed in E. coli bacteria in 1994. Since then, the gene has been chemically modified to express blue (BFP) and red (RFP) fluorescent proteins.

Pigmentation is the ability for a compound to absorb specific wavelengths of light and reflect others. The chemical basis for this is, once again, quantum mechanics. As each wavelength of light is associated with a different amount of energy, certain wavelengths of light will excite electrons and others will not. Those wavelengths that do excite electrons are absorbed by electrons of the protein. The return of the electron to its ground state in pigmented molecules does not include a large quantum level change as it does in fluorescence; instead, the return of electrons to its ground state with respect to the protein contains multiple steps, each of which releases heat. Importantly, the same electron does not execute this multi-step release; indeed, it is unfeasible for this to occur due to the quantization of energy. Instead, the electrons’ energy release trigger the excitation of other electrons, in a chain reaction eventually dissipating all of the initial energy of the photon as heat. In addition to this, some wavelengths of light are instead reflected, which occurs when the light cannot be absorbed by any electrons in the compound. This occurs when the light has an inappropriate amount of energy so that no change in quantum state for the electron would conserve energy upon photon absorption. Since white light is a combination of multiple wavelengths of visible light, a pigment will reflect some of the constituents of white light and reflect others. This causes the pigment to be visualized by the color that it reflects. Biological examples of pigments include the chlorophyll of plants giving plants their green color, the melanin of animals that determines skin color, and the hemoglobin in vertebrate red blood cells. The gene-regulated production of specific fluorescent and pigmented protein products allows for visualization of gene activity, corresponding to the presence of an activator or the absence of a repressor. This visualization is a concept integral to the identification of catechol and lead.

In order to detect catechol, we will use the downstream element BBa_K118021, whose protein product, catechol-2, 3-dioxygenase, catalyzes the conversion of catechol, in an oxygenated environment to the fluorescent yellow pigmentation molecule 2-hydroxy-cis,cis-muconic semialdehyde.

The catechol detection mechanism can be extended to detect naphthalene via the catabolism of cytosolic naphthalene into catechol. The nahR gene, on plasmid NAH7 (BBa_J61051), controls naphthalene degradation. It is organized in two operons which encodes six enzymes in nahA-F, and eight enzymes in nahG-M. The “upper operon” or nahA-F on the NAH7 plasmid degrades naphthalene to salicylate and pyruvate. Then, the “lower operon” or nahG-M encodes enzymes that degrade salicylate into intermediates of the TCA cycle. Thus, this gene breaks down naphthalene in a cell environment into detectable catechol.

In order to detect lead, we will transform the E. coli with a lead promoter (BBa_I721001) and a lead binding protein (LBP) (BBa_I721002). When lead enters the cell, it binds to the LBP, triggering a conformational change in LBP. This change causes LBP to dimerize. The resulting dimer has greater affinity for the lead promoter. Hence, the presence of lead induces transcription of downstream genes. We will place the gene coding for a red fluorescent protein (BBa_E1010) downstream of the lead promoter; this means that after lead enters the cell, a red fluorescent protein will be transcribed. This will allow observers to quickly and efficiently learn if there is a risk of lead poisoning without having to carry around testing kits.

In addition to detection of the elements and compounds listed above, our cell will also be able to unspecifically identify other toxic fracking byproducts via a nonspecific SOS cell damage element. The promoter for this gene is a combination of signature sequences insertable on sulA. These sequences bind to SOS repressor proteins that E. coli typically destroy after detecting stress from damaging toxins or radiation. This promoter (BBa_K518010) has been isolated and will be attached to a downstream element (BBa_K592009) coding for a blue-colored protein, so that bacterial cells exhibiting stress – specifically from the presence of toxic or radioactive runoff from fracking – can be easily discerned.