Difference between revisions of "Team:Queens Canada/Design"

We have taken two approaches to the development of biological sensors, or biosensors, for the measurement of Cortisol: Firstly, we have constructed a reagentless, and continuous glucocorticoid sensor which utilizes changes in Fluorescence Resonance Energy Transfer (FRET) to detect hormones. Secondly, we have begun developing a novel, and portable biological sensor which utilizes intein splicing to produce a luminescent signal, for hormone quantification. We will explore both of these methods in depths, as well as their advantages and disadvantages.

Approach 1: Fluorescence Resonance Energy Transfer

Fluorescence Resonance Energy Transfer (FRET), is a mechanism of energy transfer between light-sensitive molecules, such as fluorescent proteins. Fluorescent proteins work by absorbing light at a peak wavelight, called the excitation wavelength, and emitting light at a higher wavelength, called the emission wavelength. When two fluorescent proteins are within close proximity of each other, it is possible to excite the first fluorescent protein at its excitation wavelength, and produce emission of the wavelength from the second fluorescent protein. For example, the excitation of Cyan Fluorescent Protein is 436 nm, and its emission is 488 nm, while the excitation of Yellow Fluorescent Protein is 517 nm and its Emission is 528 nm. If Cyan Fluorescent Protein, and Yellow Fluorescent Protein are in close proximity, excitation at 436 nm will result in emissions at both 488 nm and 528 nm. The efficiency of this energy transfer process is dependent on the proximity of the two fluorophores, and can therefore be used to quantify minor changes in the structure and activity of proteins.

Our FRET Biosensor

red-green FRET Pairing

Approach 2: Inteins

Found in organisms from all domains, inteins (intervening proteins) are auto-processing proteins that function both in endogenous and exogenous contexts [1]. These proteins are involved in the cleavage and formation of peptide bonds during a unique process where they excise themselves from a polypeptide and ligate the flanking extein (external protein) [1]. This spontaneous splicing process occurs post-translationally and is most commonly observed in proteins involved in DNA transcription, replication and maintenance processes within a cell [1]. Inteins have such great potential in protein engineering because of their rapidness, the induced splicing and joining of the exteins to create a functional extein protein is much quicker than the typical transcription and translation process.

Small Molecule Triggered Intein Splicing

The mycobacterium tuberculosis RecA intein was selected for use as it has been shown to splice in a wide variety of protein contexts [2]. Small molecule triggered intein splicing allows the production of a “molecular switch” which is only activated in the presence of the designated ligand. To function, this system requires that the intein is able to bind with a high affinity to its specific ligand, and that the resulting conformational change initiates the process of protein splicing [2]. The above graphic demonstrates a scheme for directed evolution of small-molecule triggered intein splicing, applied to the estrogen receptor [3]. We will utilize the same system, but instead of the estrogen receptor ligand binding domain, we will attempt to evolve the glucocorticoid receptor to induce splicing upon binding to cortisol. For the initial application of our biosensor system, we will be using the binding of cortisol to the human glucocorticoid receptor as the initiating reaction that triggers intein splicing.

Customizable Biosensors

The ability to interchange nuclear receptor in our modular intein system, results in the potential to detect hundreds of potential biological molecules. A few examples include, estrogen, retinoic acid, and cortisol. The ability to interchange the extein spliced together upon ligand binding, results in the capability to produce a signal in countless forms. A few examples include, luminescence, growth in antibiotic media, and florescence. We decided to start by constructing the 4-Hydroxytamoxifen dependent estrogen receptor intein construct, developed by Buskirk et al. (2004). We chose to began with the construct in the Kanamcyin Resistance Context, so that we could characterize the ability of the small-molecule triggered intein splicing system in bacteria, when it had been initially demonstrated in yeast [2]. We would simultaneously construct a novel glucocorticoid receptor intein in the Kanamycin Resistance context, so that we could select bacteria which were capable of successfully performing small-molecule triggered intein splicing. If no colonies formed, we planned to perform directed evolution until the system was capable of producing a splicing event.

Split NanoLuc Luciferase Domain

Upon producing cortisol-dependent intein splicing, we can then swap out the flanking extein sequence with a quantitative reporter. We therefore chose NanoLuc® Luciferase as the extein and quantitative reporter for our construct. NanoLuc® by Promega is a luciferase derived via directed evolution from the luminous shrimp, Oplophorus gracilirostris [3]. The enzyme was obtained from deep-sea shrimp and optimized following the discovery of a novel substrate, furimazine, which allows for the production of visible light with less background activity than other luciferases [4,5]. NanoLuc® is a 19.1 kDa monomeric protein that is both soluble and ATP-independent [4]. Compared to firefly (Lampyridae) and sea pansy (Renilla) luciferases, this novel protein offers many advantages reflected by its increased stability, smaller size, and >150-fold increase in luminescence [5]. The unique characteristics of this enzyme construct combined with its high luminescence activity allow for the production of a very sensitive diagnostic assay. The split site for NanoLuc® Luciferase was chosen between the amino acids 52 and 53, as this split was previously determined to produce the highest luminescence upon successful rejoining of the N and C termini [6].

Comparing and Contrasting the two Biosensor

FRET - reagentless, continuous, small, but not portable, requires a fluorometer or a confocal laser imaging microscope. Intein - Small, portable, but irreversible reaction, and requires a luciferin substrate to produce a signal