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

Revision as of 20:34, 8 September 2018

In The Glow: Luminescent Biosensors for Hormone Detection and Diagnosis

Background

Biosensors

Biosensors are devices that are able to detect the presence of analytes and effectively convert this biological response to an electrical signal. This system consists of three components: a bioreceptor, transducer and detector. The bioreceptor is able to form substrate-specific interactions with the analyte. The transducer is then able to detect the substrate-receptor interaction and transmit this into an electrical signal, which is amplified and processed by the detector. This information is then capable of being sent to a data storage device for quantification and analytical purposes.

UPDATE THIS IMAGE PLEASE!

Ligand Binding Domain

Nuclear receptors are a family of evolutionarily conserved proteins that functions as a ligand-dependent transcription factor [1]. After binding certain ligands, the receptor undergoes a conformational change which activates them, and allows them to bind directly to DNA to alter gene transcription [1]. Circulating steroid hormones, like cortisol, are able to activate the receptor and mediate processes such as stress response, energy metabolism and immune responses [2]. The ligand binding domain of nuclear receptors generally consists of eleven alpha-helices and two beta-sheets that enable the formation of a three-layered protein structure [2]. There also exists a regulatory C-terminal helix, titled "helix 12”, that is essential for hormone binding. There are conserved residues within these helices which form critical interactions with carbon atoms of cortisol and allow for specificity within the interaction [2].
Although our team is starting with the glucocorticoid receptor, the homology in steroid hormone receptors allows for the potential ability to exchange receptor types in order to quantify a vast array of different analytes. Both of the detection methods we have developed utilize conformational changes in the nuclear receptor ligand binding domain to produce a measurable signal.

Intein Splicing Domain

Found in organisms from all domains of life, inteins (intervening proteins) are auto-processing proteins that function both in endogenous and exogenous contexts [3]. 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) [3]. 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 [3].

Intein Splicing Mechanisms

The Class 1 Splicing Mechanism: Step 1
The intein splicing mechanism consists of a series of acyl-transfer reactions that results in peptide bond cleavage at the junction between the intein and extein, followed by the formation of a new peptide bond between the N and C termini of the exteins [3]. Either a cysteine or serine residue is almost always present at the N terminus of the intein, which attacks the carbonyl carbon of the N-extein residue to produce an intermediate [3]. Next the C-extein residue undergoes nucleophilic attack, followed by cyclization at the C-terminal of the intein [3].

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 [6]. 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 [6]. For the initial application of our technology, we will be using the binding of cortisol to the human glucocorticoid receptor as the initiating reaction that triggers intein splicing.

Previous Part: 4HT Dependent Intein

The 4HT Dependent Intein splices in the presence of 4-hydroxytamoxifen which allows for the ability to control the engineered protein in a dose-dependent manner.
Our team will utilize cortisol binding to the ligand binding domain of the glucocorticoid receptor as a signal to trigger the intein excision and extein ligation process.

Split NanoLuc Luciferase Domain

NanoLuc® is an engineered protein produced by Promega following directed evolution of luciferase derived from Oplophorus gracilirostris [4]. This enzyme obtained from deep-sea shrimp was optimized following the discovery of a novel substrate, furimazine, which allows for the production of visible light with less background activity [4,5]. NanoLuc® is a 19.1 kDa monomeric protein that is both soluble and ATP-independent [4]. Compared to Firefly and Renilla luciferases, this novel protein offers many advantages reflected by its increased stability, smaller size and >150-fold increase in 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.
Based on these findings. A Split sites for NanoLuc Luciferase was chosen between the following amino acids: 52/53; The N- and C-termini were inverted and reconnected through a flexible GGGGS–GGGGS linker.

@@ Line 26: / Line 26: @@
 <h3>Biosensors</h3>
 <p>Biosensors are devices that are able to detect the presence of analytes and effectively convert this biological response to an electrical signal. This system consists of three components: a bioreceptor, transducer and detector. The bioreceptor is able to form substrate-specific interactions with the analyte. The transducer is then able to detect the substrate-receptor interaction and transmit this into an electrical signal, which is amplified and processed by the detector. This information is then capable of being sent to a data storage device for quantification and analytical purposes. </p>
+UPDATE THIS IMAGE PLEASE!
 <img src="https://static.igem.org/mediawiki/2018/7/79/T--Queens_Canada--Project_Biosensors.png" width=50% height=50% />
 </div>