Team:The Citadel-Charleston/ProjectOverview


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Overview    Peptide YY   Population Control


Summary of the Project

     The Citadel team decided to tackle a unique and exciting challenge for iGEM 2010.  In humans and many other mammalian species there exists an intricate communication network between the digestive system and the central nervous system (CNS) called the brain-gut axis.  Interactions between the two regions of the axis are performed by hormonal peptides produced by cells within the gastrointestinal tract.  These proteins are absorbed into the bloodstream, traverse the blood-brain barrier, and interact directly with neurons within the brain to regulate the intake of calories.  Inspired by this connection between the gastronomic system and the CNS, and acutely aware of the plethora of engineer-able microbes already inhabiting the human GI microbiome, The Citadel team set out to harness this potent natural system for applications to human and animal health, especially in the areas of medical diagnostics and drug delivery.  The student researchers pursued a project that would demonstrate the production in E.coli of a novel protein associated with the brain-gut axis,as well as indicate the far-reaching potential for engineered microbes to influence complex systems in remote regions of the body.  Hunger was an especially appealing target for bacterial influence, and quickly became the focus of the project.  The team identified a protein, Peptide Tyrosine Tyrosine (3-36), abbreviated PYY3-36 which is naturally generated by L cells within the intestines in response to calorie absorption after a meal.  PYY3-36  migrates across the brain-gut axis and binds to receptors within the hypothalamus, triggering the activation of pathways that result in taste aversion, a decrease in pancreatic secretion, and a reduction in appetite.  The Citadel team converted the PYY3-36 gene into the Biobrick standard format and designed a system for it's expression and control.


Genetic Circuit Design

     There are two separate modules contained within the circuit envisioned by The Citadel iGEM team.  The next few paragraphs are devoted to a explanation of how these modules were designed and what they accomplish.  Please refer to the illustrations – they  will be very helpful as you follow the description of the circuit.


The PYY 3-36 Module

     The first module is a simple means for generating the peptide of interest.  A Biobrick protein coding sequence encoding PYY3-36 is inserted into an translational unit.  A constituatively expressed promoter (the team selected J23102 for its high RPU) and a ribosome binding site (B0034) are placed upstream of the coding region (K373000), triggering the transcription of PYY3-36.  A double terminator  (B0015) signals the end ofthe sequence.  This module is as fundamental as possible, ensuring consistent expression of the target protein while allowing for interchangeability with other peptides without affecting the operation of the second module in the genetic circuit.


The Population Control Module

     The second molecule of the genetic circuit is intended to regulate the expression of PYY3-36 .  Overproduction of the peptide needs to be prevented.  To accomplish this goal, the team chose to couple cell death with a quorum sensing system.  The module is divided into two parts.  In the first part, a promoter and ribosome binding site are inserted upstream of an AHL coding sequence (F1610).  Acyl homoserine lactone (AHL) is expressed.  This molecule serves as an inter-cellular message system, diffusing through the cell membrane along concentration gradients and giving each cell a basic means of detecting its colony's size/condition.   Quorum sensing of the AHL molecule allows a cap to be placed on the population size of the engineered E.coli colony by enforcing cell death once the colony has reached a sufficient size.  This is accomplished by way of the second part of the Population Control module.
     A cell-signaling promoter (F2620) specifically active in the presence of AHL begins transcription.  Then a sensitivity tuner is inserted into the circuit.  This component acts as a precision activation mechanism, a gateway that allows downstream transcription only if the Polymerase per Second (PoPS) input falls within a specific range.  Any of the various sensitivity tuners developed by Cambridge 2009  (K274370-K274395) can be integrated into our device to achieve the desired result, population capping, and variation of which tuner is used will yield different limits.  The higher the PoPS requirement of the sensitivity tuner used, the larger a colony can grow before the countermeasure (the production of a cell poison) is engaged.  Once the sensitivity tuner's activator is synthesized, translation of the downstream regions of the circuit begins.  The poison gene CcdB (P1010) has been selected to balance the increasing number of engineered cells due to replication.  Once the requirement set by the sensitivity tuner has been met, cells begin to produce poison, preventing the colony's growth.  Thus, the team can ensure a strict limit to the amount of PYY3-36 produced.       

Successes and Failures

     The Citadel team has succeeded in formulating a system for the expression and regulation of PYY3-36 in E.coli.  We have designed a Biobrick part (K373000) that encodes the protein of interest.  This part has been been ordered synthesized, and will be made available in the Registry of Standard Biological Parts.
Due to time constraints and the natural hurdles encountered by a group of undergraduate students and professors new to biological engineering and the iGEM competition, The Citadel team has not yet been able to characterize the Biobrick parts it has built.  We hope to continue this work in the future or to inspire others to continue researching PYY3-36.