Difference between revisions of "Team:Columbia NYC/Design"

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<a href= "https://2015.igem.org/Team:Columbia_NYC"> <img src="https://static.igem.org/mediawiki/2015/c/ca/Columbia_NYC_Logo.jpg" alt="Columbia iGEM Logo" style="width:100%"> </a>
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<a href = "#"> <li> THE PROJECT
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<a href = "https://2015.igem.org/Team:Columbia_NYC/Description"> <li> DESCRIPTION </li></a>
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<a href = "https://2015.igem.org/Team:Columbia_NYC/Design"> <li> DESIGN </li></a>
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<a href = "https://2015.igem.org/Team:Columbia_NYC/Experiments"> <li> EXPERIMENTS </li></a>
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<a href = "https://2015.igem.org/Team:Columbia_NYC/Results"> <li> RESULTS </li></a>
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<a href = "https://2015.igem.org/Team:Columbia_NYC/Team"> <li> TEAM MEMBERS </li></a>
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<a href = "https://2015.igem.org/Team:Columbia_NYC/Parts"> <li> TEAM PARTS </li></a>
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<a href="https://2015.igem.org/Team:Columbia_NYC/Notebook"><li> THE NOTEBOOK </li></a>
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<a href = "https://2015.igem.org/Team:Columbia_NYC/Attributions"> <li> ATTRIBUTIONS </li></a>
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<a href = "https://2015.igem.org/Team:Columbia_NYC/Collaborations"><li> COLLABORATIONS </li></a>
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<a href = "https://2015.igem.org/Team:Columbia_NYC/Practices"><li> HUMAN PRACTICE </li></a>
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<a href="https://2015.igem.org/Team:Columbia_NYC/Safety"><li> LAB SAFETY </li></a>
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<a href = "https://2015.igem.org/Team:Columbia_NYC/Sponsors"> <li> OUR SPONSORS </a>
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<h1> DESIGN</h1>
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<h2> Project Design </h2>
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<p> With two specific bacterial species defined along with their respective signal peptides, the team then focused on experimental protocols necessary to attach the signal peptides to each of the desired gut peptides.  This was accomplished via a Ligation Cycle Reaction (LCR) in which each of the signal peptides – PhoA, PelB, Lp3050, and M6 – were combined with each of the gut peptides – GLP-1, PYY, and Ghrelin – for a total of twelve reactions.  Upon the completion of these ligations, each combination underwent a PCR with primers that had flanking regions which included a stop codon and his-tag for the gut peptides. </p>
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<p> The technique employed for testing the synthesis and secretion of the gut peptides the team used is known as a Western Blot.  Bacterial colonies were grown up and upon screening were placed in growth media dependent on the bacterial species – LB for E. coli and MRS for L. reuteri.  After a period of time, a sample of the media supernatant was saved for the Western Blot detection of gut peptide secretion.  The cells were then spun down and lysed to serve as a sample for the Western Blot detection of the gut peptide initial synthesis.  Supernatant and lysis samples for the same constructs were blotted side-by-side for a comparison of peptide concentration inside and outside of the bacterial cells. </p>
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<p> With the synthesis and secretion of the signal-gut peptide combinations characterized through the Western Blot procedures, the team’s next focus was the construction of the inter-species quorum sensing communication system necessary for a true interactive consortium of bacteria.  </p>
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<p> Research into known quorum sensing structures within our team’s desired bacterial structures led to the design of a bacterial communication system that also was able to incorporate a lysis cassette.  The LuxR/I family of quorum sensing structures is found naturally within Vibrio fischeri and facilitates a bio-density detection mechanism; our team’s system implemented a 3-oxoheanoyl-homoserine lactone (AHL) variant.  AHL is unique and indeed integral to quorum sensing for its ability to flow within and out of the cellular membrane.  The AHL is meant to travel from cell to cell, complexing with protein LuxR (a product we can constitutively express within our system) to then engage with pLux promoter regions. </p>
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<p> For the purposes of our experiments, we utilized two known pLux regions: BBa_R0061 and BBa_R0062.  In the presence of AHL and LuxR they are repressed and induced (respectively) from otherwise contrary constitutive states (that is, constitutively on or off).  These served as the basis of our quorum sensing system. </p>
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<p> Below is an outline of the entire system in the presence of AHL.  </p>
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<p> Breaking down each component, let us further analyze the system. </p>
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<h2> The AHL Factories </h2>
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<p> Consists of LuxI production; experimentally under anhydrotetracycline inducible control </p>
  
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<h2>Design</h2>
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<p> Within clinically functional system this might represent the gut colonizing species where production is controlled via pH or fatty acid sensing </p>
  
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<h2> The Target Molecule Production Lines </h2>
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By talking about your design work on this page, there is one medal criterion that you can attempt to meet, and one award that you can apply for. If your team is going for a gold medal by building a functional prototype, you should tell us what you did on this page. If you are going for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Applied Design award</a>, you should also complete this page and tell us what you did.  
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<p> Consists of constitutively expressed LuxR and inducible pLux (active transcription in the presence of AHL). Design allows any target molecule  and not just our targeted hormone (modularity is key in any factory).  </p>
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<h2> Protein Dependent Timed Cellular Lysis </h2>
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<p>In order to be considered for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Best Applied Design award</a> and/or the <a href="https://2015.igem.org/Judging/Awards#Medals">functional prototype gold medal criterion</a>, you must fill out this page.</p>
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<p>This is a prize for the team that has developed a synthetic biology product to solve a real world problem in the most elegant way. The students will have considered how well the product addresses the problem versus other potential solutions, how the product integrates or disrupts other products and processes, and how its lifecycle can more broadly impact our lives and environments in positive and negative ways.</p>
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<p> The Suicide Cassette is our take on the popular BBa_K112808 enterobacteria phage T4 lysis cassette.  This system consists of endolysin and holin under pCI control as well as a constitutivly active T4 antiholin to suppress basal expression levels </p>
  
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<p> The timer relies upon the activity Enterobacteria phage lamda repressor protein cI under control of repressible (constitutively promoting transcription) variant of pLux to generate a time delayed and protein dependent lysis. In the presence of AHL. cI production ceases and lysis ensues. </p>
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If you are working on art and design as your main project, please join the art and design track. If you are integrating art and design into the core of your main project, please apply for the award by completing this page.
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Revision as of 03:16, 19 September 2015

DESIGN

Project Design

With two specific bacterial species defined along with their respective signal peptides, the team then focused on experimental protocols necessary to attach the signal peptides to each of the desired gut peptides. This was accomplished via a Ligation Cycle Reaction (LCR) in which each of the signal peptides – PhoA, PelB, Lp3050, and M6 – were combined with each of the gut peptides – GLP-1, PYY, and Ghrelin – for a total of twelve reactions. Upon the completion of these ligations, each combination underwent a PCR with primers that had flanking regions which included a stop codon and his-tag for the gut peptides.

The technique employed for testing the synthesis and secretion of the gut peptides the team used is known as a Western Blot. Bacterial colonies were grown up and upon screening were placed in growth media dependent on the bacterial species – LB for E. coli and MRS for L. reuteri. After a period of time, a sample of the media supernatant was saved for the Western Blot detection of gut peptide secretion. The cells were then spun down and lysed to serve as a sample for the Western Blot detection of the gut peptide initial synthesis. Supernatant and lysis samples for the same constructs were blotted side-by-side for a comparison of peptide concentration inside and outside of the bacterial cells.

With the synthesis and secretion of the signal-gut peptide combinations characterized through the Western Blot procedures, the team’s next focus was the construction of the inter-species quorum sensing communication system necessary for a true interactive consortium of bacteria.

Research into known quorum sensing structures within our team’s desired bacterial structures led to the design of a bacterial communication system that also was able to incorporate a lysis cassette. The LuxR/I family of quorum sensing structures is found naturally within Vibrio fischeri and facilitates a bio-density detection mechanism; our team’s system implemented a 3-oxoheanoyl-homoserine lactone (AHL) variant. AHL is unique and indeed integral to quorum sensing for its ability to flow within and out of the cellular membrane. The AHL is meant to travel from cell to cell, complexing with protein LuxR (a product we can constitutively express within our system) to then engage with pLux promoter regions.

For the purposes of our experiments, we utilized two known pLux regions: BBa_R0061 and BBa_R0062. In the presence of AHL and LuxR they are repressed and induced (respectively) from otherwise contrary constitutive states (that is, constitutively on or off). These served as the basis of our quorum sensing system.

Below is an outline of the entire system in the presence of AHL.

Breaking down each component, let us further analyze the system.

The AHL Factories

Consists of LuxI production; experimentally under anhydrotetracycline inducible control

Within clinically functional system this might represent the gut colonizing species where production is controlled via pH or fatty acid sensing

The Target Molecule Production Lines

Consists of constitutively expressed LuxR and inducible pLux (active transcription in the presence of AHL). Design allows any target molecule and not just our targeted hormone (modularity is key in any factory).

Protein Dependent Timed Cellular Lysis

The Suicide Cassette is our take on the popular BBa_K112808 enterobacteria phage T4 lysis cassette. This system consists of endolysin and holin under pCI control as well as a constitutivly active T4 antiholin to suppress basal expression levels

The timer relies upon the activity Enterobacteria phage lamda repressor protein cI under control of repressible (constitutively promoting transcription) variant of pLux to generate a time delayed and protein dependent lysis. In the presence of AHL. cI production ceases and lysis ensues.