Team:Caltech/Notebook

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<h1 >WELCOME TO iGEM 2014! </h1>
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<p>Your team has been approved and you are ready to start the iGEM season!
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<br>On this page you can document your project, introduce your team members, document your progress <br> and share your iGEM experience with the rest of the world! </p>
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<p style="color:#E7E7E7"> <a href="https://2014.igem.org/wiki/index.php?title=Team:Caltech/Notebook&action=edit"style="color:#FFFFFF"> Click here  to edit this page!</a> </p>
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<a id='home' href="https://2014.igem.org/Team:Caltech"style="color:#000000">Home </a> </td>
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<a href="https://2014.igem.org/Team:Caltech"style="color:#000000">Home </a> </td>
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<a id='team' href="https://2014.igem.org/Team:Caltech/Team"style="color:#000000"> Team </a> </td>
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<a href="https://2014.igem.org/Team:Caltech/Team"style="color:#000000"> Team </a> </td>
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<a id='profile' href="https://igem.org/Team.cgi?year=2014&team_name=Caltech"style="color:#000000"> Official Team Profile </a></td>
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<a href="https://igem.org/Team.cgi?year=2014&team_name=Caltech"style="color:#000000"> Official Team Profile </a></td>
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<a id='project' href="https://2014.igem.org/Team:Caltech/Project"style="color:#000000"> Project</a></td>
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<a href="https://2014.igem.org/Team:Caltech/Project"style="color:#000000"> Project</a></td>
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<a id="parts" href="https://2014.igem.org/Team:Caltech/Parts"style="color:#000000"> Parts</a></td>
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<a id='modeling' href="https://2014.igem.org/Team:Caltech/TXTL"style="color:#000000"> TXTL Promoter Characterization</a></td>
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<a id='notebook' href="https://2014.igem.org/Team:Caltech/Notebook"style="color:#000000"> Notebook</a></td>
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<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; safety.style.color='white'" onMouseOut="this.bgColor='white'; safety.style.color='black'" bgColor=white><a id="safety" href="https://2014.igem.org/Team:Caltech/Safety"style=" color:#000000"> Safety </a></td>
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<a href="https://2014.igem.org/Team:Caltech/Notebook"style="color:#000000"> Notebook</a></td>
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<tr><td colspan="3"> <h3>Notebook</h3> Apparently there's a calendar feature to take care of this too???</td></tr>
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<tr> <td bgColor=#FFFFFF colspan = 3 height = 60px> <font size = +5> <center>Notebook </center> </td> </tr>
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<h4>Tuesday, 6/17/14</h4>
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<b><font size=+1>Overview</font></b><br><br>
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<b><a href='week1'>Week 1</a></b><br><br>
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<b><a href='week2'>Week 2</a></b><br><br>
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<b><a href='week3'>Week 3</a></b><br><br>
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<b><a href='week4'>Week 4</a></b><br><br>
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<b><a href='week5'>Week 5</a></b><br><br>
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<b><a href='week6'>Week 6</a></b><br><br>
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<b><a href='week7'>Week 7</a></b><br><br>
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<b><a href='week8'>Week 8</a></b><br><br>
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<b><a href='week9'>Week 9</a></b><br><br>
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<b><a href='week10'>Week 10</a></b><br><br>
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<b><a href='week11'>Week 11</a></b><br><br>
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<b><a href='week12'>Week 12</a></b><br><br>
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<b><a href='week13'>Week 13</a></b><br><br>
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<b><a href='week14'>Week 14</a></b><br><br>
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<b><a href='week15'>Week 15</a></b><br><br>
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<ul><li>Get acquainted with summer logistics and lab basics</li>
 
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<td>We got absolutely no results today!!!</td>
 
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<h4>Wednesday, 6/18/14</h4>
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<h1> Project Overview </h1>
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<p>Our project was split into 3 subprojects: AgrBDCA signal reception & Combinatorial Promoters, FsrABC/lamBCDA signal reception, and Ligand export systems.</p>
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<h4>AgrBDCA Signal Reception & Combinatorial Promoters</h4>
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AgrBDCA is a quorum sensing system found to regulate virulence factors in <i>Staphylococcus aureus</i>. In this system, agrC and agrA function as a histidine kinase receptor and a response regulator respectively. This subproject aimed to introduce agrC and agrA in functioning form into <i>E. coli</i>. The circuit used for this subproject is depicted below.
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<table width=70%><tr><td><b>Figure 1</b> The agr quorum sensing system, like most quorum sensing systems, has 2 components involved in signal reception: a histidine kinase receptor (agrC) that detects the ligand outside the cell and a response regulator (agrA) that is phosphorylated inside the cell by the activated histidine kinase receptor.</td></tr></table>
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Additionally, this subgroup focused on prototyping various combinatorial promoters--those which accept multiple inputs in order to determine an output state. This part of the subproject stemmed from the original overall project idea, which was to characterize a negative feedback loop with extracellular components and required a combinatorial promoter to control production of the ligand exported out of the cell. Since work had already begun on that project, it was continued, even after the shift in project design and focus, in hopes that it will later be incorporated again in the circuit.
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<h4>LamBCDA & FsrABC Signal Reception</h4>
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lamBCDA is another quorum sensing system native to <i>Lactobacillus plantarum</i> that shares homology with the agrBCDA quorum-sensing system in <i>S. aureus</i>. Normally, it regulates adherence to surfaces and cell morphology. The components mirror those of agrBCDA, shown in Figure 1, exactly. (i.e. lamA is the response regulator, lamC is the histidine kinase receptor, etc.)
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FsrABC is a third quorum sensing system homologous to the agrBCDA system of <i>S. aureus</i> and is also believed to regulate virulence factors involved in biofilm formation in <i>Entereococcus faecalis</i>. In this system, FsrC and FsrA function as histidine kinase receptor and response regulator respectively. FsrB is a membrane protein that is processed to form a cyclic 11-amino-acid polypeptide <!-- add a tag here that brings you down to Ligand export systems section -->(see Ligand Export Systems).
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<table width=70%><tr><td><b>Figure 2</b> The fsr quorum sensing system also has 2 components involved in signal reception: fsrC as the histidine kinase receptor and agrA as the response regulator. However, unlike the agr and lam systems, which have separate domains encoding a short peptide ligand-precursor and a membrane-protein to modify said ligand-precursor, the fsr system encodes a single membrane protein (fsrB), which is eventually cleaved to form the peptide ligand.</td></tr></table>
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<h4>Ligand Export Systems</h4>
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While the previous 2 subgroups focused primarily on the signal reception aspects of their respective quorum sensing systems, this subgroup focused on the signal transmission aspects of those quorum sensing systems. Specifically, we focused on importing into <i>E. coli</i> the genes for the peptide ligand and any "accessory" proteins (e.g. agrB, lamB) necessary for export of and modification of that peptide into its functional ligand form.
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Specifically, within the AgrBDCA system, it is believed that agrB is a membrane protein that exports and modifies a short polypeptide sequence (agrD) into a polypeptide ligand to be bound by agrC, the histidine kinase receptor. Similarly, the lamBDCA system is thought to function in a very similar way. The fsrABC's export system however, is believed to function by cleaving off a small fragment of fsrB, which is localized in the cell membrane, to become the peptide ligand (see Figure 2 above).
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<ul><li>Continue to discuss project and clean up lab space</li>
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    <li>Designed PCR primers for combinatorial promoters subproject</li>
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<td>We got absolutely no results today!!!</td>
 
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<h4>Thursday, 6/19/14</h4>
 
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<ul><li>Formulated plasmid design for the response regulation subproject</li>
 
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<td>With the exception of the lacI gene, PCR products were confirmed via gel electrophoresis</td>
 
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<h4>Friday, 6/20/14</h4>
 
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<ul><li>Purification of PCR products created yesterday</li>
 
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    <li>Gibson assembly of purified products</li>
 
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    <li>Transformation of Gibson-assembled plasmids into JM109 E. coli</li>
 
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    <li>Meeting with Prof. Murray to discuss give an update on the project</li>
 
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<td>Colonies were found and picked after transformation and incubation.</td>
 
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Latest revision as of 02:28, 30 September 2014


Home Team Official Team Profile Project Parts TXTL Promoter Characterization Notebook Safety Attributions
Notebook
Overview

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Project Overview

Our project was split into 3 subprojects: AgrBDCA signal reception & Combinatorial Promoters, FsrABC/lamBCDA signal reception, and Ligand export systems.

AgrBDCA Signal Reception & Combinatorial Promoters

AgrBDCA is a quorum sensing system found to regulate virulence factors in Staphylococcus aureus. In this system, agrC and agrA function as a histidine kinase receptor and a response regulator respectively. This subproject aimed to introduce agrC and agrA in functioning form into E. coli. The circuit used for this subproject is depicted below.


Figure 1 The agr quorum sensing system, like most quorum sensing systems, has 2 components involved in signal reception: a histidine kinase receptor (agrC) that detects the ligand outside the cell and a response regulator (agrA) that is phosphorylated inside the cell by the activated histidine kinase receptor.

Additionally, this subgroup focused on prototyping various combinatorial promoters--those which accept multiple inputs in order to determine an output state. This part of the subproject stemmed from the original overall project idea, which was to characterize a negative feedback loop with extracellular components and required a combinatorial promoter to control production of the ligand exported out of the cell. Since work had already begun on that project, it was continued, even after the shift in project design and focus, in hopes that it will later be incorporated again in the circuit.

LamBCDA & FsrABC Signal Reception

lamBCDA is another quorum sensing system native to Lactobacillus plantarum that shares homology with the agrBCDA quorum-sensing system in S. aureus. Normally, it regulates adherence to surfaces and cell morphology. The components mirror those of agrBCDA, shown in Figure 1, exactly. (i.e. lamA is the response regulator, lamC is the histidine kinase receptor, etc.)

FsrABC is a third quorum sensing system homologous to the agrBCDA system of S. aureus and is also believed to regulate virulence factors involved in biofilm formation in Entereococcus faecalis. In this system, FsrC and FsrA function as histidine kinase receptor and response regulator respectively. FsrB is a membrane protein that is processed to form a cyclic 11-amino-acid polypeptide (see Ligand Export Systems).

Fsr System
Figure 2 The fsr quorum sensing system also has 2 components involved in signal reception: fsrC as the histidine kinase receptor and agrA as the response regulator. However, unlike the agr and lam systems, which have separate domains encoding a short peptide ligand-precursor and a membrane-protein to modify said ligand-precursor, the fsr system encodes a single membrane protein (fsrB), which is eventually cleaved to form the peptide ligand.

Ligand Export Systems

While the previous 2 subgroups focused primarily on the signal reception aspects of their respective quorum sensing systems, this subgroup focused on the signal transmission aspects of those quorum sensing systems. Specifically, we focused on importing into E. coli the genes for the peptide ligand and any "accessory" proteins (e.g. agrB, lamB) necessary for export of and modification of that peptide into its functional ligand form.

Specifically, within the AgrBDCA system, it is believed that agrB is a membrane protein that exports and modifies a short polypeptide sequence (agrD) into a polypeptide ligand to be bound by agrC, the histidine kinase receptor. Similarly, the lamBDCA system is thought to function in a very similar way. The fsrABC's export system however, is believed to function by cleaving off a small fragment of fsrB, which is localized in the cell membrane, to become the peptide ligand (see Figure 2 above).