Difference between revisions of "Part:BBa K2033002"

Revision as of 09:14, 17 October 2016

isovaleryl-HSL, 3-methyl-N-[(3S)-2-oxooxolan-3-yl]butanamide Sender- BjaI

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 294
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Short Description

This part produces the AHL quorum sensing molecule isovaleryl-HSL (IV-HSL, also known as 3-methyl-N-[(3S)-2-oxooxolan-3-yl]butanamide. This AHL synthase is paired with a constitutive Tet promoter and mCherry.

Bja System

AHL quorum sensing functions within two modules. The first module, the "Sender," must be induced by certain environmental conditions, usually population density of surrounding organisms. This will begin production of the AHL by the cell, which is then detected by the second module, the "Receiver." Once a certain threshold of AHLs is breached, the Receiver will cause the expression or silencing of certain genes to achieve the desired purpose of the communication, whether it is the production of GFP or to increase growth rate.

The Bja system originates from the soil bacterium Bradyrhizobium japonicum. It produces an isovaleryl AHL, also known as 3-methyl-N-[(3S)-2-oxooxolan-3-yl]butanamide. The structure is shown below:

This AHL notably has an isovaleryl tail, which will serve as a unique binding domain for the transcription factor.

The BjaI part arises from the soil bacterium Bradyrhizobium japonicum. The designed part by Ryan Muller was cloned into competent DH5AT E. coli cells. These were ligated into the psb1C3 vector and plated, producing the following gel:

An optical density test was conducted on the produced BjaI construct to determine if the AHL is produced. The plate reader ran an 8-hour read from 580-610nm, indicating the presence of the mCherry fluorescent molecule. The AHL gene lies upstream of the mCherry gene, so successful production of mCherry is a good indicator that the AHL molecule is being produced. A positive growth curve was found for the BjaI construct over the 8-hour read.

@@ Line 18: / Line 18: @@
 This part produces the AHL quorum sensing molecule isovaleryl-HSL (IV-HSL, also known as 3-methyl-N-[(3S)-2-oxooxolan-3-yl]butanamide. This AHL synthase is paired with a constitutive Tet promoter and mCherry.
-===Introduction to AHL Quorum Sensing===
+===Bja System===
-AHLs, or N-Acyl Homoserine Lactones, are a common chemical compound produced by a wide range of bacteria to communicate. As a major variant of quorum sensing, AHLs come in many forms, although they share the same basic backbone shown below:
+AHL quorum sensing functions within two modules. The first module, the "Sender," must be induced by certain environmental conditions, usually population density of surrounding organisms. This will begin production of the AHL by the cell, which is then detected by the second module, the "Receiver." Once a certain threshold of AHLs is breached, the Receiver will cause the expression or silencing of certain genes to achieve the desired purpose of the communication, whether it is the production of GFP or to increase growth rate.
-<div style="text-align: center;">[[File:T--Arizona State--HSLMolecule.jpg]]</div>
+The Bja system originates from the soil bacterium Bradyrhizobium japonicum. It produces an isovaleryl AHL, also known as 3-methyl-N-[(3S)-2-oxooxolan-3-yl]butanamide. The structure is shown below:
-Distinguishable by its lactone ring, HSLs in quorum sensing are used as a density-dependent communication system for many bacteria that controls growth rate, virulence, and bio-luminescence among other things. The Aub system originates from an unidentified soil bacteria and is highly uncharacterized.
+<div style="text-align: center;">[[File:T--Arizona State--bjahsl3d.png|250px|]]</div>
-HSL quorum sensing functions within two modules. The first module, the "Sender," must be induced by certain environmental conditions, usually population density of surrounding organisms. This will begin production of the HSL by the cell, which is then detected by the second module, the "Receiver." Once a certain threshold of HSLs is breached, the Receiver will cause the expression or silencing of certain genes to achieve the desired purpose of the communication, whether it is the production of GFP or to increase growth rate.
+This AHL notably has an isovaleryl tail, which will serve as a unique binding domain for the transcription factor.
-===Bja System===
+The BjaI part arises from the soil bacterium Bradyrhizobium japonicum. The designed part by Ryan Muller was cloned into competent DH5AT E. coli cells. These were ligated into the psb1C3 vector and plated, producing the following gel:
-The Bja system originates from the soil bacterium Bradyrhizobium japonicum. It produces an isovaleryl AHL, also known as 3-methyl-N-[(3S)-2-oxooxolan-3-yl]butanamide. The structure is shown below:
+<div style="text-align: center;">[[File:T--Arizona State--Gel4.jpg]]</div>
-<div style="text-align: center;">[[File:T--Arizona State--bjahsl3d.png|250px|]]</div>
-This AHL notably has an isovaleryl tail, which will serve as a unique binding domain for the transcription factor.
+An optical density test was conducted on the produced BjaI construct to determine if the AHL is produced. The plate reader ran an 8-hour read from 580-610nm, indicating the presence of the mCherry fluorescent molecule. The AHL gene lies upstream of the mCherry gene, so successful production of mCherry is a good indicator that the AHL molecule is being produced. A positive growth curve was found for the BjaI construct over the 8-hour read.
+<div style="text-align: center;">[[File:T--Arizona State--BJARFP.png]]</div>