Difference between revisions of "Part:BBa K2033000"
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This is a synthase enzyme that produces N-dodecanoyl-L-homoserine lactone (C(12)-HSL).
 
This is a synthase enzyme that produces N-dodecanoyl-L-homoserine lactone (C(12)-HSL).
  
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===Introduction to HSL Quorum Sensing===
+
===Aub System Introduction===
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HSLs, or Homoserine Lactones, are a common chemical compound produced by a wide range of bacteria to communicate. As a major variant of quorum sensing, "N-Acyl homoserine lactones" 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 an AHL synthase, which is then detected by the second module, the "Receiver." 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 Aub system was discovered as a result of a metagenomic soil study, in which the system was discovered. However, because the study was part of a metagenomic soil study, the specific bacterial origin is unknown. The Aub system produces a unique AHL molecule, which is shown below:
 +
<div style="text-align: center;">[[File:T--Arizona State--aubhsl3d.png|250px|]]</div>  
 +
This AHL possesses an alkane tail, which is the primary recognition factor for AubR. The further characterization of this part is shown in the Design portion of this part.
  
−
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.  
+
===Aub System===
 +
The original AubI part was sampled from soil and is thought to be from an unknown soil-borne bacteria. The bacteria was transformed into BL21 Competent E. coli cells to produce sufficient stock for future experiments. Of the 70 colonies that were produced, three samples were taken for verification. After mini-prepping, the gel verification affirmed the identity of the sample, as shown below:
  
−
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.
+
<div style="text-align: center;">[[File:T--Arizona State--Gel4.jpg]]</div>
  
−
===Aub System===
+
<div style="text-align: center;">Lanes 3,4,5 correspond to AubI samples; Lane 1 contains the kb+ ladder.</div>
  
−
The Aub system was discovered as a result of a metagenomic soil study, in which the system was discovered. However, because the study was part of a metagenomic soil study, the specific bacterial origin is unknown. The Aub system produces a unique AHL molecule, which is shown below:
+
After gel verification and sequencing, the AubI part was retransformed in BL21(DE3) E. coli and run in a 96-well plate from 580-610nm to measure mCherry production. This produced the curve below, indicating that the AHL is being produced by the sender, since mCherry production increased over an 8hour read time. This is because both the AHL and mCherry genes are found on the Sender.
−
<div style="text-align: center;">[[File:T--Arizona State--aubhsl3d.png|250px|]]</div>  
+
 
−
This AHL possesses an alkane tail, which is the primary recognition factor for AubR. The further characterization of this part is shown in the Design portion of this part.
+
<div style="text-align: center;">[[File:T--Arizona State--AUBRFP.png]]</div>
 +
 
 +
Mass spectrometry was also used to characterize AubI. After purification of the AHL serum by HPLC, a PBwas combined with the sample and run using mass spectrometry. The comparison below between the negative control and the sample shows that a peak around 283.9 m/z appeared in the sample, which matches the predicted mass to charge ratio of the AubI AHL.  
 +
<div style="text-align: center;">[[File:T--Arizona_State--Aubmassspec.png]]</div>

Revision as of 04:55, 17 October 2016

N-dodecanoyl-L-homoserine lactone (C(12)-HSL) Sender- AubI


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 535
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 510
  • 1000
    COMPATIBLE WITH RFC[1000]


Short Description

This is a synthase enzyme that produces N-dodecanoyl-L-homoserine lactone (C(12)-HSL).

Aub System Introduction

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 an AHL synthase, which is then detected by the second module, the "Receiver." 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 Aub system was discovered as a result of a metagenomic soil study, in which the system was discovered. However, because the study was part of a metagenomic soil study, the specific bacterial origin is unknown. The Aub system produces a unique AHL molecule, which is shown below:

T--Arizona State--aubhsl3d.png

This AHL possesses an alkane tail, which is the primary recognition factor for AubR. The further characterization of this part is shown in the Design portion of this part.

Aub System

The original AubI part was sampled from soil and is thought to be from an unknown soil-borne bacteria. The bacteria was transformed into BL21 Competent E. coli cells to produce sufficient stock for future experiments. Of the 70 colonies that were produced, three samples were taken for verification. After mini-prepping, the gel verification affirmed the identity of the sample, as shown below:

T--Arizona State--Gel4.jpg
Lanes 3,4,5 correspond to AubI samples; Lane 1 contains the kb+ ladder.

After gel verification and sequencing, the AubI part was retransformed in BL21(DE3) E. coli and run in a 96-well plate from 580-610nm to measure mCherry production. This produced the curve below, indicating that the AHL is being produced by the sender, since mCherry production increased over an 8hour read time. This is because both the AHL and mCherry genes are found on the Sender.

T--Arizona State--AUBRFP.png

Mass spectrometry was also used to characterize AubI. After purification of the AHL serum by HPLC, a PBwas combined with the sample and run using mass spectrometry. The comparison below between the negative control and the sample shows that a peak around 283.9 m/z appeared in the sample, which matches the predicted mass to charge ratio of the AubI AHL.

T--Arizona State--Aubmassspec.png