Difference between revisions of "Part:BBa K2033000"
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===Short Description===
 
===Short Description===
−
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). This AHL synthase is designed to be inserted into a modular sender vector BBa_K2033011 with a constitutive Tet promoter, 2 ribosome binding sites (RBSs), an RFC10 prefix and mCherry.
  
−
===Introduction to HSL Quorum Sensing===
+
===Aub System Introduction===
−
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]]</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. This part was sequenced with the help of the Core Laboratory at ASU.
  
−
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--aubsequence1.png|600px]]</div>
 +
<div style="text-align: center;">[[File:T--Arizona State--aubsequence2.png|600px]]</div>
  
−
===Aub System===
+
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, suggesting that the AHL is being produced by the sender, since mCherry production increased over an 8-hour read time. The mCherry gene lies downstream of the AubI synthase gene, so mCherry production is a good indicator of Aub AHL production.
 +
<div style="text-align: center;">[[File:T--Arizona State--AUBRFP.png]]</div>
 +
<div style="text-align: center;">OD580-610 absorbance over time of the AubI system</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:
+
Mass spectrometry was also used to characterize AubI, in order to confirm the results from the optical density test. After purification of the AHL serum by HPLC, a DBP matrix was combined with the sample, treated with the Matrix-assisted laser desorption/ionization (MALDI) technique 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. The appearance of this peak confirms that the AHL was produced by E.coli.
−
<div style="text-align: center;">[[File:T--Arizona State--aubhsl3d.png|250px|]]</div>  
+
<div style="text-align: center;">[[File:T--Arizona_State--Aubmassspec.png]]</div>
−
This AHL possesses an alkane tail, which is the primary recognition factor for AubR.
+
<div style="text-align: center;">Left: Negative control MALDI matrix MS readings, Right: MALDI Matrix+AubI AHL MS readings</div>
 +
 
 +
Gas chromatography was also done on the E. coli cultures to confirm production of the AHL molecule by the E. coli chassis. These tests are still in progress and will be completed at a later date.
 +
 
 +
Finally, the ability of the Aub AHL to induce the well-characterized Receiver Device Bba_F2620 was tested. F2620 relies on the LuxR part to output PoPS and GFP production. First, a visual induction was performed by plating the AHL Sender in the center of the plate with a GFP positive control, negative receiver control and F2620. As shown below, Aub is able to induce F2620, as some colonies in the top right section began producing GFP. This indicates crosstalk occurred between the two systems.
 +
<div style="text-align: center;">[[File:T--Arizona_State--aubplate.png]]</div>
 +
<div style="text-align: center;">Plate with GFP+(top left), Sender(center), -Receiver(bottom) and F2620(top right)</div>
 +
 
 +
The figure below compares AubI at 10% and 50% concentrations compared with the natural AHL synthase, LuxI at 10% and 50% concentrations. AubI is shown to induce F2620, albeit to a low degree. This suggests that the Aub system will crosstalk minimally with Lux and F2620.
 +
<div style="text-align: center;">[[File:T--Arizona_State--aubind.png]]</div>
 +
<div style="text-align: center;">GFP absorbance from AubI over time</div>
 +
 
 +
===Safety===
 +
This section aims to provide safety information and suggestions about the AubI part. The greatest concern from this part is the activation of pathogens via crosstalk. According to Integrated Device Technologies, quorum sensing genes are not considered dangerous by themselves, as they do not directly cause the creation of a new pathogenic strain. They may contribute to pathogenicity, but so do synthetic promoters. So, the actual AHL molecules are the chief concern.
 +
 
 +
====Crosstalk Partners====
 +
AubI's AHL has an alkane acyl tail, and may potentially activate other pathogens. C12-HSL was shown to induce F2620 by Dr. Barry Canton (2008) and so, other organisms' with LuxR receivers, such as Pantoea stewartii and Yersinia pestis are potentially activated by AubI as well.
 +
 
 +
====Disposal====
 +
In order to properly dispose of N-dodecanoyl-DL-homoserine lactone (C(12)-HSL), the sample should be autoclaved. This AHL does not possess a beta-ketone group in the acyl tail, and so, bleach is not capable of effectively degrading it. Further details about proper AHL disposal can be found here: http://2016.igem.org/Team:Arizona_State/WhitePaper.
 +
 
 +
====Other Considerations====
 +
This AHL is not considered a hazardous substance or mixture.
 +
 
 +
===References===
 +
(1) Nasuno, E., N. Kimura, M. J. Fujita, C. H. Nakatsu, Y. Kamagata, and S. Hanada. "Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach." Applied and Environmental Microbiology 78.22 (2012): 8067-074. Web
 +
 
 +
(2) Steindler, Laura, Bertani, Iris, De Sordi, Luisa. "LasI/R and RhlI/R Quorum Sensing in a Strain of Pseudomonas aeruginosa Beneficial to Plants." Applied and Environmental Microbiology. 75.15: 5131–5140.

Latest revision as of 18:51, 26 October 2017

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). This AHL synthase is designed to be inserted into a modular sender vector BBa_K2033011 with a constitutive Tet promoter, 2 ribosome binding sites (RBSs), an RFC10 prefix and mCherry.

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. This part was sequenced with the help of the Core Laboratory at ASU.

T--Arizona State--aubsequence1.png
T--Arizona State--aubsequence2.png

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, suggesting that the AHL is being produced by the sender, since mCherry production increased over an 8-hour read time. The mCherry gene lies downstream of the AubI synthase gene, so mCherry production is a good indicator of Aub AHL production.

T--Arizona State--AUBRFP.png
OD580-610 absorbance over time of the AubI system

Mass spectrometry was also used to characterize AubI, in order to confirm the results from the optical density test. After purification of the AHL serum by HPLC, a DBP matrix was combined with the sample, treated with the Matrix-assisted laser desorption/ionization (MALDI) technique 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. The appearance of this peak confirms that the AHL was produced by E.coli.

T--Arizona State--Aubmassspec.png
Left: Negative control MALDI matrix MS readings, Right: MALDI Matrix+AubI AHL MS readings

Gas chromatography was also done on the E. coli cultures to confirm production of the AHL molecule by the E. coli chassis. These tests are still in progress and will be completed at a later date.

Finally, the ability of the Aub AHL to induce the well-characterized Receiver Device Bba_F2620 was tested. F2620 relies on the LuxR part to output PoPS and GFP production. First, a visual induction was performed by plating the AHL Sender in the center of the plate with a GFP positive control, negative receiver control and F2620. As shown below, Aub is able to induce F2620, as some colonies in the top right section began producing GFP. This indicates crosstalk occurred between the two systems.

T--Arizona State--aubplate.png
Plate with GFP+(top left), Sender(center), -Receiver(bottom) and F2620(top right)

The figure below compares AubI at 10% and 50% concentrations compared with the natural AHL synthase, LuxI at 10% and 50% concentrations. AubI is shown to induce F2620, albeit to a low degree. This suggests that the Aub system will crosstalk minimally with Lux and F2620.

T--Arizona State--aubind.png
GFP absorbance from AubI over time

Safety

This section aims to provide safety information and suggestions about the AubI part. The greatest concern from this part is the activation of pathogens via crosstalk. According to Integrated Device Technologies, quorum sensing genes are not considered dangerous by themselves, as they do not directly cause the creation of a new pathogenic strain. They may contribute to pathogenicity, but so do synthetic promoters. So, the actual AHL molecules are the chief concern.

Crosstalk Partners

AubI's AHL has an alkane acyl tail, and may potentially activate other pathogens. C12-HSL was shown to induce F2620 by Dr. Barry Canton (2008) and so, other organisms' with LuxR receivers, such as Pantoea stewartii and Yersinia pestis are potentially activated by AubI as well.

Disposal

In order to properly dispose of N-dodecanoyl-DL-homoserine lactone (C(12)-HSL), the sample should be autoclaved. This AHL does not possess a beta-ketone group in the acyl tail, and so, bleach is not capable of effectively degrading it. Further details about proper AHL disposal can be found here: http://2016.igem.org/Team:Arizona_State/WhitePaper.

Other Considerations

This AHL is not considered a hazardous substance or mixture.

References

(1) Nasuno, E., N. Kimura, M. J. Fujita, C. H. Nakatsu, Y. Kamagata, and S. Hanada. "Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach." Applied and Environmental Microbiology 78.22 (2012): 8067-074. Web

(2) Steindler, Laura, Bertani, Iris, De Sordi, Luisa. "LasI/R and RhlI/R Quorum Sensing in a Strain of Pseudomonas aeruginosa Beneficial to Plants." Applied and Environmental Microbiology. 75.15: 5131–5140.