Difference between revisions of "Part:BBa K2033002"
 
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===Short Description===
 
===Short Description===
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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.  
+
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 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.  
  
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===Introduction to AHL Quorum Sensing===
 
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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:
 
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<div style="text-align: center;">[[File:T--Arizona State--HSLMolecule.jpg]]</div>
 
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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.
 
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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.
 
  
 
===Bja System===
 
===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:
 
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--bjahsl3d.png|250px|]]</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.
 +
 +
The BjaI part arises from the soil bacterium Bradyrhizobium japonicum. The designed part by Ryan Muller was cloned into competent DH5AT E. coli cells. This part was sequenced with the help of ASU's Core Laboratory:
 +
 +
<div style="text-align: center;">[[File:T--Arizona State--bjasequence1.png|600px]]</div>
 +
<div style="text-align: center;">[[File:T--Arizona State--bjasequence2.png|600px]]</div>
 +
 +
 +
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. The initial dip in mCherry levels was likely the result of the transfer of the cells from an aerated, incubated environment to a 96-well plate. However, overall, mCherry  production increased over time, suggesting that the BjaI Synthase had been produced in E. coli.
 +
<div style="text-align: center;">[[File:T--Arizona State--BJARFP.png]]</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 Bja 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, Bja 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--bjaplate.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 BjaI at 10% and 50% concentrations compared with the natural AHL synthase, LuxI at 10% and 50% concentrations. BjaI is shown to induce F2620, albeit to a low degree. This suggests that the Bja system will crosstalk minimally with Lux and F2620.
 +
<div style="text-align: center;">[[File:T--Arizona_State--bjaind.png]]</div>
 +
<div style="text-align: center;">GFP absorbance from BjaI over time</div>
 +
===Safety===
 +
This section aims to provide safety information and suggestions about the BjaI part. The greatest concern from this part is the activation of pathogens via crosstalk. According to Integrated DNA 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====
 +
BjaI is known to be similar to the Rpa system, according to Lindemann A. (2011). Rpa has been shown to interact with Silicibacter pomeroyi, as well as the well-characterized part Bba_F2620, which was designed for the Lux system. Other crosstalk partners likely exist.
 +
 +
====Disposal====
 +
In order to properly dispose of 3-methyl-N-[(3S)-2-oxooxolan-3-yl]butanamide (isovaleryl-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====
 +
No other safety information is available for IV-HSL.
 +
 +
===References===
 +
(1) Lindemann A. "Isovaleryl-homoserine lactone, an unusual branched-chain quorum-sensing signal from the soybean symbiont Bradyrhizobium japonicum." PubMed. 108.40 (2011):16765-70.

Latest revision as of 08:09, 27 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 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.


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:

T--Arizona State--bjahsl3d.png

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. This part was sequenced with the help of ASU's Core Laboratory:

T--Arizona State--bjasequence1.png
T--Arizona State--bjasequence2.png


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. The initial dip in mCherry levels was likely the result of the transfer of the cells from an aerated, incubated environment to a 96-well plate. However, overall, mCherry production increased over time, suggesting that the BjaI Synthase had been produced in E. coli.

T--Arizona State--BJARFP.png

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 Bja 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, Bja 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--bjaplate.png
Plate with GFP+(top left), Sender(center), -Receiver(bottom) and F2620(top right)

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

T--Arizona State--bjaind.png
GFP absorbance from BjaI over time

Safety

This section aims to provide safety information and suggestions about the BjaI part. The greatest concern from this part is the activation of pathogens via crosstalk. According to Integrated DNA 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

BjaI is known to be similar to the Rpa system, according to Lindemann A. (2011). Rpa has been shown to interact with Silicibacter pomeroyi, as well as the well-characterized part Bba_F2620, which was designed for the Lux system. Other crosstalk partners likely exist.

Disposal

In order to properly dispose of 3-methyl-N-[(3S)-2-oxooxolan-3-yl]butanamide (isovaleryl-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

No other safety information is available for IV-HSL.

References

(1) Lindemann A. "Isovaleryl-homoserine lactone, an unusual branched-chain quorum-sensing signal from the soybean symbiont Bradyrhizobium japonicum." PubMed. 108.40 (2011):16765-70.