Difference between revisions of "Part:BBa K2033004"
 
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An optical density test was conducted on the produced BraI 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 BraI 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 BraI Synthase had been produced in E. coli.
 
An optical density test was conducted on the produced BraI 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 BraI 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 BraI Synthase had been produced in E. coli.
 
<div style="text-align: center;">[[File:T--Arizona State--BRARFP.png]]</div>
 
<div style="text-align: center;">[[File:T--Arizona State--BRARFP.png]]</div>
 +
<div style="text-align: center;">GFP Absorbance from Bra over Time</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 Bra 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, Bra 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--braplate.png]]</div>
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<div style="text-align: center;">Plate with GFP+(top left), Sender(center), -Receiver(bottom) and F2620(top right)</div>
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The figure below compares BraI at 10% and 50% concentrations compared with the natural AHL synthase, LuxI at 10% and 50% concentrations. BraI is shown to induce F2620, albeit to a low degree. This suggests that the Bra system will crosstalk minimally with Lux and F2620.
 +
<div style="text-align: center;">[[File:T--Arizona_State--braind.png]]</div>
 +
<div style="text-align: center;">GFP absorbance from BraI over time</div>
  
 
===Safety===
 
===Safety===
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====Crosstalk Partners====
 
====Crosstalk Partners====
−
BraI is was shown by Ahlgren (2011) to induce members of the genus Bradyrhizobium. This would include the Bja network, which we also studied. Other crosstalk partners likely exist.  
+
BraI was shown by Ahlgren (2011) to induce members of the genus Bradyrhizobium. This would include the Bja network, which we also studied. The 3-phenyl group is very similar to that of the RpaI AHL, p-Coumaroyl HSL, which was shown to induce BBa_F2620. Other LuxR homologues could also be induced by BraI. Other crosstalk partners likely exist.  
  
 
====Disposal====
 
====Disposal====

Latest revision as of 08:09, 27 October 2016

(3S)-3-[(2-oxo-3-phenylpropyl)amino]oxolan-2-one (3-phenyl-HSL) Sender- BraI


Short Description

This is a synthase enzyme that produces the AHL quorum sensing molecule (3S)-3-[(2-oxo-3-phenylpropyl)amino]oxolan-2-one

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 83
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 412
  • 1000
    COMPATIBLE WITH RFC[1000]


Short Description

This part produces the AHL quorum sensing molecule (3S)-3-[(2-oxo-3-phenylpropyl)amino]oxolan-2-one (3-phenyl-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.


Bra 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 Bra system originates from the aquatic bacterium Paraburkholderia kururiensis, a species of proteobacteria. It produces a 3-phenyl HSL, also known as (3S)-3-[(2-oxo-3-phenylpropyl)amino]oxolan-2-one. The structure is shown below:

T--Arizona State--brahsl3d.png

This AHL notably has a phenyl group on the third carbon of the acyl tail, which will serve as a unique binding domain for the transcription factor. This is very similar to the p-coumaroyl AHL produced by the Rpa system, with one less hydroxyl group.

The BraI part arises from the proteobacteria Paraburkholderia kururiensis. The designed part by Ryan Muller was cloned into competent DH5AT E. coli cells. These were ligated into the psB1C3 vector, plated, and sequenced with the help of ASU's Core Laboratory:

T--Arizona State--brasequence1.png
T--Arizona State--brasequence2.png

An optical density test was conducted on the produced BraI 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 BraI 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 BraI Synthase had been produced in E. coli.

T--Arizona State--BRARFP.png
GFP Absorbance from Bra over Time

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

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

T--Arizona State--braind.png
GFP absorbance from BraI over time

Safety

This section aims to provide safety information and suggestions about the BraI 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

BraI was shown by Ahlgren (2011) to induce members of the genus Bradyrhizobium. This would include the Bja network, which we also studied. The 3-phenyl group is very similar to that of the RpaI AHL, p-Coumaroyl HSL, which was shown to induce BBa_F2620. Other LuxR homologues could also be induced by BraI. Other crosstalk partners likely exist.

Disposal

In order to properly dispose of (3S)-3-[(2-oxo-3-phenylpropyl)amino]oxolan-2-one, 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 this AHL.

References

Suarez-Moreno, Z. R., J. Caballero-Mellado, and V. Venturi. "The New Group of Non-pathogenic Plant-associated Nitrogen-fixing Burkholderia Spp. Shares a Conserved Quorum-sensing System, Which Is Tightly Regulated by the RsaL Repressor." Microbiology 154.7 (2008): 2048-059. Web.