Difference between revisions of "Part:BBa K2033006"

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3OH-7-cis-C14-HSL Sender Device-CerI

This synthase is produced by the bacteria Rhodobacter sphaeroides and interacts with the receiver molecule CerR.

Sequence and Features

Short Description

This part produces the AHL quorum sensing molecule 3OH-7-cis-C14-HSL ((Z)-3-hydroxy-N-[(3S)-2-oxooxolan-3-yl]tetradec-7-enamide). 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.

Cer 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 Cer system originates from the aquatic proteobacteria Rhodobacter sphaeroides. It produces an 3OH-7-cis-C14-HSL, also known as ((Z)-3-hydroxy-N-[(3S)-2-oxooxolan-3-yl]tetradec-7-enamide). The structure is shown below:

This AHL notably has an unsaturated acyl tail, forming a Z-orientation double bond, which will serve as a unique binding domain for the transcription factor.

The CerI part arises from the soil bacterium Rhodobacter sphaeroides. The designed part by Ryan Muller was cloned into competent DH5AT E. coli cells. These were ligated into the psB1C3 vector and plated, as well as sequenced with help from ASU's Core Laboratory.

An optical density test was conducted on the produced CerI 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 CerI construct over the 8-hour read. Overall, mCherry production increased over time, suggesting that the CerI Synthase had been produced in E. coli.

Finally, the ability of the Cer AHL to induce the well-characterized Receiver Device Bba_F2620 was tested. F2620 relies on the LuxR part to output PoPS and GFP production. The figure below compares CerI at 10% and 50% concentrations compared with the natural AHL synthase, LuxI at 10% and 50% concentrations. CerI is shown to induce F2620 strongly. This suggests that the Cer system will crosstalk with Lux and F2620.

Safety

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

CerI produces 3OH-7-cis-C14-HSL, which is known to have 4013 Biosystem pathways in quorum sensing, as shown https://pubchem.ncbi.nlm.nih.gov/compound/5281979#section=InChI. With this level of characterization, potential crosstalk partners are well understood.

Disposal

In order to properly dispose of 3OH-7-cis-C14-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 3OH-7-cis-C14-HSL.

References

(1) Ahlgren NA. "Aryl-homoserine lactone quorum sensing in stem-nodulating photosynthetic bradyrhizobia." PubMed. 108.17 (2011):7183-8..