Difference between revisions of "Part:BBa K2033008"

Latest revision as of 07:48, 27 October 2016

N-3-oxo-tetradecanoyl-L-Homoserine lactone* Sender- SinI

This part produces the N-acyl homoserine lactones (AHLs) that acts as a signal for the Sin system. *The Sin system produces 6 known variants of AHLs.

Sequence and Features

Short Description

This part produces 6 AHL quorum sensing molecule, which are (C8-HSL) N-[(3S)-2-oxooxolan-3-yl]octanamide, (C12-HSL) N-[(3R)-2-oxooxolan-3-yl]dodecanamide, (3O-C14-HSL) 3-oxo-N-(2-oxooxolan-3-yl)tetradecanamide, (9-cis-C16-HSL) (Z)-N-[(3S)-2-oxooxolan-3-yl]hexadec-9-enamide, (3O-9-cis-C16-HSL) (Z)-N-[(3S)-2,3-dioxooxolan-3-yl]hexadec-9-enamide, (C18-HSL) N-[(3S)-2-oxooxolan-3-yl]octadecanamide. 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.

Sin 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 Sin system originates from the soil bacterium Sinorhizobium meliloti. It produces 6 variants of AHLs, of which . The structures is shown below. The alignment below displays the successfully created part, starting from the Tet promoter and ending at the SinI gene with negligible differences in identified nucleotides.

These AHLs have a variety of structures, and each acyl tail structure contributes towards unique transcription factor binding interactions.

The SinI part arises from the soil bacterium Sinorhizobium meliloti. 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:

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

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 Sin 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, Sin is not able to induce F2620, as no colonies in the top right section began producing GFP. This indicates possible orthogonality between the two systems.

The figure below compares SinI at 10% and 50% concentrations compared with the natural AHL synthase, LuxI at 10% and 50% concentrations. SinI is shown to not induce F2620. This suggests that the Sin system is orthogonal to Lux and F2620. However, because Sin produces 6 different AHLs, it is possible that E. coli does not have the correct acyl-carrier protein to produce all 6 variants, leaving open the possibility that Sin can crosstalk with F2620. Further characterization must be done to confirm orthogonality.

Safety

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

SinI is known to work together with the Exp system, which work together to regulate the original SinI host, Sinorhizobium meliloti. Over 200 genes in this bacteria are regulated by SinI. These interactions are shown below. Due to the number of AHL variants produced by this system, it is likely that many other bacteria are capable of crosstalking with the Sin system.

Disposal

In order to properly dispose of SinI AHLs, the sample should be autoclaved. Several of these AHLs have a 3-oxo acyl tail, so these (if known) can be degraded with a 10% bleach solution. However, if the specific AHL being produced by the Sin system is unknown, the solution should be autoclaved, which will fully degrade all AHLs. 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 SinI AHLs

References

Hoang, Hanh H. "The LuxR Homolog ExpR, in Combination with the Sin Quorum Sensing System, Plays a Central Role in Sinorhizobium meliloti Gene Expression." Journal of Bacteriology. 186.16 (2004): 5460-5472.