Part:BBa_K2033000

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-DL-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:

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. Of the 70 colonies that were produced, three samples were taken for verification. After mini-prepping, the gel verification affirmed the identity of the sample, as shown below:

Lanes 3,4,5 correspond to AubI samples; Lane 1 contains the kb+ ladder.

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.

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.

Left: Negative control MALDI matrix MS readings, Right: MALDI Matrix+AubI AHL MS readings

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 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

AubI's AHL has an alkane acyl tail, and may potentially activate other pathogens. RhlI also produces alkane acyl-HSLs, and has been shown by Steinfeldt (2009) to crosstalk with Pseudomonas aeruginosa, so it is feasible that AubI is able to crosstalk with pathogenic bacteria.

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.