Human Practices

Description

The lack of characterization of N-acyl homoserine lactones (AHLs) requires a comprehensive review of the safety of these molecules. The need for increased understanding extends to both the designed "Sender" parts and the AHL molecules themselves. Because quorum sensing is used by a myriad of bacterial species to induce virulence or biofilm formation, among other things, it has many implications towards activating a wide-range of bacteria. Our project aimed to investigate this broad issue by:

1. Consulting with experts in the field

2. Compiling a list of bacteria (pathogens, soil, water) that may crosstalk with AHLs produced by our Senders

3. Designing a AHL safe disposal plan

4. Writing a report that provides suggestions for future research/use of AHLs

5. Adding safety information to the Parts Pages of the Senders we constructed

Consulting with Experts

Integrated Device Technologies

We contacted Integrated Device Technologies (IDT) to gather information on the possible threats associated with the Sender sequences that our team designed. This response was gathered over email, as shown below:

They asked the following questions in regards to how safe a gene might be:

Could it be harmful to our lab personnel?
Would inserting these genes into a different species lead to a new highly pathogenic strain?
Could an accidental transfer to a different species lead to a highly dangerous pathogen?

GeneWiz

We contacted GeneWiz about the possible impact of AHL molecules on inducing quorum sensing in nature. This was done through an email response and a Skype call. The initial email response is shown below:

From the Skype call, our team aimed to clarify information about the dangers of AHLs. The following points were the biggest takeaways:

GeneWiz checks for protein sequences, but not the products that the proteins create
The possibility of AHLs activating pathogens has not been brought up before, but it’s the customer’s responsibility if they are dealing with potentially harmful chemicals/toxins.
They check for toxins and strains that are on a list provided by the FBI (the two documents are attached)

List of Pathogens that Crosstalk

According to Davis, Muller, and Haynes, there are over 100 species of bacteria that are known to produce unique AHL inducers. Our concern is that some of these bacteria are pathogenic, and that AHL exposure could cause activation of virulence or biofilm formation, among other consequences. We assembled a list of 14 pathogenic bacteria that may potentially activate due to AHL molecules generated by our Senders.

Some organisms of note include Burkholderia cepacia, which works together with other bacteria to activate Cystic Fibrosis, Nautella sp. R11, which causes bleaching in coral, and Yersinia pestis, which notably caused the Bubonic Plague.

AHL Safe Disposal Plan

After investigating the risks offered by AHLs, we developed a plan to safely dispose of AHLs. This topic has been investigated before by S.A. Borchardt, who found that "the results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity". In other words, bleach exposure was capable of destroying 3-oxo AHLS, but all other AHLs were unable to fully deteriorate.

Our disposal plan focused on standard EH&S sanitation methods, which included a 10% bleach treatment and autoclaving. We did not conduct a study on variation of bleach concentration, as the necessary dilution of bleach to break down AHLs had already been discovered by Borchardt. After isolating AHLs from a liquid culture supernatant, the samples were re-seeded and treated with bleach and ethyl acetate. These reactions were done for 10, 20 and 30 minutes and then added to a 96-well plate with F2620. The plate reader measured GFP levels over an 8-hour period. The Rpa, Lux, and Las systems were tested, producing the figures below:

According to the results, the Las system was effectively neutralized by a 10% bleach solution, with the untreated sample displaying a steady production of GFP while all 3 treatments of bleach showed negligible absorbance of GFP. This is consistent with the results found by the Borchardt paper, which showed that a minimal concentration of bleach (0.14mM) was capable of breaking down 3-oxo AHLs. However, the Lux results were not nearly as consistent, which showed decreased induction when treated with bleach, but no evidence of complete AHL deconstruction. The Lux AHL is also a 3-oxo AHL, which should have been effectively destroyed by bleach.

The Rpa, Lux, and Las system deactivation was also tested through autoclaving.