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:

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)



Twist Bioscience

We consulted with Twist Bioscience to ask about their screening process when synthesizing genes. This was done through an email chain, shown below:



The following takeaways were gathered from this email response:

• Twist follows 2010 US HHS/ASPR Screening Framework Guidance for Providers of Double Stranded DNA and the follow-on IGSC Harmonized Screening Guidance, which can be found here.
• Twist focuses its screening on organisms specially regulated by CDC Select Agent and Commerce Control List
• Twist "does not focus on screening for sequences that, only in complex combination or in a unique context (e.g. exposed to AHL signaling as you suggest), would be capable of causing harm."
• "The Intelligence Advanced Research Projects Agency (IARPA) recently funded a three year project FunGCAT focused intently on producing tools capable of estimating risk from primary DNA sequence."
• AHLs do not trigger the bioscreening protocol, as they are "second-order in that predicting the effect requires knowing the mix of organisms, conditions and metabolites present at the time."

iGEM Safety Committee

We contacted Dr. Megan Palmer, who " leads programs in safety and responsible innovation for the international Genetically Engineered Machine (iGEM) competition." After briefly discussing our project, she forwarded our questions to the iGEM Safety Committee, which sent the following response:



We learned the following from this email chain:

• Current research standards assume that AHLs degrade in water.
• Virulence factors are often only relevant after onset of infection, so the effect of AHLs prior to infection may be very weak.
• The international IAP:Global Network of Science Academies review of trends in science and technology relevant to the BWC was written in 2015, which discussed the importance of reviewing biofilm formation in potential pathogens as well as potentially inhibiting Receivers or Senders using small molecules.
• AHL quorum sensing should not be classified as dual-use research of concern(weaponized biology), and the benefits should outweigh potential dangers.
• Abide by the ‘golden rule’, keeping bacterial pathogens sensible to AHLs away when working with it.
• Overall, do these parts come from a dangerous organism? No. Do these parts do something risky? Yes - when used in a certain way, with certain other parts.

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. A standard liquid cycle was run (120°C for 15 minutes) on a liquid extraction of AHLs, which were then re-seeded and measured in the plate reader. The figures below are the result of an 8-hour read.






According to the results, autoclaving is able to degrade nearly all Rpa, Las, and Lux AHL molecules. All 3 systems produced nearly no F2620 induction after autoclave treatment. The extreme pressure and temperature generated by the autoclave was more than enough to remove any threat posed by the AHL samples.

In summary, 3-oxo AHLs may be treated by a standard 10% bleach solution for a period between 10-30 minutes for safe disposal. However, AHLs without a beta ketone group should be autoclaved to ensure full degradation of the molecules. While this experiment did not greatly expand on the knowledge gathered by the Borchardt study, we believe that these suggestions are greatly applicable, as they follow standard EH&S guidelines and would likely already be used by research institutions. Future work may include testing of AHLs for each of the sub-categories (alkane, unsaturated etc.) as well as treatment using 70% ethanol, another standard EH&S sanitation method.

White Paper

After gathering information and conducting the safe disposal experiment, we summarized our findings and provided detailed suggestions in a collective white paper. This can be found here.

Parts Pages Safety Information

At the bottom of each parts page that we submitted to the registry, we included a safety section, which lists potential crosstalk partners, proper disposal of the AHL, and other considerations.