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 white paper 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?
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 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."
- Some current researchers 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.
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:
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:
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. This includes over 68 proteobacteria that are LuxI/R homologs, which was taken from a sample size of 512 bacteria with completed genomes (Case 2008). 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 tested four AHL systems with bleach and autoclaving: Cer, Lux, Las, and Rpa. The samples were bleached for 10, 20 and 30 minutes, and then added to a 96-well plate with F2620. This protocol is located under the protocol header. For the autoclaving experiments, a standard liquid cycle was run (120°C for 15 minutes) on a liquid extraction of AHLs, which were then added to F2620 and measured in the plate reader. The figures below are the result of an 8-hour read measuring GFP levels. The Cer, Las, Lux, Rpa systems as well as Esa AHL deactivation was also tested through autoclaving. Esa was added to this test because there was room on the 96-well plate and it had shown good induction results on the previous F2620 tests.
The results demonstrate that all systems, Lux, Las, Rpa and Cer were able to effectively induce F2620 after being extracted. However, only the Las system was effectively neutralized by a 10% bleach solution. All 3 treatments of bleach showed negligible induction through the GFP/OD measurements. 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 inactivation. What is also interesting is the decreased induction by Cer after being in bleach. The Cer AHL is not a 3-oxo, but rather has an unsaturated acyl tail. However, all other QS systems retained activity after bleach.
According to the autoclave results, a standard 15 min liquid procedure is able to degrade nearly all AHLs including Rpa, Car, Las, and Lux AHL molecules. All 4 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 these AHL samples. However, Esa was still able to induce after being autoclaved. This means that further research may be required to determine an exact method to inactivate all AHLs.
In summary, our data suggests that autoclaving will ensure at least some inactivation of AHLs, while bleach may have no effect at all on the degradation of AHLs. The data we have portrayed here is not conclusive, and further testing of disposal methods and AHL activity will be required before determining an all-inclusive disposal method. However in general, the human practices project does conclude that AHL degradation is an issue that needs to be considered. That gene synthesis companies and biosafety committees have not properly considered AHLs as a potentially harmful agent, while many studies show AHLs activate virulence factors in pathogens. Also that the widespread use and release of AHLs into the environment without proper disposal may cause significant activation and issues with pathogens.
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.