All ASU team members were trained through EH&S courses in Lab Safety, Hazardous Waste Management, Fire Safety, Biological and Bloodborne Pathogens, Gas Cylinders, and Recombinant DNA. These course provided a comprehensive overview of lab safety. In addition, all lab members completed a "cloning bootcamp," in which basic cloning knowledge was assessed before moving on to other tasks. The details for the bootcamp can be found here .
The research in this lab was all completed under Standard Operating Procedures. This research was all with accordance with ASU's Biological Safety Manual, found here. The safety manual draws upon the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. The project was also presented to ASU's Institutional Biosafety Committee, where it was reviewed and feedback was received. Any safety concerns that would potentially arise would be reviewed by this committee as well.
The lab space in which wet-lab work was done was all classified as BSL1 with the exception of the tissue culture room, which was BSL2. The lab was located in the 2nd floor wet-lab at Arizona State University's ISTB4 building. Other work, such as mass spectrometry, was done at the Biodesign Institute at ASU.
The organism that was primarily used in this experiment was BL21(DE3) E. Coli. This organism can be handled in BSL1 conditions and "presents minimal potential hazard to laboratory personnel and the environment," according to the New England BioLabs SDS. The handling of this organism was done in sterile and controlled conditions, and presents little immediate danger.
One major biosafety consideration is the experimentation with quorom sensing and how new interactions between AHLs will affect communication between cells. Because AHL quorom sensing can directly affect factors like virility and growth rate, these could potentially have negative societal impacts. A deeper analysis of these impacts was done under the Human Practices tab.
All experimental organisms were contained through extensive methods. Benchtops were sterilized before and after use using 10% bleach and/or 70% ethanol. Organisms were grown in liquid culture tubes or agarose plates that were sealed off using Parafilm and stored in a 4°C cold room to slow growth. All biological waste was disposed of properly in biohazardous waste containers, which were sterilized in an autoclave before disposal. The viability of using these procedures was investigated as part of our human practices, in which we developed a safe disposal protocol to address any concerns about AHLs. The samples that were tested were characterized using Mass Spectrometry to show that the molecules had separated (lactone ring from acyl tail) and thus, were rendered inactive.
In addition, after reviewing the paper by Borchardt(2001), we learned that bleach is able to inactivate AHLs with a 3-oxo acyl tail but not those without. As a result, AHLs from systems like Lux were treated with bleach, while other AHLs without the 3-oxo acyl tail were autoclaved. These results were tested in an induction experiment measured in a plate reader for GFP expression, in which autoclaving was shown to completely inactivate all AHLs. Consequently, any cultures with unknown AHLs present were autoclaved during our experimentation.
Our chassis of choice was E. coli, which belongs in Risk Group 1. The BL21(DE3) strain was used for induction testing while DH5αT strain was used for part construction. While both strains are pathogenic, proper safe experimentation and sanitation provides an extremely low-risk environment. The parts that we designed are capable of producing AHLs, but because inserting these parts into bacteria does not directly translate to a sharp increase in pathogenicity, there is very little risk. In addition, the majority of encounters with AHLs will occur in a research lab setting. Each individual researcher will be responsible for properly disposing of AHLs while preventing exposure of the AHLs to potentially dangerous pathogens.
While the parts that we designed do not directly produce virulence factors, AHLs are capable of potentially activating pathogens and increasing virulence. Because this induction requires specific conditions to be dangerous, an investigation on these safety issues was done in our Human Practices.