Safety & Risk Assessment
In our project we tested various lipases from various sources, in order to find novel and improved enzymes compared to our reference lipase TliA (Thermostable lipase A). Some of our lipases came from potentially dangerous environments (as they are extracted directly from potentially pathogenic populations present in fatbergs) , it would therefore be a mistake to assume that the lipase extracted would be similar to our reference wouldn’t it?
A lab induction for everyone working in the lab was completed, this involved knowledge on waste disposal (bio-hazard and phenol waste), the operation of key lab equipment (autoclave and microwave) and general lab practice. Practical training has also occurred involving cloning, however, approval of the containment level 1 Risk Assessment (CAT1 RA) has to be completed before independent research can start.
A pre-existing RA for similar CAT 1 work has been proposed as a draft and various elements (such as names of participants and possible E.coli strains) have been changed. With the use of a pre-existing RA as a template, we plan on reducing risk of dispersal and injury, as the previous RA was successful.
The RA for the containment level 1 (CAT1) part of the lab has been submitted, further reviewed and is waiting approval. Approval of the CAT1 RA will allow the team to clone lipase genes into the E.coli chassis. So far, most of the parts proposed have been considered as safe (no pathogenic strains have been selected). Maximum care will be taken regardless to avoid damage.
A draft of the iGEM safety form has been produced; it includes containment level 2 work. At the moment the project is unclear, therefore the draft is very general. Hence, not much detail can be put into the strains of the E.coli chassis however, some candidate E.coli have been proposed. The use of sewage for the containment level 2 (CAT2) element of the project requires a human tissue license as faecal matter was regarded as a human sample rather than an environmental sample by our ethics committee.
A risk assessment draft for the containment level 2 part of the project has been made, based on a pre-existing study from the University of Aberystwyth with help from Dr.Justin Pachebat
An SOP (Standard Operating Procedure) to accompany the CAT2 risk assessment has been made. It includes the extraction of organisms from the fatberg, their culture in LBA to identify them and the extraction of their DNA to find some novel lipase sequences.
The CAT2 SOP has been updated from feedback after being reviewed. The sample will be incubated at room temperature rather than 37°C to increase the growth of slow growing bacteria and to find lipases that work well at room temperature (such as in a sewer, where the lipase will potentially be placed). Natamycin plates have been added to screen out fungi, as their sequencing wouldn’t be very helpful.
The CAT1 RA (which includes the insertion of lipase genes into E.coli ) has been sent for approval by the safety committee. A general RA encompassing genetic modification from a previous, similar project involving genetic modification of E.coli (Top10 variants and BL21) has been used.
The CAT1 RA has been approved by the safety committee
The CAT2 RA has been further polished to account for the changes to the SOP. After further discussion from CAT2 competent users, further changes to the SOP have been made. This includes the use 50% glycerol stocks to preserve useful lipase strains that have been identified and are not pathogenic. A screening step has been added, using both SMAC agar (which turns purple in the presence of pathogenic E.coli strains and salmonella)and natamycin plates (to remove fungi). DNA extraction using a commercial kit (DNeasy PowerSoil) has been proposed.
The organisation of the CAT2 team has also been discussed, as they will need specific vaccinations.
The CAT2 SOP has been formatted to comply with University's Guidelines, further meetings will occur with the Biosafety director and the PI of the CAT2 lab we plan on operating in to ensure maximal safety.
Additional review of the CAT 2 SOP revealed further edits are required. This includes specification of one time use equipment and the sterilisation of reusable equipment using ChemGene disinfectant. The transport of the Fatberg sample from Severn Trent water (Liverpool) to the University of Warwick has also been discussed and arranged.
A reviewer from iGEM has asked us for some clarification involving the parts we plan to use and the CAT 2 training we will have – citing biosafety cabinet usage as the main point. An extensive review of the form has been performed, and the proposed genes we plan on inserting have been added as “proposed parts” after an in-depth review of their effect in other studies which involved their insertion into E.coli strains (most parts are not naturally from E.coli ).
3 people have been selected to work in the CAT 2 lab, and immunisation details have been filed with Occupational Safety. Further edits from the Biosafety director have been proposed which involved the lysis of cells after culturing and safety details regarding their culturing at room temperature.
The CAT2 RA has been approved; Severn Trent water will therefore be contacted again so that we can schedule the arrival of the sample.
Another round of review has been performed for the CAT 2 SOP. Various changes to the experimental design have been made to reduce pseudo replication. The SOP has also been changed in the event that 300g or more of sample will be brought back.
After the approval of Severn Trent to give a fatberg sample, we have scheduled a car trip to get the sample with the appropriate measures being taken for the transport safety procedures.
An authority to import form has been filed in order to secure the fatberg sample potentially containing human tissue.
Standard Operating Procedures (SOPs)
Attached below are our SOP's compiled into a document.