The chassis we have chosen for expression of our plasmid construct is Escherichia coli BL21 DE3, which carries the T7 RNA polymerase gene that can direct high-level expression of cloned genes under the control of the T7 promoter. This is a common lab strain used routinely for protein expression, and is a non-virulent strain. BL21 does not carry the well recognized pathogenic mechanisms required by strains of E. coli causing the majority of enteric infections. It is considered to be non-pathogenic and unlikely to survive in host tissues and cause disease, and hence would not pose a health threat if it escapes the lab.
The expression of pyocin proteins from our construct is only inducible by IPTG, due to the T7-lac promoter sequence of our expression plasmid pET-28a) and hence their production and release outside lab settings is nearly impossible. Even if they do happen to get produced by chance, pyocins are not known to show any toxic effects to eukaryotic organisms. R-type pyocins are in essence mutated derivatives of a defective prophage and their mechanisms are limited to binding and penetration of Gram-negative bacterial cell walls.
Our chassis and construct do not produce any products that would pose a threat to either human/animal health or the environment. Also, we want to use the chassis only for expression and not for the actual therapeutic application. Hence, we decided that incorporation of a kill-switch was not warranted, as there is no intention to release.
For the validation of our modelled protein, we would be performing antimicrobial activity assays on a library of Acinetobacter baumannii clinical isolates and type strains which range from MDR to XDR. Acinetobacter baumannii is an opportunistic pathogen that causes infections such as pneumonia, bacteremia and urinary tract infections in immunocompromised individuals. However, they do not pose a threat to healthy people. It is hence classified as a BSL2 organism. This allows researchers to safely handle A. baumannii isolates in Class II Type B2 model biosafety cabinet, which is routinely used in our lab. Furthermore, our PI, Prof. Pathania, is an expert on Acinetobacter pathogenesis and our instructors are well versed with safety procedures for handling this bacterium. Special care would be taken when performing spot tests, broth microdilution and time-kill tests. All sensitive experiments would be performed under the supervision of our instructors.
Following strict disinfection protocols and sterile technique will effectively contain the pathogen. Handling Acinetobacter isolates does not require any special safety precautions. They are handled with standard BSL2 precautions including but not limited to wearing lab coats, gloves and possibly masks, regular decontamination and handwashing.
Once our institute reopens post-pandemic, we plan to receive a month long lab
training to learn standard practice and precautions to be taken while working
with Acinetobacter and also with carcinogens like SDS, ethidium bromide and
acrylamide. This would ensure that we minimize our risk to exposure and unintentional
spread. To keep safe in this unusual time, we also will follow social distancing and
wear masks in and outside the lab, though restrictions may have been lifted, as
coronavirus exposure would remain a threat for years to come.