This year’s Peking iGEM Team is trying to develop a highly efficient, low-cost and modularized biomaterial to deal with uranium pollution in the environment, using the methods of synthetic biology. Here we wish to share our experiences with safety control.
The produced in this project is polymer network secreted by genetically modified strains of host bacteria. The strains are restricted to the lab so that the cells produce the Uranium Reaper system are continuously under rigorous control without any possibility to contact the outside environment. Therefore, we have prevented recombinant DNA from flowing out into the environment. Even if a small fraction of bacteria leaked out, they would not express any protein without the induction with IPTG. Additionally, the bacterial hosts are laboratory strains which are likely to be highly uncompetitive compared wild bacteria in the outside environment.
During the utilization of the bio-functional polymer network, uranyl ions are enriched to a high concentration, which is a potential cause for concern. However, the resulting high concentrations of ions are collected along with the bio-functional polymer network for further processing. In this way, both the uranium and proteins are disposed of appropriately and do not remain in the outside environment.
Details are as follows
Choosing a non-pathogenic chassis
Non-pathogenic laboratory strains of E. coli and B. subtilis were the only chassis used for either molecular cloning or protein expression. All the other experiments including protein purification and functional assays were carried out in vitro without the involvement of live micro-organisms.
Choosing parts that are not harmful to humans/animals/plants
Harmful parts of any kind were not allowed. Almost all the parts in the project were modified from pre-existing iGEM parts, such as SUP, LBP and so on. The characteristics of these parts had been described in detail in previous work, and all part collections, such as the signal peptide library were derived from parts that are nonpathogenic and harmless to humans or the environment. It is thus conclusive that none of the parts would raise any safety issues according to current professional knowledge.
Substituting safer materials for dangerous materials in a proof-of-concept experiment
The original intention of the project was to develop a highly efficient, low-cost and modularized material to deal with uranium pollution in the environment. In order to prove the usefulness of the concept, we did a series of experiments using simulated pollution scenes which were constructed by dissolving uranyl nitrate hexahydrate either in TBS buffer or in environmental water samples from the Weiming Lake on campus and the Bohai Sea. We did not make any contact with genuine, and thus potentially dangerous, pollution samples from the field.
Preventing the escape of engineered bacteria
Because the bacteria used to produce and secrete the proteins were sequestered in enclosed environments, such as fermenters, and we plan on only using the proteins instead of live bacteria to deal with pollution in the environment, we did not add a kill-switch in this project. All media that had come in contact with bacteria were sterilized to prevent any escape of microbes.
According to the requirements of iGEM policy we never performed any dangerous experiments in daily bench work or faced any unusual safety issues. The bench work followed some basic regulations as below:
Duplicating a key to the laboratory without permission was strictly prohibited.
All involved participants needed to understand the experiment completely before commencing work.
All experimenters had to wear rubber gloves.
Any steps involving potential release of live microorganisms were performed in a bio-safety cabinet.
Appliances such as Bunsen burners, electric heaters and microwave ovens were not left unattended while in use.
All liquid and solid waste potentially containing living organism was sterilized.
The entire lab was sterilized using UV-light every week.
The last person to leave the lab always made sure that water, electricity, gas, and the air conditioner were shut down, and doors and windows have been locked before leaving.
For the proof-of principle test, all uranyl solutions were prepared by specially trained professional researchers in the Nuclear Environmental Chemistry Group of Peking University. What we did ourselves was only to use the simulated pollution scenes for the proof-of-principle test in the Bio-safety Level 2 iGEM lab, which means that we never made any contact with genuine, potentially dangerous pollution samples from the field.
As mentioned above, the DNA parts are absolutely safe because they encode non-hazardous proteins such as SpyCatcher, SpyTag, SUP, mSA and their combinations. The DNA parts were safely confined within PCR tubes as the Parts Registry requires.
