Team:Fudan/Safety
- Addon: ribo
- Addon: TALE
- Addon: T2
- Model: transcriptional amplifer
- Model: Notch-ligand kinetics
- Software
Safety
General safety concerns
Our ENABLE project has provided diverse real-world applications.
As a foundational advance project, the majority of the immediate users of
our project are researchers in biology labs. Components in our project are
perfectly safe to use and can be modified in standard biology laboratories
equipped with a cell-culturing room and a Class II biological safety cabinet.
To ensure our project remains safe and ethical when applied to other practical
usages in the long term, we have hosted a special debate and a creative display between our team
and the public in which several mutual points have been reached. For more information,
please visit our Human Practice page.
Laboratory practice
Because we have inserted our logic gates into mammalian cells to assay the functional outputs, we were performing our experiments both in E. coli strains and on mammalian 293T and HeLa cells. Also, we carry out our experiments in the lab of our supervisor, Liang Cai, who was also our Cell Biology course instructor. The setup in Cai Lab meets all the standards for working on Risk Group 2 organisms. Since some of our team members have never worked on mammalian cells before joining the team, we divided our lab work into two parts which are carried out by either the students who have previously done experiments on E. coli or on mammalian cells. This safety arrangement grants only the members who previously have been fully trained with the mammalian cell culture experimental skills to perform related experiments.
For further precaution, even though every team member has previous lab experience and has passed the laboratory safety knowledge test organized by our university, they were trained again by members of 2017 iGEM team on basic safety measures during the assembly of the team. Before performing experiments in Cai Lab, we were all required to follow the Good Lab Practices written by our supervisor, and a subsequent test was taken to ensure we understood the rules precisely. Before then, we were not allowed to start doing experiments on our own.
Specific safety concerns on our project
Aside from the general concerns for lab safety, we would like to reflect upon certain aspects which are related to our project such as:
Molecular cloning
Most of our E. coli experiments are done through molecular cloning. The strains of Escherichia coli we used are T-FAST
and DH5α competence bacteria, both of
which will die in the natural environment without supplied nutrients. The plasmids we transformed into the bacteria do not
contain anything related to virulence. Furthermore, our designed and encoded genes will be translated into fluorescent protein fusions and
transcription factors.
We made efforts to ensure that those bacteria were kept within control. These efforts include methods we utilized such as sealing the culture medium and plates with parafilm, sterilizing our clean benches with 75% ethanol and ultraviolet illumination before and after the operations, and washing our hands upon entering and leaving the lab, intact nitrile gloves all the time, etc.
To visualize double stranded DNA, we used ethidium bromide. However, to ensure safety, we always wear an additional pair of plastic gloves in which we take off immediately after handing materials containing ethidium bromide to avoid spreading the contamination to other regions of the lab.
Cell culturing
Before entering the cell culture room, we always make sure to exchange our shoes or to wear shoe covers, and put on nitrile gloves. All cell culture operations were done in biosafety cabinets to keep the cells in a sterile environment as well as to prevent the serum from contaminating the outside environment. We used non-self replicable retrovirus to generate stable-transfected cell lines for our experiments.
Additional lab training when coaching high school students
We hosted a lab training program as part of our Human Practice page
during the summer semester 2018. As all the students were very inexperienced, we took special care in ensuring
their safety during the program. Beside memorizing all the lab safety protocols and policies, they were required
to wear long trousers inside their protective gowns even though it was summertime; luckily the air conditioning
was working. Moreover, we ensured that they were under at least one iGEM team member supervision the whole time
throughout their stay in the lab, no matter if they were doing experiments or eating/drinking in the resting area.
Abstract
Contact-dependent signaling is critical for multicellular biological events, yet customizing contact-dependent signal transduction between cells remains challenging. Here we have developed the ENABLE toolbox, a complete set of transmembrane binary logic gates. Each gate consists of 3 layers: Receptor, Amplifier, and Combiner. We first optimized synthetic Notch receptors to enable cells to respond to different signals across the membrane reliably. These signals, individually amplified intracellularly by transcription, are further combined for computing. Our engineered zinc finger-based transcription factors perform binary computation and output designed products. In summary, we have combined spatially different signals in mammalian cells, and revealed new potentials for biological oscillators, tissue engineering, cancer treatments, bio-computing, etc. ENABLE is a toolbox for constructing contact-dependent signaling networks in mammals. The 3-layer design principle underlying ENABLE empowers any future development of transmembrane logic circuits, thus contributes a foundational advance to Synthetic Biology.