Safety

Our safety measures can be split into two parts: how we conducted the experiments and what we implemented into our design.

1. Safety within the Experiment Conduct

All the participants in this project went to Zhou Lab and followed the leadership of tutors Ms. Lin and Ms. Ma for the experimental work. Moreover, Professor Zhou guided us and supported us with technical guidance throughout our experience in the laboratory.

Before entering the laboratory for specific experimental work, each team member learned the laboratory safety norms to ensure the safety of the experiment. After the training, each team member strictly abided by the rules of conduct of the laboratory and carried out the experiment in an orderly manner.

Figure 1. Safety and Sanitation Measures of Laboratory

Figure 2. Laboratory's Guidelines for Students

Figure 3. Safety Training Prior To Experimentation for Specific Laboratory Procedures

Figure 4. Training Prior To Experimentation on Safety Conduct when Working in the Laboratory

(1) Preparation before entering the laboratory

Each team member entering the laboratory was required to wear gloves, masks, and white coats to prevent experimental agents harmful to human bodies from splashing on exposed skin.

(2) Safety guidelines after entering the laboratory

When one person conducted an experiment, others observed whether the operator's experimental steps were correct and played a supervisory role. In this way, the accuracy of the experiment was increased, the efficiency was improved, and we did not have to start over experiments.

(3) Details of the equipment used in the specific laboratory

After using the experimental drug or equipment, it was always placed back in its original place.

When taking the chemical, the same substance was weighed with the same spoon before and after. Before weighing the next substance, the measuring spoon was washed and dried with wiping.

When operating a high-temperature pressure cooker, a teacher with an operating certificate was required to operate it, and it was not allowed to be used without authorization.

Before picking nematodes, the wire needs to be sterilized with an alcohol lamp at high temperature and then picked on the Petri disha.After use, the alcohol lamp needs to be covered twice with the lid down to ensure that the flame is completely extinguished.

Figure 5. Use of an Alcohol Lamp to Sterilize the Wire at High Temperature

When operating in the UV cleaning table, we wore gloves and did not put mobile phones or other objects with bacteria into the UV cleaning table. After the operation was completed, we placed all items back in place. The UV cleaning table facilitated ultraviolet sterilization. We closed the glass window of the workbench.

Figure 6. Use of UV for Sterilization upon Putting Everything Back to its Place

When taking fluorescent photos, we turned off the lights in the room. After it was done, the computer and all of the machines on the console were turned off.

2. Safety within the project design

(1)IPTG-induced Protein Expression

IPTG can be understood as lactose and functions the same way in the inducible Lac operon. In the absence of this compound, the Lac operator will be bound by repressor protein encoded by the upstream lacI gene. But in the presence of IPTG, the repressor protein will bind to IPTG so that the operator is not hindered. It is only when we add IPTG that the transcription and translation of our “Black Box” protein can occur.

(2)Blue light-induced Mutagenesis：

In the presence of blue light, our p-mag and n-mag components originally belonging to two proteins will bind together, beginning the mutagenesis process starting at the designed T7 promoters. Meanwhile, in the absence of blue light, n-mag and p-mag will separate, separating the C-Terminal RNA Polymerase and the N-Terminal RNA Polymerase and thus stopping the protein from sliding across the DNA for controlled mutagenesis. This blue-light controllable system allows precise control of queued mutations from the T7 promoter to the T7 terminator, which can be used as a start and stop button for the process.

(3)Arabinose-induced E. coli Bacterial Suicide System

In addition to the two integrated designs, we designed a suicide system within the E. coli to be induced by the carbohydrate L-arabinose for extra safety measures. Here are the two illustrated cases with and without arabinose.

1）Without Arabinose

Figure 7. Schematic of effect on E. coli DNA parts and protein without the presence of L-arabinose

With the reversed promoter K2442103, the araC regulatory gene is transcribed and translated into a regulatory protein. Without arabinose, araC serves as a repressor by forming a dimer that binds to both the araO2 and araI1 genes that serve as control sites. This causes the formation of a DNA loop that prevents the expression of the following genes like Cas9 by blocking RNA polymerase entry. In this figure, Cas9 is followed by 3 sgRNAs (single guide RNA), namely sgRNA-A1, SgRNA-A2, and sgRNA-R. Their function is to guide Cas9 to identify and cut specific DNA sequences. Without the expression of Cas9, however, we see no effect on the ATP synthase and DNA polymerase of E. coli, and the bacteria stays alive.

2）With Arabinose

Figure 8. Schematic of effect on E. coli DNA parts and protein with the presence of L-arabinose

In the presence of arabinose, arabinose binds to the regulatory protein araC, which is allosteric and no longer causes the DNA loop formation as seen previously. RNA polymerase is able to bind to the promoter K4491007 and begin transcribing downstream genes. The Cas9 gene is expressed under the induction of arabinose. The expressed Cas9 protein will bind to multiple sgRNAs (single-guided RNA). The figure shows sgRNA-A1, sgRNA-A2 and sgRNA-R, which will guide the Cas9 protein to cut the genes associated with ATP synthase subunit C and RNA polymerase subunit beta. Cas9 triggers DNA double-stranded rupture on these target genes, resulting in the inability to express these important proteins normally. After Cas9 cuts these genes, bacteria cannot synthesize ATP or transcribe RNA, which eventually leads to bacterial death.

As arabinose can be found in nature, the gene editing of these key proteins will occur as soon as the bacteria enters the outer environment, causing bacterial death. Although it is true that this system necessitates the absence of glucose, it is unlikely that there is always a source of glucose in the environment for the organism. Additionally, one can also use arabinose to directly kill the E. coli bacteria directly upon completely lab work to eliminate the bacteria immediately. All of this guarantees that no bacteria will leave the laboratory.