Team:SDU CHINA/safety

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• Safety

Part 1 Safety Level Rating System

In China, the Biosafety laboratory means that laboratory structures and facilities, safety practices and safety equipment can ensure that workers are not contaminated by subjects infected with pathogenic microorganisms and toxins, and that the surrounding environment is unaffected. According to the harm degree of microorganism and toxin, it is divided into four levels with the lowest level of one and the highest level of four. And they are named after the safety standards that are required to equip and experiment: BSL-1 to BSL-4.

Biosafety-level 1 laboratories are generally applicable to microorganisms that have no pathogenic effect on healthy adults; the second level is applicable to microorganisms with moderate potential hazards to humans and the environment; the third level is applicable to pathogenic microorganisms or toxins that cause severe or even fatal diseases, mainly through respiratory pathways; level four is applicable to the human body with a high degree of risk, and the pathogenic microorganism or its toxin, which is unknown and has no effective vaccine or treatment method, is transmitted or transmitted by means of a vapor-sol pathway.

With the friendly allowance of the Institution of Molecular Biology and Biological Chemistry (Prof. Yuan and Prof. Ren) and the Open and Creativity Laboratory (Prof. Cao), we are able to conduct our research at Shandong University. During our experiment, we received friendly help and instruction from professors and graduate students there so that we can finish our plan as scheduled successfully. They gave us some guidance and helped us analyze how we did, which made our work more efficient and fruitful.

Part 2 General laboratory safety

All work were conducted in the BSL-2 laboratory, which required additional training and safety measures. Before entering the laboratory, we have been trained for several days. We received training from professional laboratory manager Prof. Cao and Prof. Ren. And we learned the standards of laboratory safety (See more in the attachment and Part 3).

 

Part 3 Work condition and environmentt

 

We obey the following rules of laboratory strictly: Do not eat, or smoke, clean contact lenses and make-up in the work area. Food should be stored in a special cabinet or refrigerator outside the work area. Do not use the mouth pipetting, and one can only use mechanical device pipetting. Obey standard use of sharp norms. All the operation should be as carefully as possible to avoid spill and aerosol. Use effective disinfector that is effective to pathogens when the experiment is completed and before getting off work. All cultures, reservoirs and other specified wastes should be disinfected using a viable disinfection method prior to release, such as autoclaving. Transfer to the nearest laboratory disinfection of the material should be placed in durable, leak-proof containers, sealed out of the laboratory. Dispose of the material from the system for disinfection prior to transfer, and its packaging should comply with the relevant local, state, federal regulations.

We have separated places for experiment and protocol. Before and after our work for a day, we cleaned the laboratory with proper methods, from desk, stools, floor to equipment such as centrifuge, clean bench, tubes and so on. We were careful when using the centrifuge to avoid production of aerosol and imbalance. We put reagent and equipment into certain places according to their characteristics. We prepared things we need for the next day. All the tubes, pipette tips, EP tubes, PCR small tubes and culture medium we used were disinfected with autoclaves. As for wastes, we divided them into regulate ones that were not harm to environment and can be disposed as normal rubbish, and laboratory ones that needed to be sterilized with an autoclave to make them harmless or special management, and the later were put into a specialized bag for laboratory waste and sent to certain institution for disposal.

 

Part 4 Personal Protective Equipment

 

We wore lab coats that are made of cotton, enclosed leather shoes and rubber inspection gloves all the time. When working with corrosives, acids, bases, and alcohols, safety glasses were also required. Upon leaving the lab, we used a disinfectant hand-wash.

And there are some safety equipment, including the operation of emergency showers and eyewash, as well as other emergency equipment including a fire blanket, extinguisher, and the panic button. While working with EtBr, we used thicker nitrile gloves and layers of plastic gloves to protect ourselves from this dangerous chemical. And any operation with EtBr were under control to ensure safety.

Part 5 Organisms and parts that were used

 

Organisms

Species name (including strain)	Risk Group	Risk Group Source	Disease risk to humans?
E.coli DH-5α	1	NIH	No
H460	2	NIH	No
A549	2	NIH	No
293T	1	NIH	No

 

Parts

Part number/name	Natural function of part	How did you acquire it?	How will you use it?
pSB1C3 - BBa_J04500	IPTG inducible promoter with RBS	We acquired it from iGEM DNA Distribution Kits.	Transcription in prokaryotic bacteria
pSB1C3 - BBa_B0015	Cause transcription termination	We acquired it from iGEM DNA Distribution Kits.	Transcription termination
pSB1C3 - BBa_K592012	Coding red chromoprotein	We acquired it from iGEM DNA Distribution Kits.	As a reporter gene
pSB1C3 - BBa_E0840	GFP generator	We acquired it from iGEM DNA Distribution Kits.	As a reporter gene
Promoter 1（hTERT）	Start up plasmid expression in toumor cell	Order the part DNA from a synthesis company	Start up plasmid expression in toumor cell
Promoter 2（Survivin）	Start up plasmid expression in toumor cell	Ordered the part DNA from a synthesis company	Start up plasmid expression in toumor cell
tetR	Express tetR protein	Order the part DNA from a synthesis company	Used it to inhibited the function of tetR repressible promoter
tetR represible promoter	It can't start up the expression of plasmid when it combine with tetR protein.	Order the part DNA from a synthesis company	Play a role of regulation
amilCP	Coding purple protein	Order the part DNA from a synthesis company	AS a reporter gene
eforRed	Coding red protein	Order the part DNA from a synthesis company	AS a reporter gene
neomycin	Express protein to resistant neomycin	Order the part DNA from a synthesis company	For screening

Part 6 Risk assessment

As our project is to use gene therapy as the heart to conquer non-small cells lung cancer (NSCLC), and we also hope this kind of treatments can serve as a weapon used in real clinical treatment someday, so we are very concerned about the safety of plasmid vectors we constructed when they are delivered into cells.

We believe that risks are mainly embodied in two aspects. The first one is the efficiency of our system based on the major premise plasmids triger gene expression in cells. So the sensitivity of the tumor specific promoter and the specificity of the sgRNA is fundamental. The other one is that plamids could spread through not only the tumor cells but also the normal cells, which contains potential risks.

In order to test the working condition of the whole system, validation was performed for every part. In the experiment, we have inserted the hTERT and Survivin promoters respectively to pgl-3 vectors containing double luciferases reporter genes and then transferred the constructed plasmids into cancer cells as well as normal cells respectively, and finally measured the luciferase activity of each group, which could indicated the sensitivity of the tumor specific promoters. Meanwhile, various sgRNA was connected to LentiCRISPR v2 which could be transferred into NSCLC cells and normal cells, and we could detect the protein of PD-L1 through PCR and endonuclease BsmBI. In this way, best promoter and sgRNA were chosen already. To further reduce the unpredictable of the plasmids flow, we design another knockout system based on the technique of CRISPR/Cas9, minimizing the effect of the plasmids. The concrete design is that when the plasmids are transferred into normal cells that hTERT/Survivin promoter can not start gene expression, two plasmids would achieve suicide to ensure biosafety via the sgRNA targeting the gene of Cas9.

Click on and download the safety rules