Team:SEFLS Shanghai/Safety

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Best Basic Part

SAFETY

Goal of Project

The engineered organism of our team is supposed to produce squalene at a high level of productivity.
1. Learning from the study conducted by T. Liu of Wuhan University, we create the MVA pathway in E. coli, using plasmids pMVA1 and pMVA2.
pMVA1: pBBR1MCS-1(p15A)-AtoB-HMGS-tHMGR
pMVA2: pBBR1MCS-2-MK-PMK-PMD-Idi
AtoB, HMGS, tHMGR, MK, PMK and PMD are from plasmid P35151 purchased from Addgene.
2. We make MEP pathway more effective by over expressing Idi, Dxs, IspG and IspH which are the key enzymes in MEP pathway. Plasmids are used to realize this goal.
pMEP1: pBBR1MCS-2-Dxs-IspG
pMM: pBBR1MCS-2-MK-PMK-PMD-Idi- Dxs-IspG
pET-IIAY-CN: pETDuet-1-1T7-Idi-IspA-Yss-2T7-CrtN
pET-HCN: pETDuet-1-1T7-IspH-Idi-IspA-Yss-2T7-CrtN
pUC-IIAY-CN: pUC19- Idi-IspA-Yss- CrtN
pUC-HCN: pUC19- IspH-Idi-IspA-Yss- CrtN
3. We compare the squalene synthase from different sources, namely Y. lipolytica, S. cerevisiae, B. subtilis and human being.
For analysis, we use dehydrosqualene desaturase CrtN to transform squalene into carotene to observe the production efficiency of different strains.
After we optimize the fermentation conditions of the strains with high squalene yield, we then carry out a final test using a 5-litre high-density fermentation tank.

Used Organisms

E. coli BL21(DE3), E. coli XL1-Blue, S. cerevisiae INVSC1, Y. lipolytica, Pichia Pastoris, B. subtilis, Taphylococcus aureus, in which there is no risk.
We used E. coli BL21 (DE3) and E. coli XL1-Blue as chassis, both are organisms of Risk Group 1. During the experiment we didn't add any virulence gene into our chassis. We just added the parts which are necessary for squalene production. These parts were employed in other studies and in theory will decrease the vitality of the cell, so we needn't consider the prevalence of our engineered cell. Other organisms used during this process were not added any virulence gene as well and are all in Risk Group 1.

Chasses

There isn't any risk. We used E. coli BL21 (DE3) and E. coli XL1-Blue as chassis, both are organisms of Risk Group 1. During the experiment we didn't add any virulence gene into our chassis. We just added the parts which are necessary for squalene production. These parts were employed in other studies and in theory will decrease the vitality of the cell, so we needn't consider the prevalence of our engineered cell.

Experiment

We want to increase the amount of squalene synthase and the efficiency of it in our chasis which are E.coli BL21(DE3) and E.coli XL1-Blue. Our experiments are summarized briefly here: (1) First, we make the MVA pathway in E. coli, using plasmids pMVA1 and pMVA2. (pMVA1: pBBR1MCS-1(p15A)-AtoB-HMGS-tHMGR; pMVA2: pBBR1MCS-2-MK-PMK-PMD-Idi); (2) Secondly, we create MEP pathway by expressing Idi, Dxs, IspG and IspH which are the key enzymes in MEP pathway; (3) Thirdly, we examine the squalene synthase from different sources, namely Y. lipolytica, S. cerevisiae, and B. subtilis to compare their efficiency. Towards the final stage, after the fermentation conditions with high squalene yield is optimized, fermentation test is to be carried out in a 5-litre high-density fermentation tank.
During gel electrophoresis, we use ethidium bromide as a dye, which is poisonous. We use a blade to cut agaropectin and could accidentally hurt ourselves. Besides we also use an ultraviolet lamp, which is harmful to our eyes. We use an alcohol burner in the biosafety cabinet and could accidentally burn ourselves. We use microwave oven to heat liquid and could accidentally scald ourselves.

Expected Usage

We would expect our product to be produced in a factory and used as in consumer products that ordinary people buy.
During the use of our engineered bacteria in factory, we need to make sure that the production takes place in sterile environment so that the pollution of other kinds of bacteria doesn't occur. Because theoretically, our engineered bacteria are less viable than the original E. coli. They may be killed by other kinds of bacteria or hybridize with them.

How is our experiment overseen?

PhD. Xu is the person who oversees our experiments and keeps us company during the whole experimental procedure. He makes sure that our experiment is under control. Since he has worked in the laboratory for several years, he is familiar with all the basic experiments that are included. For example, gel electrophoresis, plasmid transformation and so on. During the experiment, he keeps reminding us of the laboratory safety and emphasizes the points we should notice. For example, we have to wear two gloves when we use eb and change it when it is polluted.

Rules of Guidance

Our experiment complies with the laws and regulations that govern biosafety or biosecurity in research laboratories.

Safety Measures

We have received all the training that is necessary for our experiment, and used multiple risk management tools to minimize possible risks. To ensure safety, our experiment was carried out in a Safety Level 2 Lab We also addressed the following safety concerns in our experiment:
"During gel electrophoresis, we use ethidium bromide as a dye, which is poisonous." We wore two pairs of gloves when conducting this experiment and changed gloves each time when the gloves were polluted.
"We use a blade to cut agaropectin and could accidentally hurt ourselves. Besides we also use an ultraviolet lamp, which is harmful to our eyes." When we cut agaropectin with gloves and we see it through the glass plate to protect our eyes. PhD. Xu, our guide, stood beside us to ensure the safety.
"We use an alcohol burner in the biosafety cabinet and could accidentally burn ourselves." We kept ethanol far from the alcohol burner. Our teacher took care of us during the whole experiment and prepared fire extinguisher.
"We use microwave oven to heat liquid and could accidentally scald ourselves." Our teacher PhD. Xu took the liquid out from the microwave oven for us.

Compliance with iGEM Regulations

We have carefully checked that we strictly comply with iGEM's rules and policies during our whole experiment process.