About Our Chassis

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Our project uses Bacillus subtilis 168. strain as the major chassis for the expression of sfp, yerP, and LmrA. This strain is classified as Biosafety Level 1[1] based on the standards made by USA Center for Disease Control and Prevention. Therefore, we can use the chassis with relative ease, since the strain is non-pathogenic for healthy individuals and it poses a minimal potential hazard to our lab members and the lab environment. But we still have to follow the biosafety precautions in our school lab.

Multidrug Resistance Factor LmrA

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LmrA is an ABC transporter responsible for multidrug resistance in Lactococcus lactis. It can actively efflux a wide spectrum of clinically relevant antitumor, antimicrobial, antiviral drugs[2], including anthracyclines, vinca alkaloids, aminoglycosides, lincosamides, macrolides, quinolones, streptogramins, tetracyclines, chloramphenicol and novobiocin with energy provided by cellular ATP. Studies on the heterologous expression of lmrA in E. coli. has raised our awareness of potential biosafety issues. Transformation of the hypersensitive Escherichia coli strain CS1562 by a LmrA encoding plasmid has resulted in an increased resistance to 17 out of 21 clinically most used antibiotics[3], which is a significant result suggesting that such wild spectrum resistance is probable to be observed again in Bacillus subtilis, a Gram-positive bacteria like Lactococcus lactis, while Escherichia coli. is Gram-negative. To reduce the risk as much as we can, we deliberately ordered LmrA coding sequence, without regulatory elements, to be synthesized by IDT and tried to assemble the operon afterward in one step to avoid the unexpected expression of LmrA in E. coli strain DH5a. Therefore, we have proposed a plan using a kill-switch regulated by the absence of crude-oil related hydrocarbons in our chassis so that the potential problem of unwanted resistance may be avoided if we use LmrA instead of endogenous YerP for surfactin efflux.

Proof-of-Concept Experiment

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In our proof-of-concept experiment, we set out to verify a protocol for quantitatively determining the efficiency of oil elution by different volumes and concentrations of surfactant solutions. Having such a protocol will greatly expedite our further experiments on the effectiveness of surfactin producing bacteria by directly comparing the qualitative results generated from using pure commercial surfactant solutions and Bacillus subtilis cell suspension as eluents. To make our proof-of-concept experiment safer and greener, while retaining validity of conclusions, we chose edible oil and dish detergent as substituents for crude oil and chemical-pure surfactants.

Local Laws

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So far China has legislated to control the usage of genetically modified organisms (GMOs) in agriculture and food industries. More specifically, according to our investigation, the GMOs laws regulating the environmental effects of planting genetically modified soybean, oilseed rape, and corn are emphasized. However, China has not yet legislated against industrial application of GMOs. What our team research into was " Agricultural Genetically Modified Organisms Control Ordinance " of Chinese Order No.304 of the State Council. Under the new, stricter GMOs rules, both foreign suppliers and local importers must apply for GMOs safety certificates and labeling certificates from China's Ministry of Agriculture.

To take strict precautions against damaging environment and our health, we analyzed two acts that are related to our experiment. The third act defined GMOs as genetically processed plants, animals, microbes, and their products, including:

1. 1. Genetically modified animals, plants (seeds, breeding livestock, aquatic fingerlings), and microbes.
2. 2. Genetically modified animals, plants, and microbe products.
3. 3. Directly processed GMOs.
4. 4. Seeds, living stocks, aquatic fingerlings, pesticides, veterinary medicine, fertilizers, and chemical additives containing genetically modified ingredients.

Moreover, the thirteenth act is especially crucial to our experiment since it is about safety requirement when experimenting. The GMOs experiment should be undertaken in three processes: intermediate test, environmental release, and production test. First, the intermediate test is the small-scaled experiment which can be controlled and monitored by testers. Second, the environmental release is the middle-scaled experiment under natural conditions with safety precautions. Last, the production test is the large-scaled experiment before actually producing and exercising GMOs.

Lab Practice

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Ms. Wenfeng Liu, our secondary PI, is responsible for the safety of our labs.All of our team members strictly comply to the lab rules stated below:

1. All students, before participating in lab activities, must receive safety training.
2. A teacher MUST be present when one or more than one students are in the lab.
3. Food and water MUST not be brought into the lab.
4. Wear lab coat and gloves at all times when necessary.
5. Sign on the equipment usage record sheet before using any equipment.
6. Corrosive wastes must go to the corresponding bins. Do not throw solid wastes into the sink. Biological wastes containing microorganism must be autoclaved.
7. Do not bring any reagents, disposables, or other equipments outside the lab.
8. Wash hands after each lab session.

Besides, we paid extreme attention to using laminar flow cabinets to ensure there is no contamination either inside or outside. After we used a piece of gel, we disposed it into a sealed-up, durable plastic bag which is separated from other wastes to prevent the leakage of TAE solution and gel dyes.

References

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1. Bacillus subtilis (ATCC 23857) Product Sheet, American Type Culture Collection, Retrieved from “https://www.atcc.org/products/all/23857.aspx”
2. Lage, Hermann. (2003). ABC-transporters: Implications on drug resistance from microorganisms to human cancers. International journal of antimicrobial agents. 22. 188-99. 10.1016/S0924-8579(03)00203-6.
3. Poelarends GJ, Mazurkiewicz P, Putman M, Cool RH, Veen HW, Konings WN. An ABC-type multidrug transporter of Lactococcus lactis possesses an exceptionally broad substrate specificity. Drug Resist Updat 2000;3:330-4.