About Our Chassis
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 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
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, 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, 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.
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.
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. Genetically modified animals, plants (seeds, breeding livestock, aquatic fingerlings), and microbes.
- 2. Genetically modified animals, plants, and microbe products.
- 3. Directly processed GMOs.
- 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.
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:
- All students, before participating in lab activities, must receive safety training.
- A teacher MUST be present when one or more than one students are in the lab.
- Food and water MUST not be brought into the lab.
- Wear lab coat and gloves at all times when necessary.
- Sign on the equipment usage record sheet before using any equipment.
- Corrosive wastes must go to the corresponding bins. Do not throw solid wastes into the sink. Biological wastes containing microorganism must be autoclaved.
- Do not bring any reagents, disposables, or other equipments outside the lab.
- 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.
- Bacillus subtilis (ATCC 23857) Product Sheet, American Type Culture Collection, Retrieved from “https://www.atcc.org/products/all/23857.aspx”
- 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.
- 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.
According to our interview with a petroleum chemistry professor, we learnt that when disposing oil spill on land, it is difficult to leach out the oil because it easily permeates. However, the current large scale oil spill cleaning methods are mostly physical or chemical, such as burning and leaching. Not only are these methods challenging to conduct due to the extensiveness of ex-situ remediation, they can also possibly cause pollution or the irreparability of the contaminated soil.
The purpose for our project to utilize the environmental friendliness of bioremediation and to improve the efficiency of biodegradation. The chassis we chose, Bacillus subtilis, can produce alkane degradative enzyme, such as alkane hydroxylase and alcohol dehydrogenase. The biosurfactant producing ability is also crucial in our project. Many studies have shown that the slow release of hydrophobic organic compounds from the soil into aquatic phase is the limiting factor of biodegradation, and surfactin produced by B.subtilis increase the bioavailability of these compounds. Our genetic modification is to increase the rate of surfactin production of B.subtilis, and in turn increase the efficiency of bioremediation of soil oil spill.
Our project, in comparison with conventional oil spill disposal, is that we can perform in-situ remediation. B.subtilis produces endosperm that can survive extreme condition such as heat, desiccation and salinity, and the presence of B.subtilis will not disturb the indigenous microcosm in the soil. In-situ remediation can largely diminish the difficulty and expense of oil spill disposal, and bioremediation with B.subtilis and surfactin is one more step to the possibility of a cheap, safe and clean way for soil oil contamination.
As the aim of our project merely seeks to boost of the surfactant production efficiency of B.subtilis to solve the problem of oil leakage in soil, the experimental procedures mainly involve B.subtilis, soil and common laboratory equipment. There was no animal or human experiments performed, voluntary or involuntary, thus there is no religious conflict. The products aren’t a threat to human, proven by our experimenters who have not been affected and so exposure to the products will not cause any damage or risks to health. Therefore, it can be said that our experiments have no intention to harm and is ethical.
Safety on human health
Studies have shown that the chassis in our product, Bacillus subtilits, does not pose threats to human health under most conditions. Although B. subtilis can survive a large range of temperature including that of our bodies’, it doesn’t possess the mechanism to colonize in human bodies, except that it can temporarily inhabit the skin and gastrointestinal tract. B. subtilis itself doesn’t have virulence in its genes, therefore even if it could colonize certain part of the human bodies, it is unlikely to infect us. Similar to many species in its genus, B.subtilis can produce lecithinase, an enzyme that may cause disruption in mammalian cells membrane, but no research has revealed that this enzyme could cause human diseases; it can also produce a toxin called subtilisin, which may induce allergies to workers of frequent contact.
Safety on environmental health
In the case of animals, B.subtilis has a lower frequency of causing infections in livestocks compared to other species, but it has been shown to infect a small number of bovine and mosquito diseases. It has also rarely been reported as a plant-infecting pathogen.
To facilitate the bioremediation of petroleum-contaminated soil, we have chosen to up-regulate the synthesis and excretion of the molecule surfactin. However, surfactin, with its antibacterial properties, has shown to affect the bioactivity of indigenous bacteria in diesel/soil systems. (Whang et al 2007) This raises environmental concerns related to our product that it may disrupt the soil’s original ecosystem. Concerned with this trend, we found proof that surfactin, as a biosurfactant, is biodegradable. It shows both stability in environmental applications and degradation capacities that reduces risks of dangerous accumulations in the environment.(Lima et al 2010)
There is still a need for further investigation on the interactions of surfactin and other organisms existing in the soil and the rate of biodegradation of surfactin in diesel/soil systems though to ensure the safety of the usage of our product.
Another feature of surfactin that needs to be noted is its non-specific cytotoxicity. It can lyse both animal and pathogen cells. This is a safety concern if surfactin in soil may somehow end up in human bodies. Researches have shown though that the toxicity surfactin, however, may be only observed at high concentrations of surfactin. The no-observed-adverse-effect level (NOAEL) of surfactin for female rice was 500 mg/kg after repeated oral administration. (Seydlova et al 2011) The results from toxicity study in mice have also shown that surfactin was less toxic than other surfactants. (Seydlova et al 2011)
Punniyakotti Parthipan et al, (2007) Biosurfactant and Degradative Enzymes Mediated Crude Oil Degradation by Bacterium Bacillus subtilis A1, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5299021/
NA, (1997) Final risk assessment of Bacillus subtilis, https://www.epa.gov/sites/production/files/2015-09/documents/fra009.pdf
Dr. Cooper et al, Enhanced Production of Surfactin from Bacillus subtilis by Continuous Product Removal and Metal Cation Additions, http://aem.asm.org/content/42/3/408.full.pdf
Liang-Ming Whang et al, (2007) Application of biosurfactants, rhamnolipid, and surfactin, for enhanced biodegradation of diesel-contaminated water and soil, http://www.sciencedirect.com/science/article/pii/S0304389407007819?via%3Dihub
Taˆnia M. S. Lima et al, (2010) Biodegradability of bacterial surfactants, http://dacemirror.sci-hub.cc/journal-article/7adaf68d3fa546a10c0cea9c65f5b59f/lima2010.pdf
Seydlová, et al, (2011). Surfactin-novel solutions for global issues, https://www.researchgate.net/publication/303254133_Surfactin-novel_solutions_for_global_issues