Project Risk Assessment

Application of Our Project

Identifying potential risks of our project is vital for ensuring safety and security while implementation may occur in places outside the laboratory. Before that, a clear picture of how our product will be applied in the real world should be drawn. This year, the NYCU-Taipei iGEM team developed an alternative approach for monitoring bacterial growth status by integrating synthetic biology with novel hardware design. We expect our product to be applied in the following aspects:

  1. In the lab:

    As a fundamental tool that assists in real-time detection of bacterial growth, our engineered bacteria would be applied in laboratory settings for determining different time points within the bacterial growth curve.*

  2. In industrial manufacturing context:

    Our project would also be used in the biotechnology or bio-pharmaceutical industry that use microorganisms as an expression system for recombinant protein production, such as drugs, antibiotics, monoclonal antibodies, etc. Our indicators could be transformed into their bacteria and serve as a timer for specific industrial procedures, e.g. time for IPTG induction or harvesting.

  3. In a small enclosed device:

    A customer kit would be used for the delivery of our E. coli between laboratories. Promoters and reporter genes in our product are specifically designed for the DH5α strain, and extra modifications should be made before applying them on other microorganisms. Therefore, an instruction manual for the design and testing of fluorescent growth status indicators would be attached along with the kit to meet customer requirements.

For more information regarding our project applications, please visit our Implementation page.

Clarifying potential risks

In general, our product is used in laboratory settings. Despite the fact that our product will not cause harm to human health, there are still safety issues that we should take into consideration. After constant reflection, we came up with the following potential risks that require safety measures:

  1. Accidental environmental release:

    Fragments of our plasmids and other parts in our engineered bacteria may affect gene expression of other bacterial strains when accidentally released to the environment. Therefore, it is necessary that we apply a biocontainment strategy to prevent the release of transgenic elements through biological activities such as horizontal gene transfer.

  2. Contamination during co-culture:

    When applied in the industrial field, our indicator bacteria will be cultured with the user bacteria in the same bioreactor as an internal control. Metabolites (e.g. fluorescent proteins) released from the indicator bacteria may contaminate the fermentation product, which requires extra purification procedures to extract the final target protein. Safety measures should be adopted to minimize the interference of our indicators on user bacteria. For more information regarding safety considerations in industrial applications, please visit our Expert Consultation page.

Biosafety Measures

Two biosafety measures were taken to deal with the potential risks. We applied a CRISPR DNAi device to ensure that our engineered parts would not be introduced into other organisms upon release to the environment, and designed a disk-shaped filter device that could be intercalated within the inner wall of the bioreactor to reduce the risk of contamination.

CRISPR DNAi Device

The CRISPR DNAi device is a genetically encoded device (DNAi) packaged with a type-IE CRISPR nuclease, which responds to a specific transcriptional input and directs degradation to specific genomic regions by recognizing the PAM and proto-spacer sequence (Fig. 11). To modify this device and make it feasible for our project, we defined the following preconditions:

  1. Transcriptional input:

    Removal of our engineered bacteria from a defined culture environment activates a sensor and increases expression of an input promoter.

  2. Our defined culture environment:

    The liquid culture media and nutrient agar used to culture our engineered bacteria.

  3. Degradation output:

    The input promoter signal activates Cas system promoter PJ23117 and degrades the target plasmid DNA which includes our fluorescent growth status indicators.

Fig. 1) Schematic representation of the CRISPR DNAi device

This strategy ensures that our engineered parts would not be introduced into other organisms upon release to the environment. We chose this method due to its many advantages:

  1. DNAi can be stably carried in an engineered organism, with no impact on cell growth and plasmid stability.
  2. As an inducible genetic switch, CRISPR-based DNAi provides high specificity in knocking out plasmids or specific sensitive regions of the genome without harming the host. Other common kill switch systems such as the MazE/MazF toxin-antitoxin system induce cell death, which increases the likelihood of gene transfer between organisms after the release of intracellular DNA.
  3. This DNAi device is compatible with other biocontainment strategies. Furthermore, modularized component design allows the device to produce a degradation output responding to any user-specified set of DNA targets and transcriptional input. (It leads to bacterial death while the target is defined as genomic DNA)

Disk-Shaped Filter Device

Through our partnership with team Wego_Taipei, we were intrigued by their idea of applying a filter device to prevent leakage of their engineered bacteria into eutrophic water (for more details, please visit our Partnership page). After understanding the scale and appearance of industrial bioreactors through our interview with GeneFerm Biotechnology Co,. Ltd, we designed a disk-shaped filter device (Fig. 2) which consists of:

  1. Reverse osmosis membranes with 0.1 nm pore size prevents fluorescent proteins produced by our indicator bacteria from flowing out from the device.2
  2. Spheres formed by a mixture of sodium alginate and culture medium provide nutrients to the indicator bacteria. The spherical shape provides larger surface area for bacterial adsorption, which results in a brighter color due to the high density of our growth status indicators.

The filter device isolates both types of bacteria, but still enables minute quantities of liquid flowthrough between both membrane sides. Under the same culture medium, our indicator bacteria should demonstrate growth characteristics that resemble that of user bacteria. The filter device will be replaced after fluorescence is no longer visually observable due to leakage of fluorescent proteins from dead bacteria after loss of membrane integrity.3

Fig. 2) Schematic representation of the disk-shaped filter device

 

Laboratory Safety

Rules and Guidance we Follow

During our experiments, we will comply with the laboratory safe hygiene precautions of our institution as well as IGEM's safety and security rules. Moreover, we will always follow the "Safety Guidelines for Biosafety Level 1 to Level 3 Laboratory" from Taiwan Centers for Disease Control. Before beginning our experiments, all protocols will be checked by our instructors and they will guide us throughout all the experiments to ensure safety.

Training:

Every member in our team received biosafety and biosecurity training during 6/24-6/30, which was directed by our PIs and instructors. We learned about lab emergency prevention and response measures, operation protocols for machines, and biological waste management to make sure we can conduct experiments appropriately and safely.

Fig. 3) Emergency-shower-and-
eyewash-station

Fig. 4) Emergency kit

Fig. 5) Laminar flow hood

Waste Treatment / Inactivation Procedures:

For waste treatment, we comply with the "Waste Disposal Act" by Taiwan's Environmental Protection Administration and laboratory safe hygiene precautions of our institution. Although the E. coli strain we have chosen has little or no risk of pathogenicity, we still adopted biocontainment measures by planning a genetically-encoded device (DNAi) in our E. coli. Our engineered plasmid will be degraded once it is removed from a certain culture medium, which prevents horizontal gene transfer in case of accidental environmental leakages.

COVID Measures:

We adopted protection measures In response to the COVID-19 pandemic. In-person contact was prevented as much as possible and weekly project meetings are held online. We also paid close attention to CDC's COVID-19 guidelines to make sure all of our team members are safe and healthy.

 

    Reference

  1. Caliando, B. J., & Voigt, C. A. (2015). Targeted DNA degradation using a CRISPR device stably carried in the host genome. Nature communications, 6, 6989. https://doi.org/10.1038/ncomms7989

  2. Filter pore size is slightly smaller than that of fluorescent proteins. mCherry is 4nm in length. D. W. Piston, R. E. Campbell, R. N. Day, and M. W. Davidson, “Anthozoa Fluorescent Proteins.” [Online]. Available: http://zeiss-campus.magnet.fsu.edu/articles/probes/anthozoafps.html. [Accessed: 10-Oct-2019]

  3. Lowder, M., Unge, A., Maraha, N., Jansson, J. K., Swiggett, J., & Oliver, J. D. (2000). Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506. Applied and environmental microbiology, 66(8), 3160–3165. https://doi.org/10.1128/AEM.66.8.3160-3165.2000
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