Team:Lambert GA/Safety
S A F E T Y
Team Safety
- Wash in, wash out
- Protect eyes, mucous membranes, open cuts and wounds from contact with biohazard material
- Do not eat or drink when in the lab area
- Always use gloves and splash-proof goggles
- Tie back loose hair
- Disinfect all surfaces with 70% ethanol prior to working
- Disinfect all disposable tips, glassware, tubes by soaking in 10% bleach solution for 20 minutes and then disposing in normal waste
- Dispose of growth plates by disposing into a biohazard container which get autoclaved
- Check all equipment for good working order, no chips, torn cords, cracks. Report any issues to an instructor immediately
- When pipetting, don’t touch tip to side of container
- Don’t lay caps of tubes upside down. Use masking tape to hold to bottom of cabinets
- Clean work area with 70% ethanol after working
- Clean up all glassware and labware before leaving lab
- Place all backpacks and stools to the side of the lab to keep walkways clear
- Always know the correct procedure for disposal of lab materials
Project Safety
Recognizing the limitations of the current methods utilized for the detection of cholera the 2018 Lambert iGEM team will be building a disease detector utilizing RNA based biosensors called Toehold Switches. This will include engineering E. coli to detect Vibrio cholerae utilizing a short sequence from the gene, ctxB. The Cholera enterotoxin subunit B is a non-toxic gene that is specific to the Vibrio cholerae serotype O1 (strain ATCC 39315) allowing the target sequence to be accurate and safe enough for the detection of Cholera. By developing a RNA-based detection mechanism for Cholera, Lambert iGEM is able to address the epidemic of waterborne diseases at drastically lower costs, without sacrificing on quality or performance, to further integrate practical and efficient solutions for Synthetic Biology.
For future implications, the Lambert iGEM team will not be conducting any direct experimentation with Vibrio cholerae as stated in our check-in form. Any testing with V. cholerae will be conducted under a specialized lab that is authorized to work with these strains.
BL21 DH5-alpha E. coli is our nonpathogenic chassis and was developed for laboratory cloning use. The potential health and environmental hazard associated with BL21 DH5-alpha E. coli hazards are highly limited and can be handled in Biosafety level 1 laboratories.
For future implications, the Lambert iGEM team will not be conducting any direct experimentation with Vibrio cholerae as stated in our check-in form. Any testing with V. cholerae will be conducted under a specialized lab that is authorized to work with these strains.
BL21 DH5-alpha E. coli is our nonpathogenic chassis and was developed for laboratory cloning use. The potential health and environmental hazard associated with BL21 DH5-alpha E. coli hazards are highly limited and can be handled in Biosafety level 1 laboratories.
Hardware & Software Safety
The main safety hazards with our 3D printer is the temperature fluctuations. The nozzle is able to heat up to 220 degrees Celsius. In addition, leaving the printer unattended can be dangerous due to the 3D printer’s high temperatures which could cause a potential fire. However, our printer is advanced and possesses mechanisms to prevent this from happening the chance of this occurring is extremely rare, but if it occurs, it can cause serious damage and end up burning parts of the printer. The printer uses softwares called Crash Detection and Heat Overload to monitor the overall safety of the printer. The printer will always be monitored by a PI preventing any unwanted accidents.
CALM's SMS surveys do not collect any personal identifying data. The questions in the survey only ask for the governorate in which the responder resides and general health and sanitation questions (e.g. "how many people have you seen with cholera this week?"). We do not record or process phone numbers.
The voltage output from the ElectroPen is not hazardous to any user due to the low current (scale of nanoamperes to microamperes) equivalent to static electricity. The safety risks associated with the ElectroPen can therefore be regarded as minimal, and proper handling can include wearing gloves to avoid electric shock.
CALM's SMS surveys do not collect any personal identifying data. The questions in the survey only ask for the governorate in which the responder resides and general health and sanitation questions (e.g. "how many people have you seen with cholera this week?"). We do not record or process phone numbers.
The voltage output from the ElectroPen is not hazardous to any user due to the low current (scale of nanoamperes to microamperes) equivalent to static electricity. The safety risks associated with the ElectroPen can therefore be regarded as minimal, and proper handling can include wearing gloves to avoid electric shock.
Discarding of Cells in the Field
The Hsp33 protein is expressed by the gene hslO and confers resistance to oxidative protein unfolding among bacteria cells [1]. Oxidative protein unfolding occurs with treatment of bleach or peroxides at elevated temperatures, but the hslO protein protects the protein elongation factor Tu against it [2]. When cells are under certain stress, such as exposure to hypochlorite, Hsp33 activates in order to prevent oxidative protein unfolding [3].
The easiest way to kill the V. cholera would be to boil the water sample and biosensor cell culture, as the heat denatures protein structure and causes protein aggregation. When proteins begin to clump together, they are unable to function and the cell then dies. Boiling the biosensor cells causes the bonds to break down, resulting in the loss of function in the cell.
However, other sources suggest 1% sodium hydrochlorite and 70% ethanol will kill cells. Results show that a 1% sodium hydrochlorite solution sprayed on the surface and let sit for five minutes will effectively remove all DNA, saliva, blood, semen, and skin cells from any smooth or pitted surface when wiped down with 70% ethanol afterward. However, sodium hydrochlorite solution followed by ethanol can produce amounts of gaseous chlorine above recommended exposure levels. As a result, 1% sodium hydrochlorite followed by distilled water was tested and proven to be effective as well [4].
The easiest way to kill the V. cholera would be to boil the water sample and biosensor cell culture, as the heat denatures protein structure and causes protein aggregation. When proteins begin to clump together, they are unable to function and the cell then dies. Boiling the biosensor cells causes the bonds to break down, resulting in the loss of function in the cell.
However, other sources suggest 1% sodium hydrochlorite and 70% ethanol will kill cells. Results show that a 1% sodium hydrochlorite solution sprayed on the surface and let sit for five minutes will effectively remove all DNA, saliva, blood, semen, and skin cells from any smooth or pitted surface when wiped down with 70% ethanol afterward. However, sodium hydrochlorite solution followed by ethanol can produce amounts of gaseous chlorine above recommended exposure levels. As a result, 1% sodium hydrochlorite followed by distilled water was tested and proven to be effective as well [4].
References
[1] Wholey, W. Y., & Jakob, U. (2012, March). Hsp33 confers bleach resistance by protecting elongation factor Tu against oxidative degradation in Vibrio cholerae. Retrieved October 8, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/22296329
[2] Cholera - Vibrio cholerae infection. (2018, July 20). Retrieved October 8, 2018, from https://www.cdc.gov/cholera/diagnosis.html
[3] Nelson, E. J., Harris, J. B., Morris, J. G., Calderwood, S. B., & Camilli, A. (2009, October). Cholera transmission: The host, pathogen and bacteriophage dynamic. Retrieved October 8, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842031/
[4] Kaye N. Ballantyne, Renato Salemi, Fabio Guarino, James R. Pearson, Dale Garlepp, Stephen Fowler & Roland A.H. van Oorschot (2015) DNA contamination minimisation – finding an effective cleaning method, Australian Journal of Forensic Sciences, 47:4, 428-439, DOI: 10.1080/00450618.2015.1004195
[2] Cholera - Vibrio cholerae infection. (2018, July 20). Retrieved October 8, 2018, from https://www.cdc.gov/cholera/diagnosis.html
[3] Nelson, E. J., Harris, J. B., Morris, J. G., Calderwood, S. B., & Camilli, A. (2009, October). Cholera transmission: The host, pathogen and bacteriophage dynamic. Retrieved October 8, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842031/
[4] Kaye N. Ballantyne, Renato Salemi, Fabio Guarino, James R. Pearson, Dale Garlepp, Stephen Fowler & Roland A.H. van Oorschot (2015) DNA contamination minimisation – finding an effective cleaning method, Australian Journal of Forensic Sciences, 47:4, 428-439, DOI: 10.1080/00450618.2015.1004195