Safety

Introduction


The Maple Syrup industry is one of the oldest agricultural enterprises of North America and is of great economic and cultural significance to the United States1. In 2018, the global market value for maple syrup was at an all-time high of $1.24 billion and is forecasted to grow at a compound annual growth rate of 7% by 20232. Unfortunately, each year thousands of gallons of product go to waste and profits are lost due to low-grade “buddy” and “ropy” syrups. “Buddy” syrup, occurring due to metabolic changes in the tree, results in a cabbage-like taste and odor unsuitable for human consumption. “Ropy'' syrup, also unfit for human consumption, occurs due to bacterial contamination and overgrowth during sap and syrup processing3 . Both of these defects can currently only be detected in syrup, not sap, resulting in the production of food waste, and expenditure of time and energy. In this context, our project Saptasense aims to create three novel, low-cost, rapid, and accurate biosensors to detect three different compounds that are found in higher concentrations in buddy sap: asparagine, sarcosine, and choline. Additionally, we aim to isolate and increase production of dextran within the ropy syrup (usually a waste product of contaminated maple sap) by adding genetically engineered Escherichia coli to convert sucrose to dextran. Saptasense further explored the applications of dextran in the biomedical field through primary and secondary research and narrowed down the focus on repurposing the isolated dextran from the ropy syrup into making hydrogels. We aim to achieve this by chemically cross linking dextran with chemical agents for enhanced maple seedling germination.
Our safety analysis includes a thorough process of usage of microorganisms modified by genetic engineering, reagents ranging from Chemical Safety Level (CSL) 1 - 4, and building hardware products using electrical circuits and soldering. After careful consideration of the safety laws of University of Rochester Environmental Health and Safety (EH&S), guidelines of New York State, and iGEM safety guidelines, we revised our project to successfully achieve our goals while ensuring utmost safety of our team members, project, and the application of the project in the real world.

Description of Microorganism and Safety Level Summary


For this project, we will use non-pathogenic strains of Escherichia coli: E. coli K12 strains (including Top10, DH5alpha, and SHuffle® T7) and BL21. E. coli K12 is a gram-negative bacteria known for being a model organism and laboratory reagent widely used for targeted genome sequencing and genetic engineering. The absence of following structural features of E. coli K12 ensures its non-pathogenic behavior to humans, plants, and animals: adhesion and invasion factors, toxins, iron-transport systems, capsule, and plasmids4. Some specific strains that will be used as molecular biology host organisms include Top10 and DH5alpha. Top10 has a broad range of utilities for cloning including plant and mammalian DNA and has high transformation efficiency. DH5alpha E. coli is a strain used for general cloning and subcloning with a high transformation efficiency5. SHuffle® T7 is a strain that has been engineered to have enhanced capability of forming disulfide bonds in the cytoplasm6. Finally, E. coli BL21 is a widely used strain of E. coli used in high-level expression of recombinant proteins. It harbors a DE3 bacteriophage which carries T7 RNA polymerase gene under the lacUV5 promoter7.
Escherichia coli strains used for this project are categorized as a non-pathogenic, Biosafety Level 1 organism by the U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and National Institutes of Health and is listed in the white list of iGEM safety standards.

Biological parts and its safety


To successfully achieve the goals of this project, we are designing and engineering 8 biological parts consisting of a total of 8 cloned plasmids. The summary of these parts is outlined in table 1:

Table 1: Summary of Saptasense biological parts
Biological part Origin and biochemical function Biobrick Plasmid Cloning strain Expression strain
Glucose oxidase Origin - Aspergillus niger; Function - Catalyzes the oxidation of D-glucose to D-gluconolactone and hydrogen peroxide; Mutations function - Sacl site aids in protein fusions, His6tag addition is responsible for protein purification, and stabilizing mutations BBa_K2238000 (Exisiting biobrick constructed by iGEM17_ManhattanCol_Bronx team) pSB1C3 DH5alpha, Top10 BL21, SHuffle® T7
Stabilising mutations - T53K, A79M, R188N, G317S, E417Q DH5alpha, Top10 BL21, SHuffle® T7
Cysteine mutations- Pro192Cys, His201Cys DH5alpha, Top10 BL21, SHuffle® T7
Combined mutations- T53K, A79M, R188N, G317S, E417Q, Pro192Cys, His201Cys DH5alpha, Top10 BL21, SHuffle® T7
Dextransucrase Origin - Leuconostoc mesenteroides; Function - Catalyzes the transfer of glucosyl residues from sucrose to dextran polymer and liberates fructose. DexYG DH5alpha, Top10 BL21
Choline oxidase Origin - Arthobacter globiformis; Function - Catalyzes glycine betaine biosynthesis from choline via betaine aldehyde. CodA DH5alpha, Top10 BL21
Agglutination Assay Origin - Escherichia coli O81 (strain ED1a)Function - EibA binds to IgG antibodies and EibD binds to IgG and IgA antibodies to immobilize them to the surface of bacterial cells EibA DH5alpha, Top10 BL21
EibD DH5alpha, Top10 BL21


The safety of the above mentioned biological parts were carefully considered and the conclusion of each of them can be summarized as follows:
  1. Glucose oxidase, a subset of oxidoreductase enzymes, catalyzes transfer of electrons from an oxidant to a reductant8. It is widely used in commercial processes like improving color and taste, persistence of food materials (hence its categorization as a food enzyme), and eliminating oxygen from different beverages. Overall, it is identified as a low risk enzyme and the biobrick intended from this part will also be in compliance with the safety standards and fall under low risk category9.
  2. Dextransucrase (DexYG) is an enzyme that facilitates the transfer of glucosyl residues from sucrose to dextran polymer and liberates fructose. Dextran produced by DexYG has 95% alpha1,6 linkages in the main chain and 5% alpha1,3 brank linkages, making this dextran very stable and highly expressive. Dextran is used in managing and treating various clinical conditions like hemorrhage, shock, surgical procedures, and others. In the food industry, dextran is used in texture improvement, and therefore overall it’s identified as a low risk enzyme producing low risk products from low risk reactants and thus falls within the safety standards under low risk category.
  3. Choline Oxidase is a bifunctional enzyme that catalyzes glycine betaine biosynthesis from choline via betaine aldehyde. It has been widely used to genetically engineer water to alter the osmotic stress resistance in economically important crops that lack efficient glycine betaine biosynthetic pathways10. According to the Global Harmonized System (GHS) it is considered a category 2 risk enzyme as it consists of Tris base and Horseradish Peroxide. Even though it falls under health hazard 2, it has been approved to be safe to use in the food and crop industry11.
  4. Eib (used for our Agglutination Assay) is an immunoglobulin-binding protein that binds to IgA and IgG antibodies with super affinity (E.coli cells) and immobilizes the antibodies to the surface of the bacterial cells. Eib is widely used with different constructs -A, -C, -D, -E, -F, to bind to bacterial surfaces used in various medical purposes . For our project we are primarily focusing on Eib-A and Eib-D due to its efficient binding to both IgA and IgG antibodies12. This is an important protein labeled safe to use.

Chemical Reagents and its Safety


In addition to the biological parts, our project involved using several different chemicals to perform a variety of experiments. The chemical reagents in addition to the biological reagents were documented in a list that highlighted all the safety information associated with each reagent (acquired from safety data sheets) and how to manage the risk associated with each of them. For any reagent above a safety level of 2, a statement of purpose/intent was thoroughly written and evaluated by the team to conclude that the reagent was absolutely necessary and no lower hazardous materials could be used as a replacement. Once the team was satisfied with the list of chemical reagents and its pertaining information, this document (‘The Safety Proposal’) was revised and reviewed by our principal investigator and teaching assistants. Following this, the safety proposal was forwarded to our institutional biosafety office EH&S. The EH&S biosafety officers reviewed our safety proposal and commented on chemicals that should be mitigated if possible. As a result of this review, we modified a few of the experimental designs and methodology to mitigate the use of high hazard chemicals by either getting rid of them completely or finding less hazardous alternatives, for instance: p-phenylenediamine (used to reduce the rate of photobleaching under fluorescent microscopy) was replaced with Nucblue. We also changed the design of the sarcosine-aptasensor to synthesize the biosensor with cross linking chitosan with glutaraldehyde and aptasensor instead of synthesizing BSA-gold nano-clusters to link with the aptasensor. Such modifications were a result of a responsible communication plan between the teaching assistants, the principal investigator, institute’s teaching lab manager, institution's biosafety office, and the team to minimize risk and maximize safety. At the end of this review, there were very few high hazard chemical reagents that couldn’t be mitigated and had to be used by the team for this project. These chemicals are summarized in Table 2. All of the chemicals were stored in appropriate storage spaces, used only during business hours, and safety precautions were rigorously followed.

Table 2: Summary of reagents under chemical safety level(CSL) 3 and 4
Reagent Number Reagent Safety Level Safety Steps
1 Dithiothreitol (DTT)
CSL-3: Corrosive and moderate chemical or physical hazard
  1. Always wear personal protective equipment: gloves, eye protection/face shield, lab coat.
  2. Wash hands thoroughly after handling.
2 Anthrone
CSL-3: Moderate chemical or physical hazard
3 Trichloroacetic acid (10% w/v)
CSL-3: Corrosive and a moderate health hazard
4 Pyrrole
CSL-3: Flammable, acutely toxic, and corrosive
5 Glutaraldehyde
CSL-3: Moderate health hazard and corrosive
CSL-4: High chemical or physical and environmental hazard
  1. Always wear personal protective equipment: gloves, eye protection/face shield, lab coat.
  2. Wash hands thoroughly after handling.
  3. A chemical fume hood must be used at all times when handling this material.
Chemical reagents highlighted above are all the reagents that were used in the lab under chemical safety level 3 and 4. The chemical hazard symbols are in the safety level column (Table 2) with a description of the specific hazard and a corresponding safety precaution column [13]. Safety hazards for reagents used under chemical safety level 3 and 4 primarily include:
  1. Corrosive chemicals (Reagents - 1, 3, 4, and 5): These reagents may cause burns to skin, damage eyes and corrode metals if in contact with the skin, eyes, or metal.
  2. Moderate chemical and physical hazard (Reagents -1, 2, and 5): May irritate the skin and exhibit minor toxicity.
  3. Health hazard (Reagents 3 and 5) - Long term exposure could cause serious long term health effects, in case of direct skin contact and ingestion contact a health provider immediately.
  4. Flammable (Reagent 4) - Flammable chemical when exposed to heat, fire, or flammable gases when reacting with water.
  5. Acutely toxic chemicals (Reagent 4) - Chemical indicates life-threatening effects in case of ingestion.
  6. Environmental Hazard (Reagent 5) - Reagent indicated substances that are toxic to aquatic organisms and may cause long-lasting environmental effects if disposed off irresponsibly.

Even though the other reagents used fell under chemical safety category 1 and 2, safety precautions were just as rigorously taken while working with them as they were taken with safety level 3 and 4 chemicals. To ensure that every team member was aware and educated about the different safety levels, precautions, and action steps in case of an accident, the team completed various safety training sessions before working in the lab (for more information on the trainings, view the “Safety Training” subheading below).

Hardware and its Safety


Safety while working with hardware equipment was also established and followed as rigorously as the wet lab safety standards. Even though during the entirety of this project no harmful hardware equipment was used, all tools and electrical components were evaluated thoroughly and are summarized in table 3 below.

Table 3. Summary of Hardware Equipments and its safety
Hardware Technique Equipment Used Safety Concern Safety Precaution
Building a circuit Potentiostat
  1. Wires
  2. Op-amps
  3. Resistors and potentiometers
  4. Conductors and Insulators
  5. Breadboards
  6. Battery and power source
 Can cause burns because of electrocution when the power is left on, wet hands contact a running circuit, or damaged wiring.
  1. Power off battery or voltage source when not in use
  2. Be careful to not to touch plumbing or gas pipes while working with wires
  3. Using the right outlets and switches
  4. Do not overload any electrical outlets
  5. Double checking the power switches of all equipment before adding it to the board
Soldering
  1. Soldering iron
  2. Soldering gun
  3. Solder
  4. Solder flux
 May cause burns while using the heated soldering iron.
  • Wear heat resistant gloves while working with the soldering iron
  • Be careful when soldering and make sure that there are no distractions
  • This technique will be done under the supervision of the hardware TA.
Cyclic voltammetry (depositing layers on SPEs)
  1. Potentiostat
  2. Electrochemical cells
 While running the fluids for cyclic voltammetry, there can be electric failures and may cause burns and bruises.
  • Avoid interrupting the run
  • Use rubber gloves in case interruption is necessary
  • Don’t overload the circuit
  • Keep water away from the circuitry.

Safety Training


Team Sapatasense has 12 highly-driven members who have worked towards this project by actively doing safe wet lab and hardware work. To ensure safety, all biological, chemical, electrical, and environmental rules were followed in accordance with the New York State and University of Rochester safety guidelines. Additional safety trainings were completed by all members of the team which included Covid-specific lab training, general Covid safety training, and Biological and Chemical Lab safety training. These trainings played a role in educating the members about various aspects of safety within the lab and while working with hazardous chemicals, important personnel information incase of accidents, differences between biosafety levels and their respective safety standards, how to and when to use different biosafety equipments, methods of disinfection and sterilization, how to follow proper emergency procedures, physical and data biosecurity conditions, and fire safety. The hardware team members completed an additional electrical safety training educating them about different electrical safety standards, how to prevent electrical hazards, safe soldering techniques and other emergency procedures.


Following the completion of all the online trainings, the team had a three day intensive in-person training called the ‘Biological Boot-Camp,’ where the teaching assistants trained all members of the wet lab team on various essential biological methods and safety standards, including 3A Assembly, growing bacterial cultures, making glycerol stocks, miniprepping plasmids, restriction digests, electrophoresis and gel purification, and autoclave safety training. At the end of this camp, all members had equivalent base-line knowledge of safety standards and procedures. To eliminate any further risks, the team signed an agreement with University of Rochester’s Laboratory Management team highlighting all the rules and regulations that will be followed while the team is in the lab. For example,at no point in time is a member going to be alone in the lab (will always work in pairs or more), can only work with reagents with a safety level of more than 2 during business hours, will only work in the lab when at least one emergency personnel is awake and in close proximity, every protocol that needs to be performed will be reviewed by personnel with appropriate knowledge and expertise before the team will have permission to perform them, and all materials and reagents will be stored appropriately with clear indication of its hazardous nature.

Concluding Remarks


Overall, the safety of this team and the project was of the utmost importance. Thus, a combination of reviewed safety proposals, permissions and reviews from the expertise of the team Head Faculty Advisor, The Environmental Health and Safety Officers, and the teaching assistants, ensured a safe working environment for all team members.

References


  1. “Home Page: Agency of Agriculture Food and Markets.”Agency of Agriculture Food and Markets, https://agriculture.vermont.gov/.
  2. “Global Maple Syrup Market 2019-2023: Health Benefits of Maple Syrup to Boost Demand: Technavio.” Global Maple Syrup Market 2019-2023 | Health Benefits of Maple Syrup to Boost Demand | Technavio | Business Wire, 26 Nov. 2018, https://www.businesswire.com/news/home/20181126005272/en/Global-%20Maple-Syrup-Market-2019-2023-Health-Benefits
  3. “2022 Schools - Maple Syrup Grading School - University of Maine Cooperative Extension.” Maple Syrup Grading School, https://extension.umaine.edu/maple-grading-school/2022-schools/.
  4. Blount, Zachary D. “The Unexhausted Potential of E. Coli.” ELife, ELife Sciences Publications, Ltd, 25 Mar. 2015, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4373459/.
  5. “Top 10 vs DH5α? - Topo Ta Cloning Question ... (Jan/16/2007 ).” Top 10 vs DH5α? - Molecular Biology, http://www.protocol-online.org/biology-forums/posts/23777.html.
  6. Biolabs, New England. “Shuffle® Express Competent E. Coli.” NEB, https://www.neb.com/products/c3028-shuffle-express-competent-e-coli#Product%20Information.
  7. Rosano, Germán L., and Eduardo A. Ceccarelli. “Recombinant Protein Expression in Escherichia Coli: Advances and Challenges.” Frontiers, Frontiers, 1 Jan. 1AD, https://www.frontiersin.org/articles/10.3389/fmicb.2014.00172/full.
  8. Khatami SH;Vakili O;Ahmadi N;Soltani Fard E;Mousavi P;Khalvati B;Maleksabet A;Savardashtaki A;Taheri-Anganeh M;Movahedpour A; “Glucose Oxidase: Applications, Sources, and Recombinant Production.” Biotechnology and Applied Biochemistry, U.S. National Library of Medicine, https://pubmed.ncbi.nlm.nih.gov/33840140/.
  9. “Glucose Oxidase/Peroxidase Reagent for Use with Enzymatic Assay Kits Gago20, sta20: Sigma-Aldrich.” Sigma, https://www.sigmaaldrich.com/US/en/product/sigma/g3660.
  10. “Choline Oxidase.” Choline Oxidase - an Overview | ScienceDirect Topics, https://www.sciencedirect.com/topics/neuroscience/choline-oxidase.
  11. Page 1/7 Safety Data Sheet - Cayman Chem. https://cdn.caymanchem.com/cdn/msds/15100m.pdf.
  12. A;, Leo JC;Goldman. “The Immunoglobulin-Binding EIB Proteins from Escherichia Coli Are Receptors for IGG FC.” Molecular Immunology, U.S. National Library of Medicine, https://pubmed.ncbi.nlm.nih.gov/19303642/.
  13. Hazard Communication Standard Pictogram - Occupational Safety and ...https://www.osha.gov/sites/default/files/publications/OSHA3491QuickCardPictogram.pdf.