Team:Uniandes Colombia/WetLab

iGEM Uniandes-Colombia

Team Project Wet Lab Dry Lab Hardware Human Practices Collaborations -

Wet Lab

Safety
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Protocols
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Notebook
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Biosafety



Biosafety Provisions

Institutional Regulations

Our project safety procedures are governed by the general regulations of the University regarding the proper standard rules and safety guidelines for laboratory practices. Teaching and research laboratories in the university are specially regulated accordingly to the dependence they belong to. In our case, the Faculty of Sciences Department of Biological Sciences and Department of Physics. The specific guidelines for the laboratory work at the Department of Biological Sciences are available online (in Spanish only) Biosafety Guidelines. Our main workspaces are the LAMFU and the Biophysics facilities, BSL2 research laboratories, but also we occasionally work on several BSL1 teaching laboratories (Department of Biological Sciences).



Institutional Biosafety Committees

There is the Parietal Committee for Occupational Health (COPASO), regulated by the Colombian Constitution (Resolution Number 2013, Year 1989). The information regarding the functions and regulations of the COPASO committee is available online (Spanish only). There is also the Bioethical Committee of the Faculty of Sciences, to whom we have presented our project proposal. Although we are still waiting for a response from the committee, our research methods and safety precautions are well within the regulations of the Committee.



Biosafety Training & Work Precautions

All team members were required to attend safety training focused on: • General biosafety principles. • The proper use of containment equipment (Biological safety (laminar flow) cabinets and delimited working space when required) as well as personal protective equipment (PPE) (White coat and gloves). • Risk assessment of experiments involving: Recombinant DNA, ethidium bromide, other biohazardous materials. • Proper Biological waste disposal. Our main concern was to work with the appropriate safety guidelines for working in both research and teaching laboratories (BL1-BL2) in order to minimize exposure to hazardous agents. We also work with the participating laboratories´ procedures to minimize waste and encourage use of reusable glassware instead of disposable plastic.



Safety Considerations:

Hazardous reagents

and Biohazards


Health Risks & Personal Safety

Working in the lab always has implicit risks, but we have taken action to minimize them. Some of the precautions we take are listed here: • The microorganisms are grown in large petri dishes that are sealed completely during bacterial growth, thus preventing their escape. • Equipment such as biological safety cabinet class I and laminar flow cabinets are used regularly to minimize the possibility of spread of microorganisms by recently trained students. • All students involved with the project were informed of the established security arrangement and signed a commitment to strictly comply with the rules. • Also, prior to the beginning of the experimental procedures, all of the team members were given a course on biosafety, focused mainly on biological hazards, chemical reagents and general BSL-1 laboratory safety concerns and security procedures. The system design tests are conducted within a laboratory of biosafety level 1, and we use the following materials and microorganisms strains:



Known toxic chemical reagents and hazardous physical agents:

Ethidium bromide

Since ethidium bromide is a very toxic agent, we have changed our nucleic acid staining molecules to SYBR® Safe. This claims to be significantly less toxic and better for the environment. Indeed, according to the manufacturer, it may be disposed directly into wastewater drainage systems (not a risk we cared to take, though).


Ultraviolet light

It is a type of electromagnetic radiation with a shorter wavelength shorter than that of visible light. We use UV in the biological safety and laminar flow cabinets for sterilization and decontamination purposes. Also, UV radiation is used for visualization of the stained DNA in the electrophoretic gels. In order to prevent accidental exposition to UV radiation, precautions are taken and when necessary, special safety lenses are used. We always use UV-blocking shields when visualizing electrophoretic gels in UV light. All other toxic and chemicals will be handled to avoid direct contact, and observing the proper safety procedures, additionally, all chemicals reagents and biological materials will be disposed only in the designated biohazard receptacles, following the regulations for Guidelines for Integral Solid Waste Management of the Medical and Occupational Health Department at the Universidad de los Andes.





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Protocols



Agarose gel

  1. Weigh 0,3 g of agarose.
  2. Add 30 mL of TAE 1X.
  3. Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved.
  4. While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight, and that the “comb” is well placed.
  5. When homogeneous, add 2 µL of SYBR SAFE DNA Gel Stain to the solution and mix well.
  6. Pour the solution into the bed and clear all its bubbles with a tip. Put a piece of paper on top of it and let it polymerize.
  7. Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 4 µL of molecular weight marker into the first well.
Note: For big gels use 0,7 g of agarose, 70 mL of TAE 1x and 3 µL of SYBR SAFE Gel Stain.


LB medium (1L liquid)

  • 10 g tryptone
  • 10 g NaCl
  • 5 g yeast extract
  • Water



LB medium (solid, 1L = 50 dishes)

  • 15 g agar agar
  • 10 g tryptone
  • 10 g NaCl
  • 5 g yeast extract
  • Water

For selective medium, suplement with antibiotic as appropiate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin ).


Electrocompetent cells

  1. Divide the ON culture in 50 mL falcon tubes (we always used ''E. coli'' TOP10).
  2. Centrifuge 8000 rpm x 10 min.
  3. Discard supernatant.
  4. Resuspend everything with water in two falcons.
  5. Centrifuge again.
  6. Discard supernatant and resuspend with water. Wash with water two more times.
  7. Centrifuge again.
  8. Discard supernatant.
  9. Resuspend with glycerol with water. Glycerol 10 %.
  10. Centrifugue.
  11. Repeat steps 8-10.
  12. Discard supernatant and divide what is left in eppendorfs.
CAUTION: Everything must be done on ice (0-3°C) (reactants, centrifuge, transport, containers).


Genome extraction

For bacterial genome extraction we used Easy DNA Kit, Invitrogen according to manufacturer's instructions.


Plasmids extraction

For bacterial plasmid extraction we used GenElute Plasmid Miniprep Kit, Sigma Aldrich according to manufacturer's instructions.


Transformation by electroporation

  1. Mix 40 µL of electrocompetent cells with 4 µL of DNA (we used iGEM BioBricks resuspended in 20 µL of miliQ water ).
  2. Incube the cuvettes for electroporation on ice and the above mix as well.
  3. Electroporate into the cuvette.
  4. Add (as quick as possible) 200 µL of LB medium.
  5. Incubate for 30 min at 37 °C .
  6. Plate bacteria over sumplemented LB medium.
  7. Incubate at 37 °C, 24 h.
  8. Use isolated colonies to check the correct insertion.



Biobrick Assembly

The digestion and ligation of the BioBricks were carried out using the Biobrick Assembly Kit, New England Biolabs Inc. The protocols for both procedures can be found here.

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