Team:TU Eindhoven/Safety/Biosafety

Biosafety

Lab safety

One of the most important thing when working in the lab is the safety of the team members and the other persons working in the lab. Therefore it is important to follow the rules of the laboratory.

Work in the lab is done with genetically modified organism (GMO) material or with cells, therefore it is not possible to simply enter the laboratory without permission. So the team needed a safety training first. In this safety training a lab tour was given in which topics were discussed like the safety equipment, personal equipment, safety measures, waste disposal and biosafety rules or guidelines concerns working with GMOs. After the training the team received permission to enter the lab.

In Europe there are some biosafety regulations, which were also followed in the lab. These can be found here. At the Eindhoven University of Technology there is one main person responsible for the biological safety. Her name is Moniek de Liefde – van Beest. She is the biosafety officer of the TU Eindhoven. There are also principal investigators (PI). A PI is responsible for the biosafety and lab safety at his or her appointed laboratories.

The biosafety guidelines for the laboratory are as follows: when working with GMOs, it is important to be careful, work clean and hygienically, know how to work with GMOs and know the safe microbiological techniques (SMT). SMT ground rules and other biosafety rules at the lab are:

1. Always work according to the rules.
2. Keep doors and windows closed.
3. Gather everything and place it orderly.
4. Keep everything clean and tidy, make sure enough disinfectant is present.
5. Always wear a marked and closed lab coat, which is not permitted outside the lab. The lab coats have to be sterilized by autoclaving before sending it to the laundry.
6. Don’t wear any watches or jewelry, or keep them covered by gloves or lab coat. Bags, laptops etc. are not allowed and can be stored outside the lab in lockers.
7. Avoid any contact between your hands and your face. Don’t eat, drink or smoke inside the laboratory. Storing food or drinks is also not allowed.
8. Avoid formation of aerosols. Mix and centrifuge in closed tubes. The use of a needle is allowed only when no other method is available.
9. Pipetting with the mouth is not allowed; use the available equipment (pipette boy or pipette bulb).
10. Always disinfect your working space at the start and at the end of your activities.
11. After a contamination of your working space (e.g. when you spill any biological material) disinfect the working space.
12. After working with biological agents and when leaving the laboratory, always wash your hands with water and soap.
13. All re-usable materials that were in contact with biological materials have to be sterilized before being washed or discarded.
14. Solid biological material is gathered in the red biohazard bags, transferred in blue containers and disposed of as special garbage. Liquid biological waste has to be autoclaved before it can be discarded.
15. All accidents and spills, and all activities with GMOs or human material have to be reported in the appropriate logbook.

There are also rules concerning waste disposal: all solid biological waste (ML-I and ML-II) has to be collected in red biohazard bags and transferred to the leak-free blue hospital containers. After closing, labeling and disinfecting the exterior of the container with 70% ethanol it can be disposed of as special waste. Solid waste contaminated with phages can also be collected in the blue hospital containers. Liquid biological waste has to be autoclaved (20 min. at 121 degree Celsius) and can be discarded in the sewer afterwards. Liquid waste contaminated with phages has to be disinfected in a bottle with concentrated chlorine at least overnight. Glass and instruments which have been in contact with biological agents must also be autoclaved and can be cleaned in the dishwasher, subsequently. Glass and instruments which have been in contact with phages must be disinfected overnight with a chlorine solution, and then washed with water, cleaned in the dishwasher and autoclaved. Chemical waste has to be collected in the chemical trashcans in the lab, transferred in the ‘wisseldrum’ near the autoclave and disposed of when it is full.

Biosafety of the Organisms and Parts

The organisms that were used during the project are E. coli (K-12) (Nova Blue), E.coli (B) Bl21 (DE3) and E. coli (K-12) (XL10-Gold). The E. coli K12 strain is a so called ‘lab strain’. This strain cannot survive in the human digestive system and is not able to produce toxins. The Risk group of this strain is 1, and all it derives are also risk group 1 like the used NovaBlue and XL10-Gold strains [1]. The E. coli B and its derivatives, like the used BL21 (DE3) strain, are classified as risk group 1 [4].


The parts that we used are BBa_K1492000, BBa_K1492001 and BBa_K1492002 and are listed in Table 1. For more information check our Biobrick pages (COMPx, COMPy and tRNA Synthetase) and our Safety spreadsheet.

Table 1. Parts and their names we used. The table also includes the original function of the part and the organism of origin and its corresponding risk group.

Biosafety During the Project

In the project non-pathogenic laboratory strains as chassis organism were used; these strains do not produce any toxins. This reduces the risks to the safety and health of team members and other people working in the lab. The strains are not able to survive outside the laboratory or in the human digestive system and can therefore not do any harm to the environment. Besides this, they can easily be killed with 70% ethanol.

The donor organisms of the parts are listed as risk group 1, except for the E. coli which is a risk group 2 organism. However the plasmid that is used for BBa_K1492000 is derived from the DH5alpha strain, which is listed as a risk group 1 organism [5]. In addition the functions of the parts are not harmful to humans. After we have coated the bacteria, they are able to survive under harsh conditions, but they still cannot do any harm, because of their non-pathogenic properties, so they would not pose a threat to team members.

In short due to the use of these non-pathogenic strains as chassis organisms and the use of safe parts from risk group 1 donor organisms the project currently does not raise any safety issues.

Future Risks and Biosafety

Depending on the type of coating, the bacteria will be able to survive in harsh conditions, such as the immune system or in industrial reactors.

Even though the used E. coli strains are not harmful, the bacterial growth still has to be controlled. Or else it can result in uncontrollable bacterial growth. However, it is still uncertain if this will happen, the polymer coating might suppress the cell division or the bacteria might suffer from necrosis, due to this coating.

The coating is made by use of the SPAAC click reaction in the controlled environment of the lab. Because of this, the bacteria will not be able to create a new coatings after cell division, since this requires the addition of the specific molecules once again. Possible harsh conditions will therefore kill the divided bacteria, because these bacteria are unprotected. With exception of the biological synthesis of a zwitterionic antifouling protein coating where the divided bacteria will be able to make their own coating, protecting them from the harsh conditions of the human body.

Another big future risk is that a zwitterionic antifouling protein coating can be applied in other bacteria that are listed in a risk group higher than 2. This could be a potential risk for the safety and health of the general public and environment. If in this case pathogenic bacteria are used, the project can be maliciously mis-used by individuals, groups or countries.

This would not really be the case in Click Coli, firstly because it requires the use of two plasmids (orthogonal tRNA synthethase and COMPx). Firstly, the double transformation in bacteria could be a limiting factor and secondly an unnatural amino acid is added before expression. The used strain has an amber suppression, which means that the termination is suppressed at the Amber Stop Codon. Since this is not naturally occurring in most bacterial strains it is an extra safety feature. The orthogonal synthase only enables the incorporation of the unnatural amino acid at the place given by the Amber Stop Codon (TAG). Thirdly the coating is made by a chemical click reaction of the azide group with DBCO functionalized molecules and is not able to produce these DBCO functionalized molecules itself. These together reduce the chance of azide group coating with DBCO functionalized molecules on the outer membrane in other bacteria to very close to zero. But still, there is always a chance of success.

Because of these possible risks for mainly the zwitterionic antifouling protein coating, we thought of mechanisms to kill the bacteria and how we could implement these in our idea. Therefore we introduced a kill switch concept, which is elaborated on the Kill Switch Page.

Non-Biological Safety

Next to biosafety, an important aspect of working in the lab is the safety regarding to non-biological safety, such as hazardous chemicals. To be sure that we work safely with chemicals, it is important to read the material safety data sheets (MSDS) of the chemicals before using them. Most of the chemicals we used are not hazardous but some of them are. These are listed below:

1. Trichloro(1H,1H,2H,H2-perfluoro-ocyl)silane is a chemical that is used in the silanization process of our silicon wafer for microfluidics during photolithography. This chemical is corrosive so it is important to wear gloves and to work in the fume hood.


2. Ammoniumpersulfate (APS) is a chemical we use in our microfluidics droplet device in combination with TEMED to initiate the polymerization of acrylamide to polyacrylamide droplets. This chemical is harmful for the respiratory system so it is important to work in the fume hood.


3. Tetramethylethyleendiamine (TEMED) is a chemical we used in our microfluidics droplet device in combination with APS to catalyze the polymerization of acrylamide to polyacrylamide droplets. When using this chemical it is important to work in the fume hood due to its hazardous fumes, but also wear gloves due to its corrosive properties.


4. Acrylamide is used for the production of polyacrylamide droplets in a microfluidics droplet device, and it is also used for polyacrylamide gels for gel extraction. Acrylamide is toxic and hazardous and requires nitrile gloves and a fume hood.


5. Hexane is a chemical that is used for the separation of the droplets from the oil phase, due to its miscibility with FC oil. Hexane is very hazardous, it is important to always work in a fume hood and wear nitrile gloves when handling hexane.


6. Aphidicolin in DMSO: Aphidicolin is a polymerase inhibitor that can inhibit some DNA polymerase which we used for our rolling circle amplification. Due to this inhibition and because it is dissolved in DMSO, which can penetrate easily through skin, it is important to avoid contact with the hands, so it is important to wear nitrile gloves.

Safety Forms

For more safety information check our Safety Forms (Our Lab Form and Safety Form)

Bibliography

[1] http://www.dsmz.de/catalogues/details/culture/DSM-3586.html

[2] http://www.dsmz.de/catalogues/details/culture/DSM-800.html?tx_dsmzresources_pi5%5BreturnPid%5D=304

[3] http://www.absa.org/riskgroups/bacteriasearch.php?genus=escherichia

[4] A.P. Bauer, S.M. Dieckmann, W. Ludwig, K.H. Schleifer. Rapid identification of Escherichia coli safety and laboratory strain lineages based on Multiplex-PCR. FEMS microbiology letters. 269:36-40 (2007). DOI:10.1111/j.1574-6968.2006.00594.x

[5] http://www.dsmz.de/catalogues/details/culture/DSM-6897.html