Safety

Why safety is important

iGEM has two major guidelines for laboratory work during a project:

No organisms of riskgroups 3 or 4 should be used.
No genetically modified organisms should be released outside the lab.

The first guideline does not apply since all our parts and organisms are classified as riskgroup 1 at the highest. However, we modify a species, C. reinhardtii, which can be applied to the second guideline. This rule becomes especially crucial if the organism is classified as a GMO.

The question therefore follows; should our projcet be considered as GMO?

Yes we do have a genetically modified eukaryotic organism, however despite it being genetically modifed, by defintion it is not to be considered as GMO since we have been using CRISPR/Cas9 system to cut a gene without inserting new DNA. The FDA [1], as well as the swedish agriculture department [2] considers the use of CRISPR/Cas9 not to be GMO if you use the system only to cut a gene.

Safe Project Design

At LiU iGEM our main concern has always been safety. The decisions involving project design has therefore carefully been analyzed to minimize the amount of potential risks. The chassis we are using are a non-pathogenic XL-1 blue E.coli strain and all the parts included are unharmful to humans and other organisms. The main project challenge was to ensure that our genetically modified C.reinhardtii would not pose any threat to other wild type algae. That is being satisfied by the fact that Cas9 and the sgRNA will severely weaken the modified C.reinhardtii to the point that it will not be able to reproduce. This is especially true since our goal is to eventually modify the chloroplast, which, when modfied will severly weaken C.reinhardtii to grow outside in the wild. To assure that no modified C.reinhardtii would escape into the wild this system will be activated by light, so that ordinary sunlight would be sufficient to shut down the organism.

Safe Lab Work

Saftey is paramount when working in the laboratory, and this project is no expection. Before each lab a risk assesment was written down. Since we were using risk group 1 organisms we were working on a lab bench and always wearing lab coats, saftey glasses and latex/nitrile gloves. Even if the algae did not belong to any risk group, they were treated as though they are risk group 1 organisms. The waste of both bacteria and algae were collected in separate waste containers and then sterilized through autoclavation. This was also true for all the solid waste that had touched algae or bacteria. To ensure no mutated bacteria or algae got outside the lab, each laborant sterilized their hands aswell as the lab bench they were working with.

For chemicals that had a pungent odor (i.e acetic acid), or was poisonous (i.e hygromycin), or for any other type of hazards, we handled these chemicals under a fume hood with nitrile gloves and saftey glasses with outmost care. The most dangerous chemical that we worked with, ethidium bromide, was handled with extreme care. Residues of Hutners trace elements, which contained the heavy metals cobalt(II) and copper(II) were disposed of in a waste container for heavy metals.

Safe Shipment

It is of outmost importance that the products that you ship for sequencing or to the registry are transported in a safe and secure manner. Careful considerations must be taken when it comes to shipping of biological parts, it would be rather careless and unfriendly for the environment if a gene or a biological part got damaged or leaked out due to improper care during transport. Since our parts where shipped after the wiki freeze we can not tell about any specifics about how the shipment went for the parts to iGEM:s headquarters. But, since our parts were sequenced in Germany the shipment to that destination will be discussed here.

When our parts were ready to be sequenced we contacted our secondary PI, which had contacts with a sequence company and asked for help with sending it to the company. The amount of genetic material that was shipped each time were approximately 10-100 micrograms/microliter and the volume was 50 microliter. The genetic material was transported in an eppendorf tube which was sealed in a special case, then it was posted using air mail. The results usually came after 1 week, which was the standard time, indicating there were no problems with shipment regarding the sequencing matter.

MSDS for the chemicals used during the project

[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=SE&language=EN-generic&productNumber=H3274&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fh3274%3Flang%3Den Hygromycin]
[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=SE&language=EN-generic&productNumber=C0378&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fc0378%3Flang%3Den Chloramphenicol]
Kanamycin
[http://www.sigmaaldrich.com/catalog/product/sial/252859?lang=en&region=SE TRIS]
[http://www.sciencelab.com/msds.php?msdsId=9922769 Glacial acetic acid]
[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=SE&language=EN-generic&productNumber=P5655&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fsearch%3Fterm%3D7778-77-0%26interface%3DCAS%2520No.%26N%3D0%2B%26mode%3Dpartialmax%26lang%3Den%26region%3DSE%26focus%3Dproduct Potassium monophosphate]
[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=SE&language=EN-generic&productNumber=RES20765-A7&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fsearch%3Fterm%3DK2HPO4%26interface%3DAll%26N%3D0%26mode%3Dpartialmax%26lang%3Den%26region%3DSE%26focus%3Dproduct Potassium diphosphate]
[http://www.sciencelab.com/msds.php?msdsId=9923980 EDTA]
[http://cdn2.lasecsa.co.za/pdf/sds/Zinc%20Sulphate%20-%20MSDS.pdf Zinc sulphate]
[http://www.sciencelab.com/msds.php?msdsId=9927105 Boric acid]
[http://www.sciencelab.com/msds.php?msdsId=9924583 Maganese(II)chloride]
[http://www.sciencelab.com/msds.php?msdsId=9923522 Cobalt(II)chloride]
Copper(II)sulphate
[http://www.sciencelab.com/msds.php?msdsId=9922926 Ammonium molybdate]
Iron(II)sulphate
Sodium chloride
Sodium hydroxide
[http://www.sciencelab.com/msds.php?msdsId=9927402 Potassium chloride]
[http://www.sciencelab.com/msds.php?msdsId=9927230 Potassium hydroxide]
[http://www.sciencelab.com/msds.php?msdsId=9927219 Magnesium sulphate]
[http://www.sciencelab.com/msds.php?msdsId=9927477 Calcium chloride]
Ammonium chloride
[http://www.sciencelab.com/msds.php?msdsId=9927063 Agarose]
[http://www.sciencelab.com/msds.php?msdsId=9927667 Ethidium bromide]

References

1. FDA public meeting (October 30th 2015), Modernizing the regulatory system for Biotechnology products (obtained at the webpage http://www.fda.gov/downloads/newsevents/meetingsconferencesworkshops/ucm477576.pdf 2016-10-17)

2. Jordbruksverket, Defintion av GMO,(obtained at this following webpage, please note that this page is in swedish! http://www.jordbruksverket.se/amnesomraden/odling/genteknikgmo/definitionavgmo.4.4b3f0532150f4b827c71f4ca.html 2016-10-17)

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