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Team:HUNGENIOUS/Safety
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| + | </font><font style="font-size:21px;position:absolute;left:400px;top:90px;" color="#000000" face="Baskerville Old Face"><b><u>Safety</u></b></font><font style="font-size:24px" color="#000000" face="Baskerville Old Face"><br> | ||
| + | <br><br><br> | ||
| + | <b>Personal Safety</b><br> | ||
| + | <u>Safety Education</u><br> | ||
| + | At the beginning of the lab work the team has participated in a thorough biosafety education held by our mentors. The main idea of this course was to clarify that working in a molecular biology laboratory has many other important aspects beyond the scientific research work. We went through all the possible personal, public and environmental risks and possible coping strategies, as well. We have learned how to use the fire extinguisher, how to use chemically and/or biologically unsafe materials and how to handle safely all the manually or electronically controlled equipment. | ||
| + | <br> | ||
| + | <u>Biosafe “Dress Code”</u><br> | ||
| + | During all experiments, all participants had to wear gloves, which were dropped into the aforementioned container, and lab coats which were used only in the appropriate lab. All the procedures (PCR reactions, microbial cultures, transformations, etc.) were supervised carefully by at least one adult with the same “biosafe dress code”.<br> | ||
| + | <b>Regulations</b><br> | ||
| + | We have used microorganisms belonging only into BSL1 level. The only exception is <i>Chromobacterium violaceum</i> which is itself a BSL2 bacterium; however we have used only parts of its DNA (chitinase gene). The product of this gene is not connected with the <i>Chromobacterium violaceum</i>’s pathogenic effect. In this case we can state that only BSL1 bacteria were used as living organisms. | ||
| + | Concerning genetically modified organisms, we had to fulfil the requirements of the rather strict Hungarian State regulations. (You can read more on these regulations here: http://gmo.kormany.hu/hungarian-legislation) Concerning these regulations, GMO as living organism can only be produced and/or transferred by approved institutions. Because of this, all the transformational part of our project was carried out in the buildings of the Biological Research Centre of Hungarian Academy of Sciences which is a BSL2/3 and moreover an approved institution for producing and dealing with GMO. | ||
| + | <b>Microorganisms Used by Our Groups</b><br> | ||
| + | Name DNA | ||
| + | /living cells Biosafety | ||
| + | Level ATCC No. Source Used for | ||
| + | Chromobacterium | ||
| + | violaceum DNA-part BSL2 12472 DSMZ amplification template of chitinase gene | ||
| + | Escherichia coli | ||
| + | DH5 alpha | ||
| + | http://ecoliwiki.net/colipedia/index.php/DH5_alpha | ||
| + | living cells BSL1 67878 BRC transformation | ||
| + | Escherichia coli Nissle 1917 living cells BSL1 8739 BRC transformation | ||
| + | Bacillus thuringiensis | ||
| + | NCAIM - 01262 living cells / freeze dried BSL1 33679 NCAIM chitinase assay | ||
| + | Bacillus thuringiensis | ||
| + | NCAIM - 01292 living cells / freeze dried BSL1 10792 NCAIM chitinase assay | ||
| + | |||
| + | <br> | ||
| + | <b>Biosafety Arrangements<br> | ||
| + | </b> | ||
| + | <u>Public and natural environment safety</u><br> | ||
| + | During the experiments having been carried out in the school all the rooms of the laboratory, where our work took place, were signed with biohazard sign, with a title “BIOHAZRD” and in Hungarian “BIOLOGIAI VESZELY”. | ||
| + | <br> | ||
| + | All the disposable materials, (pipette tips, Eppendorf- and PCR-tubes, inoculating loops, etc.) connecting with microorganisms were put into a biohazard container, in which there was a strong oxidative disinfectant. This type of handling fulfils the Hungarian requirements in the cases of BSL1 materials. All the bacterial colonies and also the fluid cultures were handled with the same oxidative disinfectant. | ||
| + | <br> | ||
| + | All the DNA sample and disposable materials (pipette tips, Eppendorf- and PCR-tubes, inoculating loops…) connecting with it, were dropped into another container in which the strong acidic environment damaged it. All the containers are going to be transported by an officially approved firm which will continue the safety procedures with our used materials according to the Hungarian regulations. | ||
| + | <br> | ||
| + | <b>Chemical Safety<br> | ||
| + | </b> | ||
| + | </font><font style="font-size:13px" color="#000000" face="Baskerville Old Face"><br> | ||
| + | <br> | ||
| + | </font><font style="font-size:20px" color="#000000" face="Baskerville Old Face"><b>Screening Chitinase-activity</b><br> | ||
| + | We found this method in the article of N. Murthy, B. Bleakley: Simplified Method of Preparing Colloidal Chitin Used For Screening of Chitinase- Producing Microorganisms.<br> | ||
| + | We would like to use this method as a chitinase assay. In order to check that the chitinase gene was infiltrated and expressed in the genetically modified bacteria, we inoculate the bacteria onto colloidal chitin agar. ,,The colloidal chitin agar (pH 7.0+/-0.2) prepared had the following ingredients (in g/L): (K<sub>2</sub>HPO<sub>4,</sub> 0.7; KH<sub>2</sub>PO<sub>4,</sub> 0.3; MgSO<sub>4</sub> X 5H<sub>2</sub>O, 0.5; FeSO<sub>4</sub> X 7H<sub>2</sub>0, 0.01; ZnSO<sub>4,</sub> 0.001; MnCl<sub>2,</sub> 0.001), amended with 2.0 % moist colloidal chitin. Bacto Agar (Difco) was added at 2.0% as a solidifying agent. The medium was sterilized by autoclaving at STP (standard temperature and pressure: 15 psi, 20 minutes, 121 ° C) and poured into sterile Petri plates (approximately 20 ml per plate).”<br> | ||
| + | If the chitinase gene expresses, we’ll see clearing zones around the colonies on the colloidal chitin agar, indicating the production of chitinase. From the ratio of the clearing zone to colony diameter, we can conclude the efficiency of the gene expression.</font><font style="font-size:16px" color="#000000" face="Baskerville Old Face"><br> | ||
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Revision as of 21:24, 20 June 2014
Safety
Personal Safety
Safety Education
At the beginning of the lab work the team has participated in a thorough biosafety education held by our mentors. The main idea of this course was to clarify that working in a molecular biology laboratory has many other important aspects beyond the scientific research work. We went through all the possible personal, public and environmental risks and possible coping strategies, as well. We have learned how to use the fire extinguisher, how to use chemically and/or biologically unsafe materials and how to handle safely all the manually or electronically controlled equipment.
Biosafe “Dress Code”
During all experiments, all participants had to wear gloves, which were dropped into the aforementioned container, and lab coats which were used only in the appropriate lab. All the procedures (PCR reactions, microbial cultures, transformations, etc.) were supervised carefully by at least one adult with the same “biosafe dress code”.
Regulations
We have used microorganisms belonging only into BSL1 level. The only exception is Chromobacterium violaceum which is itself a BSL2 bacterium; however we have used only parts of its DNA (chitinase gene). The product of this gene is not connected with the Chromobacterium violaceum’s pathogenic effect. In this case we can state that only BSL1 bacteria were used as living organisms. Concerning genetically modified organisms, we had to fulfil the requirements of the rather strict Hungarian State regulations. (You can read more on these regulations here: http://gmo.kormany.hu/hungarian-legislation) Concerning these regulations, GMO as living organism can only be produced and/or transferred by approved institutions. Because of this, all the transformational part of our project was carried out in the buildings of the Biological Research Centre of Hungarian Academy of Sciences which is a BSL2/3 and moreover an approved institution for producing and dealing with GMO. Microorganisms Used by Our Groups
Name DNA /living cells Biosafety Level ATCC No. Source Used for Chromobacterium violaceum DNA-part BSL2 12472 DSMZ amplification template of chitinase gene Escherichia coli DH5 alpha http://ecoliwiki.net/colipedia/index.php/DH5_alpha living cells BSL1 67878 BRC transformation Escherichia coli Nissle 1917 living cells BSL1 8739 BRC transformation Bacillus thuringiensis NCAIM - 01262 living cells / freeze dried BSL1 33679 NCAIM chitinase assay Bacillus thuringiensis NCAIM - 01292 living cells / freeze dried BSL1 10792 NCAIM chitinase assay
Biosafety Arrangements
Public and natural environment safety
During the experiments having been carried out in the school all the rooms of the laboratory, where our work took place, were signed with biohazard sign, with a title “BIOHAZRD” and in Hungarian “BIOLOGIAI VESZELY”.
All the disposable materials, (pipette tips, Eppendorf- and PCR-tubes, inoculating loops, etc.) connecting with microorganisms were put into a biohazard container, in which there was a strong oxidative disinfectant. This type of handling fulfils the Hungarian requirements in the cases of BSL1 materials. All the bacterial colonies and also the fluid cultures were handled with the same oxidative disinfectant.
All the DNA sample and disposable materials (pipette tips, Eppendorf- and PCR-tubes, inoculating loops…) connecting with it, were dropped into another container in which the strong acidic environment damaged it. All the containers are going to be transported by an officially approved firm which will continue the safety procedures with our used materials according to the Hungarian regulations.
Chemical Safety
Screening Chitinase-activity
We found this method in the article of N. Murthy, B. Bleakley: Simplified Method of Preparing Colloidal Chitin Used For Screening of Chitinase- Producing Microorganisms.
We would like to use this method as a chitinase assay. In order to check that the chitinase gene was infiltrated and expressed in the genetically modified bacteria, we inoculate the bacteria onto colloidal chitin agar. ,,The colloidal chitin agar (pH 7.0+/-0.2) prepared had the following ingredients (in g/L): (K2HPO4, 0.7; KH2PO4, 0.3; MgSO4 X 5H2O, 0.5; FeSO4 X 7H20, 0.01; ZnSO4, 0.001; MnCl2, 0.001), amended with 2.0 % moist colloidal chitin. Bacto Agar (Difco) was added at 2.0% as a solidifying agent. The medium was sterilized by autoclaving at STP (standard temperature and pressure: 15 psi, 20 minutes, 121 ° C) and poured into sterile Petri plates (approximately 20 ml per plate).”
If the chitinase gene expresses, we’ll see clearing zones around the colonies on the colloidal chitin agar, indicating the production of chitinase. From the ratio of the clearing zone to colony diameter, we can conclude the efficiency of the gene expression.
