Team:Goettingen/Safety

From 2013.igem.org

The beast and its Achilles heel:

A novel target to fight multi-resistant pathogenic bacteria

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3 1. Please describe the chassis organism(s) you will be using for this project. lf you will be using more than one chassis organism, provide information on each of them:
4 2. Highest Risk Group Listed:
5 3. List and describe all new or modified coding regions you will be using in your project. (If you use parts from the 2013 iGEM Distribution without modifying them, you do not need to list those parts.)
6 4. Do the biological materials used in your lab work pose any of the following risks? Please describe.
7 5. If your project moved from a small-scale lab study to become widely used as a commercial/industrial product, what new risks might arise? (Consider the different categories of risks that are listed in parts a-d of the previous question.) Also, what risks might arise if the knowledge you generate or the methods you develop became widely available? (Note: This is meant to be a somewhat open-ended discussion question.)
8 6. Does your project include any design features to address safety risks? (For example: kill switches, auxotrophic chassis, etc.) Note that including such features is not mandatory to participate in iGEM, but many groups choose to include them.
9 7. What safety training have you received (or plan to receive in the future)? Provide a brief description, and a link to your institution’s safety training requirements, if available.
10 8. Under what biosafety provisions will / do you work?
- 10.1 Satefy Questions Part 2, Listeria monocytogenes
11 1. Organism name and strain name or number.
12 2. Organism Risk Group:
13 3. If you are using this organism as a chassis, write "chassis". If you are using a genetic part from the organism, give the name of the part and a brief description of what it does and why you are using it.
14 4. How did you physically acquire the organism or part?
15 5. What potential safety/health risks to team members, other people at your institution, or the general public could arise from your use of this organism/part?
16 6. What measures do you intend to take to ensure that your project is safe for team members, other people at your institution, and the general public?
17 7. If you are using only a part from the organism, and you believe the part by itself is not dangerous, explain why you believe it is not dangerous.
18 8. Why do you need to use this organism/part? Is there an organism/part from a less dangerous Risk Group that would accomplish the same purpose?
19 9. Is the organism/part listed under the Australia Group guidelines, or otherwise restricted for transport? If so, how will your team ship this part to iGEM and the Jamborees?
20 10. Please describe the BioSafety Level of the lab in which the team works, or description of safety features of lab (Refer to Basic Safety form, question 8. d.). If you are using organisms with a BSL level greater than you lab, please explain any additional safety precautions you are taking.
- 20.1 Satefy Questions Part 2, Mycobacterium smegmatis
21 1. Organism name and strain name or number.
22 2. Organism Risk Group:
23 3. If you are using this organism as a chassis, write "chassis". If you are using a genetic part from the organism, give the name of the part and a brief description of what it does and why you are using it.
24 4. How did you physically acquire the organism or part?
25 5. What potential safety/health risks to team members, other people at your institution, or the general public could arise from your use of this organism/part?
26 6. What measures do you intend to take to ensure that your project is safe for team members, other people at your institution, and the general public?
27 7. If you are using only a part from the organism, and you believe the part by itself is not dangerous, explain why you believe it is not dangerous.
28 8. Why do you need to use this organism/part? Is there an organism/part from a less dangerous Risk Group that would accomplish the same purpose?
29 9. Is the organism/part listed under the Australia Group guidelines, or otherwise restricted for transport? If so, how will your team ship this part to iGEM and the Jamborees?
30 10. Please describe the BioSafety Level of the lab in which the team works, or description of safety features of lab (Refer to Basic Safety form, question 8. d.). If you are using organisms with a BSL level greater than you lab, please explain any additional safety precautions you are taking.
31 Faculty Advisor Name:
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Safety forms were approved on October 3rd, 2013 by the iGEM Safety Committee.

Basic Safety Questions

1. Please describe the chassis organism(s) you will be using for this project. lf you will be using more than one chassis organism, provide information on each of them:

Species	Strain no/name	Risk Group	Risk group source link	Disease risk to humans? If so, which disease?
E.coli (K12)	XL1 blue	1	www.absa.org/riskgroups/bacteria search.php?genus=&species=coli	Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys.
E.coli (K12)	DH5 alpha	1	www.absa.org/riskgroups/bacteria search.php?genus=&species=coli	Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys.
E.coli(B)	T7 express(NEB)	1	www.absa.org/riskgroups/bacteria search.php?genus=&species=coli	Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys.
E.coli(B)	BL21	1	www.absa.org/riskgroups/bacteria search.php?genus=&species=coli	Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys.

2. Highest Risk Group Listed:

Risk group 1

3. List and describe all new or modified coding regions you will be using in your project. (If you use parts from the 2013 iGEM Distribution without modifying them, you do not need to list those parts.)

Part number.	Where did you get the physical DNA for this part (which lab,synthesis company, etc)	What species does this part originally come from?	What is the Risk Group of the species?	What is the function of this part, in its parent species?
BBa_K1045000	Cloned from purchased Mycobacteria genome	Mycobacterium smegmatis	2	The operator DNA sequence that DarR binds
BBa_K1045001	Cloned from purchased Mycobacteria genome	Mycobacterium smegmatis	2	Code regulatory protein DarR, which binds c-di-AMP
BBa_K1045002	Cloned from Bacillus genome	Bacillus subtilis	1	The c-di-AMP sensing riboswitch YdaO
BBa_K1045003	Cloned from Listeria monocytogenes genome	Listeria monocytogenes	2	Diadenylate cyclase domain of L. monocytogenes, cdaA
BBa_K1045004	Cloned from the Bacillus substilis genome	Bacillus substilis	1	YdaO Riboswitch with native Promoter and RBS
BBa_K1045005	Cloned from the Bacillus substilis genome	Bacillus substilis	1	YdaO Riboswitch with native RBS
BBa_K1045006	Cloned from the Bacillus substilis genome	Bacillus substilis	1	YdaO Riboswitch with native Promoter
BBa_K1045007	Cloned from the Bacillus substilis genome	Bacillus substilis	1	YdaO Riboswitch only
BBa_K1045009	From 2013 distribution kit	E.coli	1	Terminator
BBa_K1045010	From 2013 distribution kit	E.coli	1	Ribsome binding site
BBa_K1045011	From 2013 distribution kit	E.coli	1	Promoter
BBa_K1045012	Assembled with BBa_K1045000 and BBa_J23110
BBa_K1045013	Aseembled with BBa_K1045012 and BBa_E0240
BBa_K1045014	Assembled with BBa_K1045011 and BBa_K1045013,
BBa_K1045015	Assembled with BBa_K1045014, BBa_K1045010 and BBa_E0240
BBa_K1045016	Assembled with BBa_K1045001 and BBa_K1045009,
BBa_K1045017	Assembled with BBa_K1045015 and BBa_K1045016

4. Do the biological materials used in your lab work pose any of the following risks? Please describe.

a. Risks to the safety and health of team members or others working in the lab?

The organism we used in the lab E.coli (K12) DH5 alpha, could be pathogenicto human beings. lt may cause irritation to skin, eyes, and respiratorytract and affect kidneys.

b. Risks to the safety and health of the general public, if released by design or by accident?

The E.coli strain we used do have pathogenicity to human. But the relatively low pathogenicity, short life and vulnerability to environment factors ofthe used strain have limited the risk to general public.

c. Risks to the environment, if released by design or by accident?

The E.coli strains we used during the project are transformed with plasmids containing antibiotic markers The release of those transformed strains may cause problems like the leakage of resistance genes to wild bacterial flora.

d. Risks to security through malicious misuse by individuals, groups, or countries?

The strains we used during our project do not contain hazardous genetic compartments. The risk through malicious misuse is quite low

5. If your project moved from a small-scale lab study to become widely used as a commercial/industrial product, what new risks might arise? (Consider the different categories of risks that are listed in parts a-d of the previous question.) Also, what risks might arise if the knowledge you generate or the methods you develop became widely available? (Note: This is meant to be a somewhat open-ended discussion question.)

The system we are trying to build is mainly meant for drug screening in pharmaceutical industry. The scale of production will always be limited.

6. Does your project include any design features to address safety risks? (For example: kill switches, auxotrophic chassis, etc.) Note that including such features is not mandatory to participate in iGEM, but many groups choose to include them.

No. But as the most used model organism in molecular biology, the risk of E.coli is relatively low.

7. What safety training have you received (or plan to receive in the future)? Provide a brief description, and a link to your institution’s safety training requirements, if available.

Before the project started, we all received instructions concerning the safety problems, such as how to protect ourselves in the lab, how to handle the biological materials properly to lower the risk to public and environment.

8. Under what biosafety provisions will / do you work?

a. Please provide a link to your institution biosafety guidelines.

www.uni-goettingen.de/en/401.html
(this website has links to all safety concerning documents, but all in german)

b. Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? Describe any concerns they raised with your project, and any changes you made to your project planbased on their review.

www.uni-goettingen.de/de/401.html

c. Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible.

www.baua.de/en/Homepage.html

d. According to the WHO Biosafety Manual, what is the BioSafety Level rating of your lab? (Check the summary table on page 3, and the fuller description that starts on page 9.) If your lab does not fit neatly into category 1, 2, 3, or 4, please describe its safety features [see 2013.igem.org/Safety for help].

The biosafety level of our lab is Categroy 1.

e. What is the Risk Group of your chassis organism(s), as you stated in question 1? If it does not match the BSL rating of your laboratory, please explain what additional safety measures you are taking.

Risk Group 1.

Satefy Questions Part 2, Listeria monocytogenes

Those questions are answered because we have parts originated from organisms from Risk Group 2. They are Listeria monocytogenes and Mycobacterium smegmatis.

1. Organism name and strain name or number.

Listeria monocytogenes

2. Organism Risk Group:

3. If you are using this organism as a chassis, write "chassis". If you are using a genetic part from the organism, give the name of the part and a brief description of what it does and why you are using it.

BBa_K1045003 Diadenylate cyclase domain of L. monocytogenes, DacA (CdaA, Lmo2120) We would like to look into diadenylate cyclase(DAC) of gram-positive pathogen, as potential target for novel antibiotics.

4. How did you physically acquire the organism or part?

Cloned from purchased Listeria genome

5. What potential safety/health risks to team members, other people at your institution, or the general public could arise from your use of this organism/part?

We actually didn't work with Listeria monocytogenes. Our parts were cloned from purchased genome. And the parts themselves are not hazardous nor toxic since mammals and a few gram-negative bacteria don't use c-di-AMP as a signal molecule.

6. What measures do you intend to take to ensure that your project is safe for team members, other people at your institution, and the general public?

We follow strictly the S1 protocol to avoid any risks.

7. If you are using only a part from the organism, and you believe the part by itself is not dangerous, explain why you believe it is not dangerous.

This part is non-hazardous nor toxic while mammals and gram-negative bacteria don't use c-di-AMP as a signal molecule. And this is the reason why c-di-AMP is a possible target for novel antibiotics

8. Why do you need to use this organism/part? Is there an organism/part from a less dangerous Risk Group that would accomplish the same purpose?

We would like to look into diadenylate cyclase(DAC) of gram-positive pathogen, as potential target for novel antibiotics. There are definitely DAC in less dangerous organism like B.substilis, but DAC from B.substilis is rather difficult to purify, and our final goal is to get a 3D structure.

9. Is the organism/part listed under the Australia Group guidelines, or otherwise restricted for transport? If so, how will your team ship this part to iGEM and the Jamborees?

No.

10. Please describe the BioSafety Level of the lab in which the team works, or description of safety features of lab (Refer to Basic Safety form, question 8. d.). If you are using organisms with a BSL level greater than you lab, please explain any additional safety precautions you are taking.

BSL1

Satefy Questions Part 2, Mycobacterium smegmatis

1. Organism name and strain name or number.

Mycobacterium smegmatis

2. Organism Risk Group:

3. If you are using this organism as a chassis, write "chassis". If you are using a genetic part from the organism, give the name of the part and a brief description of what it does and why you are using it.

BBa_K1045000 The operator DNA sequence that DarR binds, DarR operator

BBa_K1045001 Code regulatory protein DarR, which binds c-di-AMP

DarR is reported to be a transcriptional inhibitor in Mycobacterium smegmatis and c-di-AMP stimulates its binding to its operator sequence, acting as a co-inhibitor. (Zhang et al., 2012) We have built a reporter system of c-di-AMP based on that.

4. How did you physically acquire the organism or part?

Cloned from purchased Mycobacteria genome

5. What potential safety/health risks to team members, other people at your institution, or the general public could arise from your use of this organism/part?

We actually didn't work with Mycrobacterium segmatic. Our parts were cloned from purchased genome. And the parts themselves are not hazardous nor toxic.

6. What measures do you intend to take to ensure that your project is safe for team members, other people at your institution, and the general public?

We follow strictly the S1 protocol to avoid any risks.

7. If you are using only a part from the organism, and you believe the part by itself is not dangerous, explain why you believe it is not dangerous.

BBa_K1045000 is only a 14bp palindrome operator sequence.

BBa_K1045001 encodes a transcriptional inhibitor DarR. DarR has no catalytic function and is not toxic.

8. Why do you need to use this organism/part? Is there an organism/part from a less dangerous Risk Group that would accomplish the same purpose?

Because DarR is the very recently discovered regulatory protein which responds to c-di-AMP.There is no another protein in a less dangerous organism discovered yet.

9. Is the organism/part listed under the Australia Group guidelines, or otherwise restricted for transport? If so, how will your team ship this part to iGEM and the Jamborees?

No.

10. Please describe the BioSafety Level of the lab in which the team works, or description of safety features of lab (Refer to Basic Safety form, question 8. d.). If you are using organisms with a BSL level greater than you lab, please explain any additional safety precautions you are taking.

BSL1

Faculty Advisor Name:

Jörg Stülke

Retrieved from "http://2013.igem.org/Team:Goettingen/Safety"

Team:Goettingen/Safety

From 2013.igem.org

Contents

Navigation:

Safety forms were approved on October 3rd, 2013 by the iGEM Safety Committee.

Basic Safety Questions

1. Please describe the chassis organism(s) you will be using for this project. lf you will be using more than one chassis organism, provide information on each of them:

2. Highest Risk Group Listed:

3. List and describe all new or modified coding regions you will be using in your project. (If you use parts from the 2013 iGEM Distribution without modifying them, you do not need to list those parts.)

4. Do the biological materials used in your lab work pose any of the following risks? Please describe.

a. Risks to the safety and health of team members or others working in the lab?

b. Risks to the safety and health of the general public, if released by design or by accident?

c. Risks to the environment, if released by design or by accident?

d. Risks to security through malicious misuse by individuals, groups, or countries?

6. Does your project include any design features to address safety risks? (For example: kill switches, auxotrophic chassis, etc.) Note that including such features is not mandatory to participate in iGEM, but many groups choose to include them.

7. What safety training have you received (or plan to receive in the future)? Provide a brief description, and a link to your institution’s safety training requirements, if available.

8. Under what biosafety provisions will / do you work?

a. Please provide a link to your institution biosafety guidelines.

b. Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? Describe any concerns they raised with your project, and any changes you made to your project planbased on their review.

c. Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible.

e. What is the Risk Group of your chassis organism(s), as you stated in question 1? If it does not match the BSL rating of your laboratory, please explain what additional safety measures you are taking.

Satefy Questions Part 2, Listeria monocytogenes

1. Organism name and strain name or number.

2. Organism Risk Group:

3. If you are using this organism as a chassis, write "chassis". If you are using a genetic part from the organism, give the name of the part and a brief description of what it does and why you are using it.

4. How did you physically acquire the organism or part?

5. What potential safety/health risks to team members, other people at your institution, or the general public could arise from your use of this organism/part?

6. What measures do you intend to take to ensure that your project is safe for team members, other people at your institution, and the general public?

7. If you are using only a part from the organism, and you believe the part by itself is not dangerous, explain why you believe it is not dangerous.

8. Why do you need to use this organism/part? Is there an organism/part from a less dangerous Risk Group that would accomplish the same purpose?

9. Is the organism/part listed under the Australia Group guidelines, or otherwise restricted for transport? If so, how will your team ship this part to iGEM and the Jamborees?

10. Please describe the BioSafety Level of the lab in which the team works, or description of safety features of lab (Refer to Basic Safety form, question 8. d.). If you are using organisms with a BSL level greater than you lab, please explain any additional safety precautions you are taking.

Satefy Questions Part 2, Mycobacterium smegmatis

1. Organism name and strain name or number.

2. Organism Risk Group:

3. If you are using this organism as a chassis, write "chassis". If you are using a genetic part from the organism, give the name of the part and a brief description of what it does and why you are using it.

4. How did you physically acquire the organism or part?

5. What potential safety/health risks to team members, other people at your institution, or the general public could arise from your use of this organism/part?

6. What measures do you intend to take to ensure that your project is safe for team members, other people at your institution, and the general public?

7. If you are using only a part from the organism, and you believe the part by itself is not dangerous, explain why you believe it is not dangerous.

8. Why do you need to use this organism/part? Is there an organism/part from a less dangerous Risk Group that would accomplish the same purpose?

9. Is the organism/part listed under the Australia Group guidelines, or otherwise restricted for transport? If so, how will your team ship this part to iGEM and the Jamborees?

10. Please describe the BioSafety Level of the lab in which the team works, or description of safety features of lab (Refer to Basic Safety form, question 8. d.). If you are using organisms with a BSL level greater than you lab, please explain any additional safety precautions you are taking.

Faculty Advisor Name: