Team:Macquarie Australia/Safety

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Safety

The safety of team members and staff in the laboratory was a priority while participating in our iGEM project. Potential risks posed to those working in the lab environment were identified and strategies to mitigate those risks were implemented, in accordance to local and international regulatory standards.

Safety Standards:

• Biosafety at Macquarie Institutional Biosafety Committee
• Biosafety at Macquarie University Faculty of Science
• Chemical Safety at Macquarie University
• Gene technology act

Prior to the execution of our project the team participated in a lab induction involving a review of safe working procedure for lab equipment and biosafety. Training included the safe disposal of waste and sharps and the handling of biological spills. The lab induction detailed the criteria of appropriate lab clothing and shoes and the use of personal protective equipment. Lab supervisors are trained in first aid and the lab fitted with multiple first aid stations. There was a Laboratory Safety and Procedures manual and chemical safety (MSDS's, risk assessment) available in the lab for reference at all times.

The Biosafety committee of Macquarie University is responsible for overseeing biological safety. The iGEM project conducted was approved by the Biosafety committee due to compliance with its guidelines.

The Australian federal government has legislative guidelines to regulate Australian Biosafety, with the enforcement of these guidelines enacted by the Office of the Gene Technology Regulator within the Department of Health. The Office provides a full list of relevant Australian legislation which may be found on their Legislation page. This iGEM project met the standards and requirements set by our internal safety committee, domestic requirements, and international guidelines.

Chassis Organism

Species name (including strain)	Risk Group	Risk Group Source	Disease risk to humans?
Escherichia coli DH5 Alpha	Level 1	Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition	Minimal health risk; the DH5 Alpha E.coli strain being used for the project is non-pathogenic. The New England Biolabs (NEB) safety data sheet states the DH5 Alpha strain is not known to consistently cause disease in immunocompromised individuals.

DNA Parts

All genes were sourced from Chlamydomonas reinhardtii and synthesised.

Three composite parts incorporated parts from previous Macquarie_Australia teams:

• BBa_K1640000
• Part ChlH (BBa_K1080001) has been revised for 2015.
• BBa_K1640001
• Part ChlM (BBa_K1326001) has been revised for 2015.
• BBa_K1640002

Part number / name	Natural function of part	How will we use it?	Notes
BBa_K1640000	ChlI1 and ChlD genes transcibe proteins that self-assemble to form a functional ChlI1:ChlD complex. The protein transcribed by the gene ChlH inserts magnesium into protoporphyrin IX. ChlI1 and ChlD genes transcibe proteins that self-assemble to form a functional ChlI1:ChlD complex. The protein transcribed by the gene ChlH inserts magnesium into protoporphyrin IX.	We will use this part to form part of the chlorophyll biosynthesis pathway.	Composed of Lac promoter (BBa_R0010), ChlI1 (BBa_K1080000), ChlD (BBa_K1080002), Gun 4 (BBa_K1080003) and ChlH (BBa_K1080001) parts.
BBa_K1640001	CTH1, Plastocyanin, and YCF54 are involved in the oxidative cyclase pathway. ChlM methylates Mg-protoporphyrin IX, facilitating the highly-regulated catalysis of Mg-chelatase. CTH1 catalyses the conversion of Mg-protoporphyrin IX monomethyl into divinyl protochlorophyllide, interacting with YCF54 and Plastocyanin. ChlH inserts magnesium into protoporphyrin IX.	We will use this part to form part of the chlorophyll biosynthesis pathway.	Composed of the lac promoter (BBa_R0010), CTH1 (BBa_K1080020), YCF54 (BBa_K1080023), Plasto (BBa_K1080014), and ChlM (BBa_K1326001).
BBa_K1640002	DVR1 reduces divinyl protochlorophyllide, POR converts protochlorophyllide to chlorophyllide, ChlG adds the geranylgeranyl pyrophosphate chain to the chlorophyllide molecule, and ChlP reduces the double bonds on GGPP. The final product is chlorophyll-a.	We will use this part to form part of the chlorophyll biosynthesis pathway.	Composed of the lac promoter (BBa_R0010), POR (BBa_K1080015), DVR1 (BBa_K1080012), ChlP (BBa_K1080008), and ChlG (BBa_K1080009).
BBa_K1640003	The psbD gene that comprises this part encodes the D2 protein, which is one of the two proteins that comprise the Photosystem II reaction centre core.	We will use this part to test a minimal Photosystem 2 biosynthesis pathway.	As of 27 August 2015 this part has been ordered but the sequence not confirmed.
BBa_K1640004	The psbC gene that comprises this part codes a subunit that makes up the Photosystem II protein P680. The P680 complex is photosystem II primary electron donor.	We will use this part to test a minimal Photosystem 2 biosynthesis pathway.	As of 27 August 2015 this part has been ordered but the sequence not confirmed.
BBa_K1640020	The psbA gene that comprises this part encodes the D1 protein, which is one of the two proteins that comprise the Photosystem II reaction centre core.	We will use this part to test a minimal Photosystem 2 biosynthesis pathway.	As of 27 August 2015 this part has been ordered but the sequence not confirmed.
BBa_K1640006	This part is composed of psbE, psbL and psbJ genes. The protein encoded by psbE binds heme. The psbJ protein subunit is required for correct Photosystem II assembly. The psbJ protein is required for the stability of the Mn cluster in the Oxygen Evolution Complex.	We will use this part to test a minimal Photosystem 2 biosynthesis pathway.	As of 27 August 2015 this part has been ordered but the sequence not confirmed.
BBa_K1640007	This part is composed of the psbT and psbB genes. The psbT protein's function is not well known however it appears to be involved in the dimerization of Photosystem II and is essential for photosynthetic activity.	We will use this part to test a minimal Photosystem 2 biosynthesis pathway.	As of 27 August 2015 this part has been ordered but the sequence not confirmed.
BBa_K1640008	This part is composed of the psbM, psbZ, and psbH genes. The psbM protein subunit is positioned at the monomer-monomer interface. The psbZ protein controls the interaction of Photosystem II cores with the light-harvesting antenna. The psbH protein is required for stability and assembly of the photosystem II complex.	We will use this part to form a composite and test a minimal Photosystem 2 biosynthesis pathway.	As of 27 August 2015 this part has been ordered but the sequence not confirmed.
BBa_K1640009	This part is composed of psbW and psbK genes. The psbW protein stabilizes dimeric Photosystem II. The psbK protein is also required for stability and assembly of Photosystem II.	We will use this part to form a composite and test a minimal Photosystem 2 biosynthesis pathway.	As of 27 August 2015 this part has been ordered but the sequence not confirmed.
BBa_K1640010	The psbO gene that comprises this part encodes a manganese stabilising protein.	We will use this part to form a composite and test a minimal Photosystem 2 biosynthesis pathway.	As of 27 August 2015 this part has been ordered but the sequence not confirmed.
BBa_K1640011	The psbP gene that comprises this part transcribes a protein that optimizes the availability of Ca2+ and Cl- cofactors in the Oxygen Evolving Complex in PSII to maintain the active Manganese cluster.	We will use this part to form a composite and test a minimal Photosystem 2 biosynthesis pathway.	As of 27 August 2015 this part has been ordered but the sequence not confirmed.
BBa_K1640012	This part is composed of the psbQ and psbR genes. The psbQ protein is an oxygen enhancer protein. The psbR protein is an important link in the Photosystem II core complex for stable assembly of the Oxygen Evolving Complex.	We will use this part to form a composite and test a minimal Photosystem 2 biosynthesis pathway.	As of 27 August 2015 this part has been ordered but the sequence not confirmed.
BBa_K1640013	Encodes the PSII reaction centre D2 (Psb-D), PSII reaction centre D1 (Psb-A), and the PSII core monomer CP43 subunit (Psb-C).	We will use this composite part to test a minimal Photosystem 2 biosynthesis pathway.	PSII Operon 1.
BBa_K1640014	Encodes Α-subunit of the PSII cytochrome b559 (Psb-E), and subunits L (Psb-L), and J (Psb-J) of the CP43-less PSII core monomer.	We will use this composite part to test a minimal Photosystem 2 biosynthesis pathway.	PSII Operon 2.
BBa_K1640015	Encodes subunit T of the CP43-less PSII core monomer (Psb-T) and CP47 subunit of the PSII reaction centre (Psb-B).	We will use this composite part to test a minimal Photosystem 2 biosynthesis pathway.	PSII Operon 3.
BBa_K1640016	Encodes subunits M (Psb-M), Z (Psb-Z), H (psb-H), W (Psb-W), K (Psb-K) of the CP43-less PSII core monomer.	We will use this composite part to test a minimal Photosystem 2 biosynthesis pathway.	PSII Operon 4.
BBa_K1640017	Encodes subunits O (Psb-O), P (Psb-P), Q (Psb-Q) of the PSII oxygen evolving complex, and subunit R of the CP43-less PSII core monomer (Psb-R).	We will use this composite part to test a minimal Photosystem 2 biosynthesis pathway.	PSII Operon 5.