Team:Hong Kong HKUST/safety

Biobrick Safety

All organisms are biosafety level 1 with the minimal potential hazard to laboratory personnel and the environment. This year, our team is planning to construct a total of 7 novel basic biological parts, and to build complex devices by making use of these parts together with parts available from the Part Registry. Our BioBricks have been considered safe for use and circulation. The parts and their safety evaluations are as follow.

aceA and aceB

Our team is cloning isocitrate lyase (aceA) and and malate synthase (aceB) from E. coli strain BW25113. They are enzymes involved in the glyoxylate cycle in plants and bacteria. Dean et al. at UCLA introduced aceA and aceB genes into human hepatocytes to investigate fatty acid metabolism, and no hazardous effect was reported¹. Based on current literature, we believe the two genes will not raise any significant safety issues.

Mitochondria Leader Sequence

Mitochondrial Leader Sequence (MLS) is a short signaling peptide that directs proteins to the mitochondria in mammalian cells. Its functionality has been tested in human hepatocytes by Bai et al. and no harmful effect of MLS has been reported². Our team cloned the coding DNA sequence of MLS from a commercial plasmid – pCMV/myc/mito (Invitrogen) – and fused it with aceA and aceB genes so the MLS can guide the two resulting enzymes into mitochondria. Again based on the paper by Dean et al., and considering the signaling function of this part, we consider this BioBrick safe.

Promoter of Human Elongation Factor-1 Alpha (pEF-1α), Promoter of Peroxisome Proliferator-Activated Receptor–Alpha (pPPARα), Liver Fatty Acid Binding Protein 1 (FABP1) and Glucose Regulated Protein 78 (GPR78)

Promoters of human elongation factor-1 alpha (EF-1 alpha), peroxisome proliferator-activated receptor–alpha (PPAR-alpha), liver fatty acid binding protein 1 (FABP1) and glucose regulated protein (GRP78) are of human origins, and they govern basic metabolic reactions. Based on their functions and origin, we believe these BioBricks pose no threats to the public.

Bacterial Fatty acid metabolism regular protein

Fatty acid metabolism regular protein (fadR) is a bacterial transcription factor that regulates lipid metabolism of fatty acid biosynthesis and beta-oxidation. fadR and fatty acid dependent promoter (pFadBA) are obtained from the 2013 distribution kit and iGEM headquarters, submitted by the NTU-Taida 2012 team. fadR and pFadBA are native to E. coli and no study has been conducted in introducing them into a mammalian system. Based on the native functions of fadR and pFadBA, we believe that these two BioBricks will not raise any safety issues when introduced into a heterologous system.

Researcher Safety

This year, our team was authorized to work with two non-pathogenic bacterial strains, the Escherichia coli DH10B and SURE strains. Both these strains are regularly employed in research, industry and study. Biosafety Level 1 standard regulations were strictly followed while using these strains.

The characterization part of our project involved human liver HepG2 and HEK 293FT cell lines. Only three of our trained members were allowed to enter the Tissue Culture Room and conducted experiments with HEK 293FT and HepG2 cells in level 2 biosafety cabinets. They received special training about biosafety and clearly understood potential risks and methods of resolution. Gloves and lab coats were worn while researchers were in lab to avoid contamination and corrosion.

Additionally, some toxic or mutagenic substances were involved in procedures like genomic DNA extraction, cell fixation and fatty acid quantification. Phenol, chloroform, formaldehyde, BF₃, n-heptane, diethyl ether and petroleum ether were used in the experiments mentioned above. Our team members who were dealing with these chemicals received full supervision from lab technicians. Gloves, lab coats and protective goggles were worn during these procedures.

Environmental Safety

No biological system is leaked into the environment and measures are taken to limit the spread of potentially harmful genes.

E. coli DH10B and SURE were used exclusively when building our constructs. The SURE Competent cells are classified to be a non-hazardous substance. All DH10B cells are classified F- and thus do not possess the Fertility factor. The risk of these cells acting as a donor in horizontal gene transfer via conjugation is greatly reduced.

The E. coli DH10B bacterial cell strain possesses an amino acid leucine metabolism deficiency that reduces its competitiveness in the wild. Such phenotypes reduce the chance these bacterial strains have when surviving beyond the lab.

Prior to disposal, harmful waste (toxic and/or biohazardous) was clearly separated and sterilized, either by application of bleach, or autoclaving.

University Safety

Hong Kong Laws & Guidelines

The Cartegena Protocol on Biosafety under the Convention on Biological Diversity (the Protocol) was extended to the Hong Kong Special Administrative Region (HKSAR) in May 2011. Under the Chapter 607, the Genetically Modified Organisms (Control of Release) Ordinance (the Ordinance), release of Genetically Modified Organisms (GMOs) knowingly without approval from the Deputy Director or an Assistant Director of the Agriculture, Fisheries and Conservation Department is considered an offence. The full text of Cap. 607 can be downloaded by following this link.

No part of the project is directed for release into the environment. HKUST iGEM 2013 team experiments are concerned strictly with molecular cloning and testing of recombinant technology biomolecules. Application for approval to perform such a procedure under the Ordinance is therefore not required.

Laboratories in Hong Kong follow Guidelines on Biosafety in the Clinical Laboratory, a publication of the Centre of Health Protection under the Department of Health. The full text of the Guidelines can be accessed here. As our laboratory is operated by the university’s Division of Life Science, the activities performed within the lab adhere to the above Guidelines.

HKUST’s HSEO

The enforcement of safety regulations is handled by the university’s Health, Safety & Environment Office (HSEO). The HSEO also provide safety training for staff and students for safe laboratory.

References

¹Dean Jason T., et al. “Resistance to Diet-Induced Obesity in Mice with Synthetic Glyoxylate Shunt.”
²Bai, Jingxiang, et al. "Overexpression of Catalase in Cytosolic or Mitochondrial Compartment Protects HepG2 Cells against Oxidative Injury." Journal of Biological Chemistry. (1999): n. page. Web.