Our BioBricks, especially those containing the gene coding for the mature region of mouse Bone Morphogenetic Protein 2 (BMP-2), all possess a degree of risk. In mammals, BMP-2 is known to elicit a wide variety of biological effects on tissues; the most well known include induction of bone and cardiac cell differentiation. Being a defining member of the Transforming Growth Factor Beta (TGF-β) pathway, it also plays important roles in cell proliferation. Hence, induction of excess BMP-2 may lead to undesirable tissue behavior in mammalian systems.

Documented adverse effects of recombinant human BMP-2 include formation of cyst-like bony structures and soft swelling with hematomas when the gene product is applied in spinal fusion therapy. Further animal tests using mice with spine defects indicate that the occurrence and severity of these effects are positively correlated with BMP-2 dosage. In addition, as BMP-2 receptor transcription has been found to be up-regulated in certain cancer types (for example pancreatic cancer), confined delivery of the chemokine is critical despite its documented capacity for retarding cell growth and inducing cell death in colon carcinoma cells.

See relevant documents by following links below:
High Doses of Bone Morphogenetic Protein 2 Induce Structurally Abnormal Bone and Inflammation In Vivo
Bone Morphogenetic Protein Signalling and Growth Suppression in Colon Cancer

We highly recommend that future teams intending to use this gene simultaneously incorporate methods to control production of BMP-2 to minimize its contact with researchers or release into the environment. Our strategy described in the 'Regulation and Control' module may be viewed as an initial attempt to achieve this.

BioBricks for expression of the lytC protein cell wall binding domain with the RPMrel phage display peptide attached at its C-terminus results in RPMrel peptides anchored to the chassis cell wall. It is known that phages displaying this peptide bind preferentially to the highly tumorigenic HT29 colorectal cell line by at least 10-fold than to the less tumorigenic HCT116 colorectal cell line. As expression of this peptide in bacterial cells is novel, steps should be taken to minimize the chance of its horizontal transfer to pathogenic bacterial species, which could result in increased infection activity of that species. Integration of this gene into the bacterial genome, an approach taken by our team, would be one way to work towards reducing horizontal gene transfer.

Please refer to the document below:
Isolation of a Colon Tumor Specific Binding Peptide Using Phage Display Selection

Elements of our toxin-antitoxin system for controlling cell lysis contain the ydcDE operon of Bacillus subtilis. The ydcE gene encodes an endoribonuclease that cleaves multiple regions of cellular mRNA, while the gene ydcD has been shown to inhibit this endonuclease activity in vivo. Our system employs these same genes, whose expressions are made controllable by equipping them with different promoters: pTms for ydcD and pXyl for ydcE. As an investigation in macaque monkeys shows that the expression of E. coli ydcE homolog (mazF) results in neither tissue damage nor antigen-specific antibody production, we consider the ydcE gene product to be non-toxic to humans.

Please refer to this document.

Construction and characterization of our project’s assorted constructs involved work with two non-pathogenic bacterial strains: Escherichia coli DH10B and Bacillus subtilis 168; both strains commonly used in research, education, and industry. In the manipulation of these strains, Biosafety Level 1 standard regulations were strictly followed. Our characterization also required the use of human colon carcinoma HT29 cells, which were handled only by trained team members using a designated tissue culture hood with HEPA filters. Biosafety Level 2 standard safety requirements were observed throughout the entire practice. Gloves and laboratory coats were worn in all laboratory work.

Notably toxic and/or mutagenic substances used in the lab include phenol, chloroform, and ethidium bromide. They were used only by members of the team who had received safety training to handle the substances properly according to their potential health hazards.

Working on B. subtilis involves a risk of endospore inhalation, as B. subtilis is known to form spores under stress. However the low toxicity of the organism to humans suggests that the occurrence of debilitating infection is extremely rare.

Hypersensitive responses to the subtilisin enzyme excreted by B. subtilis found in detergent form, however, are more likely. Standard precautions for handling microorganisms such as proper wearing of gloves to prevent direct contact are observed at all times to reduce this risk.

Of the few documented cases of B. subtilis infection, the vast majority involved severely immunocompromised patients. Should one of our researchers enters such a state, he or she will not be allowed into the lab and will be expected to rest and seek treatment.

The ultimate aim of our team’s biological system is to perform a task acting as an anti-cancer agent within the human digestive tract. Direct interaction with live human cells is a requirement of its function thus demanding particular considerations for safety. Similar precautions to protect cancer patients for whom the treatment is intended will also help in protecting the public in the off-chance our system enters the environment beyond the lab.

Firstly it must be made clear that no patient exhibiting immunodeficiency or under immunosuppression should be recommended for this treatment or any subsequent approved derivative of this treatment. There is the likelihood these patients will suffer from B. subtilis infection.

B. subtilis is a known normal gut commensal and is considered a bacterial species conferring minimal risks to human. It produces no particles considered toxic to humans. Enzymes produced by B. subtilis, including carbohydrases and proteases contributing to its function as a part of the gut microbiome, are classified ‘Generally Recognized As Safe’ (GRAS) by the FDA. The species has also been proven to function as a probiotic when consumed in certain food stuffs, most notably fermented soy bean. No recombined component of our biological system is known to confer negative effects on gut microbiome function.

Two regulatory functions were put in place to control BMP-2 production and minimize its potential negative effects. Firstly, the BMP-2 construct makes use of a xylose-inducible promoter. This induction system greatly reduces the chance of BMP-2 production in any unintended circumstance. Secondly, a cap is placed on maximum BMP-2 production per cell by incorporating a toxin-antitoxin cassette with toxin transcription in direct correlation with BMP-2 transcription. This cap is intended to prevent the onset of adverse effects caused by excessive BMP-2 signalling. More information on these regulatory functions can be found at the module 3.

Steps were taken to limit the spread of potentially harmful genes should our biological system be leaked into the environment.

Both bacterial cell strains used (E. coli DH10B and B. subtilis 168) possess an amino acid metabolism deficiency that reduce their competitiveness in the wild. DH10B exhibits leucine deficiency while 168 exhibits tryptophan deficiency. Such phenotypes reduce the chance these bacterial strains have when surviving beyond the lab.

E. coli DH10B was used exclusively when building our constructs. 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.

To limit the potential of BMP-2 horizontal gene transfer, the antitoxin component of our toxin-antitoxin cassette has been designed into an integration plasmid with antibiotic resistance selectivity for integration. This means that while the genes for BMP-2 and toxin expression exist in plasmid form and can potentially be transferred, the recipient cell cannot obtain the antitoxin and thus will be killed.

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

Laws & Guidelines
The Cartegena Protocol on Biosafety under the Convention on Biological Diversity (CBD) was implemented by China’s central government in the year 2000 and was extended to the Hong Kong Special Administrative Region (HKSAR) in May 2011. Adoption of the Protocol was facilitated by introduction of Chapter 607, the Genetically Modified Organisms (Control of Release) Ordinance (the Ordinance) in March of the same year. Under the Ordinance, release of Genetically Modified Organisms (GMOs) knowingly without approval from the Director, 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.

As activities being conducted by the HKUST iGEM 2012 team are concerned strictly with molecular cloning and the testing of recombinant technology biomolecules, no part of the project is destined for release into the environment. Application for approval to perform such a procedure under the Ordinance is therefore not required.

While every citizen in Hong Kong is subject to the Ordinance, laboratories in Hong Kong are to refer to the more detailed Guidelines on Biosafety in the Clinical Laboratory (the Guidelines), a publication of the Centre of Health Protection under the Department of Health. The full text of the Guidelines can be accessed at: Guidelines on Biosafety in the Clinical Laboratory

As our laboratory was constructed and is operated by the university’s Division of Life Science, the design of the lab, as well as activities performed within the lab, adhere to the above Guidelines.

HKUST’s HSEO
The creation and enforcement of safety regulations is handled by the university’s Health, Safety & Environment Office (HSEO). The HSEO also acts in a support function to promote biosafety and other safe laboratory practices by providing safety training for staff and students.

Special measures for promoting the biosafety of our biological system employed by our team this year are nothing revolutionary. But we hope that future teams developing a system for which production of a signalling biomolecule is the aim always make efforts to control dosage. Linking well-characterized promoters to toxin/anti-toxin ‘switches’ gives us greater control of dosage, allowing us to place a somewhat reliable and quantifiable upper limit on biomolecule production.

We did not find well-characterized inducible promoters for B. subtilis on the Registry. Our team therefore hopes to add some useful data about candidate promoters selected for our employed toxin/anti-toxin cassette. Furthermore, we strongly support efforts that seek to provide the synthetic biology community with more detailed knowledge on the transcriptional and translational efficiency of parts. Such knowledge would help the community employ more dosage-sensitive tools, increasing the repertoire of biological machinery. This year’s (2012’s) Carnegie Mellon University iGEM team has been working on a non-invasive, non-destructive characterization system that may help us take steps toward this end. See their wiki here.