The following Biobrick BBa_K2053002 has been characterized thoroughly by experiments. This fusion protein is at the heart of our patch. It combines Silification, cellulose binding and anti-body fixation. Please follow our project design Science page for details.
In our Mos(kit)o project, we used Escherichia coli (DH5-α and BL21(DE3) strains) as our chassis organism for protein expression, as well as specific protein domains, documented in the iGEM Parts Registry, and found in following species: Staphylococcus aureus protein A, cellulose-binding domain of Clostridium cellulovorans, and silica-binding domain from a published phage screen. We also searched for the existence of a kinase for improving the silification reaction from Thalassiosira pseudonana. The fusion protein, created by assembling these domains in different orders, or independently, is first produced by the bacteria in the laboratory, and then purified and incorporated into the biodetection patch. The before-mentioned patch is based on biomaterials (biosilica and cellulose). Once complete, the patch and the mosquito trap are installed in the environment.
•In Staphylococcus aureus, the protein A (BpA) binds to constant fragments of IgG antibodies in order to inhibit opsonophagocytosis, and might play a role as an adhesin during the initiation of intravascular infection by mediating attachment of S. aureus to proteins at the site of damage to the endothelium. This protein is neither a toxin nor an enzyme that synthesizes a toxin. This protein is a staphylococcal virulence factor involved in immune evasion and adherence of bacteria onto endothelium. In order to make an immunodetection-based vector-borne pathogens sensor, we will use this part as a binder for specific antibodies. We do not require the whole protein, thus we will only use the antibody-binding domain of this protein: the B domain. Based on the registry part sequence, we will perform gblock oligonucleotide synthesis (iGEM pre-existing part: BBa_K103003). • The cellulose-binding protein is secreted by Clostridium cellulovorans to bind cellulose fibers and coordinate cellulase enzymes. The catabolic process of cellulose by this bacterium results in increase of glucose, an essential nutrient for bacterium growth. This protein is neither a toxin nor an enzyme that synthesizes a toxin. Moreover, the coordination of cellulose enzymes by this protein is not a problem, because these enzymes are absent in humans. In order to make a cellulose-based vector-borne pathogens sensor, we will use this part as a binder of a cellulose matrix. We do not require the whole protein, that is why we will only use the cellulose-binding domain of this protein. Based on theregistry part sequence, we will perform gblock oligonucleotide synthesis (iGEM pre-existing part: BBa_K863110). • The silica-binding domain from phage screen is able to condense silicic acid to form biosilica. This protein is neither a toxin nor an enzyme that synthezises a toxin. In order to make a silica-based vector-borne pathogens sensor, we will use this part as a binder of silicic acid to enhance biosilica formation and increase the rigidity of the cellulose-base matrix. Based on the registry part sequence, we will perform gblock oligonucleotide synthesis (iGEM pre-existing part: BBa_K1028000). • Since the silica-binding peptide (Si4), the cellulose-binding domain of cellulose-binding protein (CBPa), and the B domain of staphylococcal protein A are individually non toxic, we assume that the combination of them (newly iGEM 2016 deposited part: BBa_K2053002) will also be non toxic, although this remains to be tested in the lab. • The silafin kinase TpsTK1 (newly iGEM 2016 deposited part: BBa_K2053000 is involved in the biomineralization of silafins. They are quite abundant in the diatoms, and help in enhancing the phosphorylation of the silificating peptides.There is no bibliographic evidence of toxicity (Pubmed, Google search).
These components used in our experiments are classified as biosafety level 1 organisms; all are harmless and were used under well-established protocols and with proper guidance and safety equipment. The safety levels of our laboratory are BHSL1 and 2. These biosafety levels are suitable for work involving potentially biohazardous agents to personnel and the environment.
All lab work and experiments were done according to laboratory safety policy of the Institut Pasteur. Students had a course on common hazard and exposure risks in the lab, including chemical and biological hazards that are found at the Institut Pasteur research laboratories. The course taught us how to prevent exposure to these hazards and emergency response procedures in case of exposure. The course also covered our lab waste handling procedures as well as useful information on methods to ensure the research laboratory is free from common physical hazards. The training was performed by Dr Deshmukh Gopaul.
N.B: Since not all team members have had wet lab experience before, all the lab work was done under the supervision and guidance of at least one of our authorized senior team members and/or advisors. Secondly, the immunodetection tests that required experiments with infected mosquitoes were conducted by one of our coaches in a biosafety level 3 laboratory. Mosquitoes, rated pathogen free, involved in the other experiments came from an insectarium in Institut Pasteur, none were taken from the environment.
Safe Lab Work From chemicals to electrical equipment, laboratories present a plethora of safety hazards, which is why it is so vital to understand the importance of lab safety. The main safety aspects of our daily routine in the lab are the following:
• Personal safety: the experiments in the lab were done with gloves, safety glasses, closed leather shoes and lab coats. • Sterilizing equipment and materials. • Disinfecting work areas before and after use. • Using a disinfectant soap to wash your hands before and after working with microorganisms. Non-disinfectant soap will remove surface bacteria and can be used if disinfectant soap is not available. Gloves may be worn as extra protection. • Not eating or drinking in the lab, nor storing food in areas where microorganisms are stored. • Labeling everything clearly. All cultures, chemicals, disinfectant, and media should be clearly and securely labeled with names and dates. If they are hazardous, we label them with proper warning and hazardous information. • Storing chemicals according to risk categories, flammables, corrosives in specialized cabinets. • Autoclaving or disinfecting all waste material. • Cleaning up accidental spills with care.
The laws and regulations that govern biosafety in research laboratories at the Institut Pasteur are ISO 15189 and ISO/CEI 17025. ISO 15189 is an international standard published by the International Organization for Standardization that specifies requirements for quality and competence particular to medical laboratories. ISO/CEI 17025, is a standard that specifies the general requirements for the competence of calibration and testing laboratories. This standard regarding any laboratory analysis and testing is the repository used during the accreditation audits.
• The laboratory supervisor must enforce the institutional policies that control access to the laboratory. Students are required to show their vaccination status, compatible for Institut Pasteur, before being allowed to enter lab and perform experiments. • Security badges are electronic access controlled to enter the campus and specific lab areas. • Specific BHSL 1/2 clearance is confered to the Education center Labs. • Persons must wash their hands before and after working with potentially hazardous materials and before leaving the laboratory. • Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas. • Mouth pipetting is prohibited, mechanical pipetting devices must be used. • Perform all procedures to minimize the creation of splashes and/or aerosols. • Individual safety equipment, including gloves, lab coat and safety goggles are required. • Group safety equipment like fume hoods, laminar flow benches are available. • Decontaminate work surfaces after completion of work and after any spill or splash of potentially infectious material with appropriate disinfectant. • Decontaminate all cultures, stocks, and other potentially infectious materials before disposal using an effective method.
