Made media: YPD, L-AMP, CSM, and CSM-His
NEB5-alpha (NEB #C2988J or #C2987I): fhuA2 ?(argF-lacZ)U169 phoA glnV44 F80? (lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17
BY4741 (ATCC #201388): MATa his3-?0 leu2-?0 met15-?0 ura3-?0
BY4742 (ATCC #201389): MATa his3-?0 leu2-?0 lys2-?0 ura3-?0
Transformations were carried out on competent E.Coli using the procedure available on the NEB website.
DNA extraction from cells was performed using a Qiagen QIAprep Spin Miniprep Kit (Cat. no. 27104). Included procedure was followed.
Digestions were performed using EcoRI, XbaI, SpeI and PstI using recommended NEB procedure.
Gel extraction of digested DNA was performed using a Qiagen QIAEX II Gel Extraction Kit (Cat. no. 20021). Included procedure was followed.
Ligations were performed following procedure available on the NEB website. Total mass of DNA for ligations was 100ng with a 1:3 to 1:4 ratio of vector to insert.
The Rose-Hulman iGEM team has considered safety a top priority since the early stages of project development. As a result, projects that could pose a significant risk were dismissed. Our laboratory space is considered a Basic Biosafety Level 1 laboratory but it does have a few characteristics of higher categories. It has controlled access, biohazard signs and waste disposal bins, and an autoclave. All participating students received standard safety and good laboratory practice training as part of their academic laboratory course-work. The Instructor provided more specific safety instruction, as necessary. Proper attire was worn at all times in the laboratory. Gloves and glasses were donned as warranted. The only organisms used in our project are common laboratory strains of Saccharomyces cerevisiae (BY4741 and BY4742) and Escherichia coli (NEB5alpa), which are both considered Risk Group 1 microorganisms according to the Laboratory Biosafety Manual published by the World Health Organization. Group 1 organisms are defined as “no or low individual and community risk, a microorganism that is unlikely to cause human or animal disease.” Aseptic technique is used whenever working with these organisms and any contaminated wastes are sterilized by autoclaving or destroyed by commercial pyrolysis. All laboratory chemicals are stored, handled and used as recommended by the manufacturer, and they are disposed of in accordance with national, state, and local regulations and recommendations. The laboratory space and contents are not accessible by unauthorized personnel. All microbial strains, including bacteria rendered antibiotic resistant by transformation, harbor nutritional auxotrophies or other mutations that mitigate the risk of their growing outside of the laboratory or causing disease in healthy humans or animals. Furthermore, none of our recombinant constructs produce any known contagion or toxin.
None of the BioBrick parts utilized or constructed are known to pose any safety issues. Furthermore, they are well contained by the microbes that harbor them. The risk of unintended transfer to any other organism is minimal.
Rose-Hulman does not have a biosafety group, committee or review board other than an Animal Care and Use Committee, which oversees animal research. Safety training and laboratory waste disposal are facilitated by an Environmental Health and Safety Officer, who also serves as a resource for faculty and students.
The possibility of designing mutually dependent strain/vector systems for routine manipulation and storage of BioBrick parts should be explored.
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