July 31, 2012
Made media: YPD, L-AMP, CSM, and CSM-His
Members: Ben, Bobby, Alex, Kristen, Devon, Dr. A.
August 1, 2012
Streaked out plasmid-bearing E. coli strains (pRS416 & pRS413) and yeast strains
Members: Ben, Bobby, Alex, Kristen, Devon, Dr. A.
August 2, 2012
Checked for E. coli colonies: success. Made liquid L-AMP media, innoculated small cultures.
Members: Ben, Bobby, Alex, Kristen, Devon, Dr. A.
August 3, 2012
Qiagen minipreps
Members: Ben, Bobby, Alex, Kristen, Devon, Dr. Dave.
August 6, 2012
Made stock solutions for transformation procedures
Members: Adam, Kristen, Dr. A
August 7, 2012
Transformed E. coli with our red construct
Members: Bobby, Adam, Devon, Kristen, Ben, Dr. A
August 8, 2012
Inoculated liquid media with transformed E.Coli
Members: Bobby, Adam
August 9, 2012
Ran Mini-Preps of pRS413, pRS416, and Red construct bearing E. Coli, stored extracted DNA
Members: Bobby, Adam
August 10, 2012
Ran quick gel of extracted DNA
Members: Bobby, Adam
August 11, 2012
Digested extracted DNA, isolated pRS413 & pRS416 fragments from gel
Members: Bobby, Adam
August 12, 2012
Isolated red construct fragments on gel, poured chloramphenicol plates
Members: Bobby, Adam
August 13, 2012
Digested extracted DNA, isolated pRS413, pRS416, and Red Construct from gel, Transformed E. Coli with NTNU construct
Members: Bobby, Adam, Dr. A
August 14, 2012
Ligated pRS413 and red construct, transformed E. Coli with ligated plasmid, inoculated liquid media with NTNU E. Coli
Members: Bobby, Adam
August 15, 2012
Mini-Preps of NTNU construct, confirmed presence on gel, Digested pRS413, pRS416, and Red Construct, poured ampicillin plates
Members: Bobby, Adam
August 16, 2012
Isolated pRS413, pRS416, and Red Construct from gel, began storing strains, DNA, and equipment
Members: Bobby, Adam
August 17, 2012
Ran digestion of pRS413, pRS416, and Red Construct, stored remaining strains, DNA, and equipment
Members: Bobby, Adam, Dr. A
August 28, 2012
Transformed E.Coli with BBa_E0422 reporter
Members: Bobby, Adam, Dr. A
August 29, 2012
Digested and isolated Red Construct and pRS413 from gel, Inoculated liquid media with BBa_E0422 reporter
Members: Adam
August 30, 2012
Digested pRS413 and Red Construct, Isolated from gel and Ligated, Transformed E. Coli.
Members: Adam
August 31, 2012
Digested pRS413 and Red Construct
Members: Bobby, Adam, Dr. A
September 1, 2012
Started cultures of pRS413 and the Red construct form frozen cultures
Members: Adam
September 2, 2012
Inoculated liquid media with colonies from plates of pRS413 and Red Construct strains
Members: Adam
September 3, 2012
Ran mini-preps of liquid colonies
Members: Adam
September 4, 2012
Ran diagnostic gel to check amount of DNA extracted
Members: Adam
September 5, 2012
Purified and concentrated DNA samples
Members: Adam
September 6, 2012
Ran diagnostic gel to check for purification success
Members: Adam
September 7, 2012
Ran BioBrick Assembly protocol on NTNU lld promoter and BBa_E0422, Transformed E.Coli with ligation mixture
Members: Adam, Dr. A
September 8, 2012
Inoculated liquid cultures with transformed E.Coli
Members: Adam
September 10, 2012
Digested and isolated Red Construct and pRS413 from gel, Ran mini-preps of liquid cultures
Members: Adam
September 11, 2012
Ran gel of lld/0422 hybrid, Gel showed presence of desired product
Members: Adam
September 12, 2012
Inoculated liquid cultures and plates with transformed E.Coli
Members: Adam
September 13, 2012
Performed ligation on Red Construct, Transformed E.Coli with blue construct
Members: Adam
September 14, 2012
Inoculated liquid cultures with blue construct transformants
Members: Adam
September 15, 2012
Ran mini-preps on colonies with blue constructs
Members: Adam and Bobby
September 16, 2012
Digested Blue construct and pRS413, isolated desired fragments from gel
Members: Adam
September 17, 2012
Ligated Blue construct and pRS413 fragments, transformed E.Coli
Members: Adam
September 18, 2012
Inoculated Liquid Media with Blue construct/pRS413 fusion bearing E.Coli
Members: Adam
September 19, 2012
NTNU construct testing via fluorescence, Mini-Preps of Blue construct/pRS413 fusion bearing E.Coli
Members: Bobby, Alex, Adam
September 20, 2012
Diagnostic gels of Blue Construct/pRS413 mini-preps
Members: Adam
September 22, 2012
Inoculated liquid media with Blue Construct/pRS413 E. Coli, prepared stock solutions
Members: Adam, Dr. A
September 23, 2012
Mini-Preps of E.Coli, Ran gel of prep, made liquid media
Members: Adam
September 24, 2012
Ran digestion of mini-prep DNA, plated Yeast
Members: Adam
September 25, 2012
Gel of digestion result, isolated fragments from gel
Members: Adam
September 26, 2012
Ligated Blue Fragment/pRS413, transformed E.Coli
Members: Adam
September 27, 2012
Inoculated LB-AMP broth with transformed E.Coli, plated Yeast
Members: Adam
September 28, 2012
Inoculated LB-CARB broth with transformed E.Coli
Members: Adam
September 29, 2012
Inoculated LB-CARB broth with transformed E.Coli
Members: Adam
September 30, 2012
Performed Mini-preps of transformed E.Coli, Began yeast transformation, plated additional yeast cultures.
Members: Adam
October 1, 2012
Plated Yeast Transformants
Members: Adam
October 3, 2012
Transformed yeast found growing on plates
Members: Adam
The official RHIT Safety page can be found
here.
Researcher, Public, and Environmental Safety
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.
BioBrick Safety
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.
Biosafety Group
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.
Future iGEM Safety
The possibility of designing mutually dependent strain/vector systems for routine manipulation and storage of BioBrick parts should be explored.
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