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
Researcher, Public, and Environmental Safety
The Rose-Hulman iGEM team has kept safety a top priority since the very early stages of the project. As a result, any project that might have posed as a significant threat to safety was quickly dismissed. During the planning phase of Checkmate, team members brainstormed lab procedures as well as guidelines for plasmid construction to provide a safe and effective workplace. Despite best efforts though, no project is without safety concerns. Some of the lab methods require using potentially hazardous chemicals that could cause injury to any one of the researchers. Another potential safety risk for researchers results from dealing with microbial agents. While it is unlikely that a researcher would develop an infection from the strains he or she is working with, contagion is still a possible safety concern. To combat these risks, the team was sure to follow all safety protocols, from aseptic techniques to complete labeling. Proper lab attire was worn at all times in the lab.
When considering public safety, it is important to consider every aspect and intermediate step of the project. In Checkmate, the strains of yeast used contain a knockout for an essential amino acid. As a result of this knockout, it is highly unlikely that this yeast could ever escape the lab and be released into the public. However, not all the species involved in the project are so easily controlled. The selection scheme for maintaining the plasmid in E. Coli involved providing the bacteria with antibiotic resistance. In an age where multiple drug resistant bacteria are on the rise and responsible for killing several thousand people a year, the potential of this bacteria escaping and passing this plasmid on to another bacteria cannot be ignored. When handling E. Coli, the team used aseptic techniques and sterilized their equipment.
Similar to the public safety concerns, most of the environmental safety concerns stem from chemicals or biological agents leaving the lab and being exposed to the environment outside the lab. Improper disposal or contamination of the environment by a couple of Checkmate’s chemicals could prove deleterious to outside environments. Once again, sterilization and aseptic techniques prevented any calamity.
BioBrick Safety
To ensure the safety of our BioBricks, we consulted the BioBrick standard for regulation of restriction sites when designed the DNA sequence. In addition, *SENTENCE TALKING ABOUT VECTOR KNOCKOUT OF KEY AMINO ACID. By characterizing the BioBricks to their fullest potential, it will allow other participates of iGEM to use our BioBricks in their intended way, optimizing the efficiency of their projects.
Biosafety Group
Rose-Hulman Institute of Technology, due to a small applied biology program, does not have a biosafety group. Instead, safety procedures are integrated into classes by professors. The United States of America has many biosafety rules about working with human cells, but projects that work without human cells, such as Checkmate, are not regulated by these rules because they do not pose the same threat as projects that use human cells.
Future Safety
Future iGEM teams can be safe by following appropriate lab safety procedures. When considering their project, future iGEM teams need to be aware that they are creating something new, and should proceed with caution, always keeping safety at the forefront. Specifically, one way to be sure that iGEM projects cannot be a danger to the public or the environment is to include some form of self-destruct. This would make the cells unable to live in a non-lab environment. The DNA could be degenerative over time, or a strain could be dependent on a vital nutrient only found in a laboratory setting.