<p>For the duration of our project we used <i> E. coli </i> (Top10) and <i> B. subtilis</i> (super competent)which are both level one organisms. The safety precautions stated on Material Safety Data Sheets were taken into consideration when using chemical and personal protective equipment was enforced at all times within the lab. Lastly, all undergraduate students were supervised at all times by graduate supervisors or the lab technician.</p>
<p>For the duration of our project we used <i> E. coli </i> (Top10) and <i> B. subtilis</i> (super competent)which are both level one organisms. The safety precautions stated on Material Safety Data Sheets were taken into consideration when using chemical and personal protective equipment was enforced at all times within the lab. Lastly, all undergraduate students were supervised at all times by graduate supervisors or the lab technician.</p>
Figure 1. Our Lab technicianFigure 1. Ainna modeling her PPE
Safety and containment were a top priority for our team. We wanted to ensure all of our team members met the training requirements as outlined in the University of Calgary’s safety policy. Mandatory training comprised of a biosafety and Workplace Hazardous Materials Information Systems (WHMIS) course. Furthermore, all team members participated in a two week molecular biology workshop in which students got to safely practice important lab techniques.
For the duration of our project we used E. coli (Top10) and B. subtilis (super competent)which are both level one organisms. The safety precautions stated on Material Safety Data Sheets were taken into consideration when using chemical and personal protective equipment was enforced at all times within the lab. Lastly, all undergraduate students were supervised at all times by graduate supervisors or the lab technician.
Containment Considerations
B. subtilis is a gram positive bacterium which naturally resides in the soil and the human gut. During conditions of energy starvation B. subtilis undergoes sporulation. For our project, we used a super competent strain of B. subtilis referred to as the “com K strain” because naturally transformation could be induced when desired by simply flooding the bacteria with a xylose solution.
Our primary containment mechanism is auxotrophy. We are inserting our reporter gene into the thrC locus of the B. subtilis genome which encodes for an enzyme required to make threonine. Disruption of the thrC locus will cause our bacteria to exhibit auxotrophy. Therefore, the bacterium will only survive if it is supplemented with a media containing threonine. Since B. subtilis is commonly found in the environment and the human gut, we believe that it poses little risk to humans and other species. However, by making our engineered bacteria auxotrophic we can ensure that it will be unable to survive if it escapes the bounds of our device.
In addition, we designed our prototype taking into consideration the target region. We wanted to ensure that the design of our device limited the need for personal protective equipment and that it could easily be transported. Our device will house the B. subtilis spores in different compartments which will be tightly secured to the base of the device. These spores will only be activated when desired by using sterile water. Freeze-dried Proteins required for IsoPCR (isothermal Polymerase Chain Reaction) will be activated using a rehydration solution. The person handling the device will NOT come into contact with the components within the device. The coloured output will clearly be visible through the transparent covers on the chambers containing the bacteria.
Accuracy
A lot of time was spent on ensuring our device was accurate. False positive and false negatives can lead to severe consequences therefore we decided to incorporate an amplification step using target specific primers. In addition, we decided to take advantage of homologous recombination which would prevent other pieces of DNA from being incorporated into the B. subtilis genome. To learn about the specifics please visit the following link: Sample Preparation
Safety Forms
Please follow the links below to view our safety forms!
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