Team:Calgary/Collaborations

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Collaborations

Canadian iGEM Newsletter

This year, the iGEM Calgary 2017 Team set a goal to achieve increased collaborativity of teams in countries throughout the world, starting with Canada. During our journey, we have reached out to many organizations as a part of our public outreach. For more details, please visit our Engagement and Education pages. To help promote communication between teams, we pioneered the first Canadian iGEM Newsletter. The main purpose of this project is to foster an avenue through which teams can share their project updates and seek collaborative assistance. Because this newsletter can be distributed by all participating teams, universities and public communities across Canada can be engaged. Additionally, we can also increase public awareness of iGEM, as well as genetic engineering in general.

Canadian iGEM Newsletter Volume 1 Issue 1

Wet-Lab Collaboration

Through the connections we made in the process of publishing the Canadian iGEM Newsletter, we were able to connect with the iGEM McMasterU team. One of their team members, Dhanyasri Maddiboina, had an internship in Calgary during the summer. Some members of our team was able to meet her and, together, we were able to bring a potential collaboration between the two collegiate teams to fruition. This year, the iGEM McMasterU team is working on a project called Glowzyme, a fluorogenic DNAzyme that cleaves RNA and detects pathogens. As a proof of concept, they synthesized a DNAzyme that generates a fluorescence when Escherichia coli is present. Because our bioplastic synthesis system utilizes E. coli on a Mars colony, the DNAzyme may be integrated into our containment system as a method for leakage detection. By assessing the possibility of incorporating the DNAzyme into our system, we can evaluate the properties of the DNAzyme and provide possible areas of improvement to iGEM McMasterU.

Procedure

To carry out this collaboration, we followed the protocol provided by iGEM McMasterU. The purpose of this experiment was to act as a proof of concept that the DNAzyme will cleave specifically in the presence of E. coli and will not cleave in the presence of other bacteria.

Materials Used:

• 60mm plates
• M9 plating media
• M9 liquid media
• E. coli DH5α
• Bacillus subtilis WB800
• 8μM DNAzyme

100mL of M9 liquid media for overnight inoculations were prepared in 87.79mL of autoclaved ddH₂O by combing 10mL of autoclaved 2xM9 salts, 200μL 1M MgSO₄, and 10μL 1M of CaCl₂. These solutions were autoclaved separately before being added to the ddH₂O. 2mL of filter sterilized 20% glucose was also added to the mixture. Two overnights of E. coli DH5α and two of B. subtilis were made using 3mL of the liquid media. The culture tubes were shaken for 24 hours at 37°C. After the inoculation period, the cell density of the overnights were measured using OD600.

As preparation for making the M9 plating media, 2.988g of agar powder was dissolved in 100mL of ddH₂O and autoclaved. 100mL of the M9 plating media was then made by combining 49.8mL of the agar solution, 49.8mL 2xM9 salt, 99.5μL 1M MgSO₄, and 9.95μL of 1M CaCl₂. 398μL of filter sterilized 20% glucose was also added. The M9, MgSO₄, and CaCl₂ salts were previously autoclaved and the plating media was made using aseptic technique. 2.5mL of the media was pipetted into four 60mm plates. Eight 60mm plates were also made using higher amounts of the media (~5mL).

Quadrants were drawn on the 60mm plates containing M9 media. On one set of plates, one quadrant was labelled DH5α for E. coli, another DNAzyme, the two remaining sections were named negative controls. A second set of plates were also labelled. The first quadrant was named B. Sub, as abbreviation for B. subtilis, and another as DNAzyme. The remaining two quadrants were negative controls. 30uL of E. coli DH5α in liquid media was pipetted to the quadrant labelled DH5α and a bacterial spreader was used to distribute the liquid culture within the quadrant. This was repeated with B. subtitles on the plate with the labelled B. sub quadrant. The plates were then incubated upside down for 36 hours at 37°C.

The next day, the DNAzyme, which was sent by team iGEM McMasterU in pellet form, was diluted to the concentration of 8μM using 12.58μL of autoclaved ddH₂O. 5μL of the DNAzyme solution was pipetted onto the E. coli DH5α and B. subtilis quadrants. 1μL of DNAzyme was also pipetted onto the quadrants labelled as DNAzyme. The plates were incubated for 1 hour at 37°C. A picture of the plates were taken under UV light.

Results and Discussion

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Conclusion

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