Team:Calgary/Collaborations
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 increase public awareness of iGEM and synthetic biology.
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 WB800 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 WB800, and another as DNAzyme. The remaining two quadrants were negative controls. 30μL 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 process was repeated with the B. subtitles WB800 cultures 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 12.50μL of autoclaved ddH2O. 5μL of the DNAzyme solution was pipetted onto the E. coli DH5α and B. subtilis WB800 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
In our first attempt, two sets of overnight cultures of E. coli DH5α and B. subtilis WB800 were made using different colonies. The cell density of these overnights were then calculated from OD600 measurements. This conversion was done using the Cell Culture Concentration from OD600 Calculator from Agilent Genomics.
| Overnight Culture | Absorbance | Cell Density (cells/mL) |
|---|---|---|
| E. coli DH5α Colony 1 | 0.157 | 1.26 x 108 |
| E. coli DH5α Colony 2 | 0.671 | 5.37 x 108 |
| B. subtilis WB800 Colony 1 | 0.130 | 1.04 x 108 |
| B. subtilis WB800 Colony 2 | 0.042 | 3.36 x 107 |
In order to reach the optimal density of 106cells/mL suggested by iGEM McMasterU, the E. coli DH5α Colony 1 culture was diluted with 23.2mL of M9 liquid media, and the B. subtilis WB800 Colony 1 culture was diluted with 18.8mL of M9 liquid media. These two liquid cultures were chosen due to its proximity to having a cell density of 106cells/mL but having a density high enough to allow for enough left-over culture for back-up use. 30μL of the E. coli DH5α Colony 1 and B. subtilis WB800 Colony 1 overnight cultures were then plated in their respective quadrants on one set of plates. After the 36 hours in the incubator, no growth was found on the two plates. As such, two E. coli DH5α Colony 1 plates and two B. subtilis WB800 Colony 1 were re-made. However, there was still no growth was found on both sets of plates the following day.
As an attempt to troubleshoot, it was suggested to us by our Advisor, David Feehan, that the agar plates may have been too thin to allow for proper bacterial growth. Thus, a new set of overnight cultures of E. coli DH5α and B. subtilis WB800 were made and was plated on a thicker agar plate (~5mL agar). To further encourage bacterial growth, the overnights were not diluted to a cell density of 106cells/mL prior to plating. The OD600 measurements of the new overnight cultures are as follows:
| Overnight Culture | Absorbance | Cell Density (cells/mL) |
|---|---|---|
| E. coli DH5α Colony 3 | x | x x 108 |
| B. subtilis WB800 Colony 3 | x | x x 108 |
After incubating the plates for 24hours, scattered colonies can be seen in both the E. coli DH5α and B. subtilis WB800 quadrants. However, the plates were allowed to incubate for another 24hours to encourage the formation of more colonies and growth lawns.
Then, 5μL of the diluted 8μM DNAzyme was pipetted onto the E. coli DH5α and B. subtitles WB800 quadrants. 1μL of DNAzyme was also pipetted onto the quadrants labelled as DNAzyme. The plates were incubated for 1 hour at 37°C and was viewed using UV light.
Summary and Conclusion
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