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Collaborations

Canadian iGEM Newsletter

This year, the iGEM Calgary 2017 Team set a goal to increase collaborations amongst teams across our country and did not limit ourselves to teams from iGEM. During our journey, we reached out to many organizations as a part of our public outreach. For more details, please visit our Engagement page. To help promote communication between teams, we pioneered the first Canadian iGEM Newsletter. The purpose of this initiative was to foster an avenue through which teams can share their project updates and seek collaborative assistance. Because this newsletter was distributed by all participating teams, universities and public communities across Canada can be engaged. Additionally, we increased 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 this summer. Our team was able to meet with her, and together we brought a between these 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 fluoresces when E. coli is present. Because our bioplastic synthesis system utilizes E. coli on Mars, the DNAzyme may be integrated into our containment system as a method for containment. 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. Check out their Collaboration page here.

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:

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

100 mL of M9 liquid media for overnight inoculations were prepared in 87.79 mL of autoclaved ddH₂O by combing 10 mL of autoclaved 2 x M9 salts, 200 μL of 1 M MgSO₄, and 10 μL of 1 M of CaCl₂. These solutions were autoclaved separately before being added to the ddH₂O. 2 mL 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 3 mL of the liquid media. The culture tubes were shaken for 24 hours at 37°C. After the incubation period, the cell density of the overnights as measured using a wavelength of 600 nm.

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

Quadrants were drawn on the plates containing M9 media. On one set of plates, one quadrant was labelled DH5α for E. coli, another DNAzyme, and the two remaining sections were named negative controls. A second set of plates were also labelled. The first quadrant was named B. Sub (B. subtilis) WB800, and another as DNAzyme. The remaining two quadrants were negative controls. 30 μL 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. subtilis 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 8 μM using 12.50 μL of autoclaved ddH₂O. 5 μL of the DNAzyme solution was pipetted onto the E. coli DH5α and B. subtilis WB800 quadrants. 1 μL 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 was 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 was then calculated from OD600 measurements. This conversion was done using the Cell Culture Concentration from OD600 Calculator from Agilent Genomics.

Overnight Culture	Absorbance at 600 nm	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 23.2 mL of M9 liquid media, and the B. subtilis WB800 Colony 1 culture was diluted with 18.8 mL 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. 30 μL 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.

Our TA, David Feehan, suggested to us 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 at 600 nm	Cell Density (cells/mL)
E. coli DH5α Colony 3	0.821	6.57 x 10⁸
B. subtilis WB800 Colony 3	0.419	3.35 x 10⁸

After incubating the plates for 24 hours, scattered colonies could be seen in both the E. coli DH5α and B. subtilis WB800 quadrants. However, the plates were allowed to incubate for another 24 hours to encourage the formation of more colonies and growth lawns.

Figure 1. E. coli DH5α and B. subtilis WB800 plates with labelled quadrants after 48 hours of incubation.

Then, 5 μL of the diluted 8 μM DNAzyme was pipetted onto the E. coli DH5α and B. subtitles WB800 quadrants. 1 μL of DNAzyme was also pipetted onto the quadrants labelled as DNAzyme. The plates were incubated for 1 hour at 37°C and were viewed using UV light.

Figure 2. Final results of the E. coli DH5α and B. subtilis WB800 plates after the addition of DNAzyme. No bright fluorescence can be detected throughout the plates. The areas circled in red indicate the location in which the DNAzyme was dispensed on the quadrants containing the growth of E. coli DH5α (left) and B. subtilis WB800 (right).

Conclusion

We did not see the expected results, despite our troubleshooting efforts; therefore, further troubleshooting of this collaboration with iGEM McMasterU is required, as we did not observe the expected result. Fluorescence should only be detected within the quadrant containing E. coli DH5α and not in any of the other sections. As shown in Figure 2, no fluorescence can be seen in any quadrants of the two plates. There is a slight change in agar colour that can be seen in the circled area of the section labelled as B. Sub, for B. subtilis WB800. However, it is unclear whether this change in colour was caused by the DNAzyme or other factors. Due to limited amounts of DNAzyme sent by iGEM McMasterU, we were unable perform this experiment again.

We could look into using a Typhoon Imager, which was the suggested viewing device, in order to better match the McMaster University protocol. Due to the constraint of time, we were unable to find a laboratory that had this equipment available for use by us. By using this technology, we would be able to better troubleshoot this protocol by analyzing results under specific parameters, such as absorbance, that a traditional UV light cannot provide.

Throughout the process of this wet lab collaboration, we were able to test the DNAzyme, named GLOWzyme, synthesized by iGEM McMasterU, and assess the plausibility of incorporating it into our PHB system on Mars. Further testing is required to determine the efficiency of the DNAzyme to detect the presence of E. coli, which will provide insight as to whether we should integrate GLOWzyme into our system or look for other possible detection methods.

In addition, if the GLOWzyme system is to be incorporated into our project, there are two design elements that will need to be addressed. Firstly, the DNAzyme system has to be engineered in a way where little effort from the astronauts is required to maintain its working capacity. Also, a secondary system will be required to be hybridized to the GLOWzyme system such that when an E. coli leak is detected by the GLOWzyme, another system will be able to alert the crew so that they can respond in a proper and timely manner.

Team:Calgary/Collaborations