Collaboration with University of Calgary iGEM - Wet Lab Collaboration

Overview

This summer, Calgary iGEM reached out to us expressing interest over our project and about beginning a collaboration between our teams. Their goal is to develop a process for bioplastic production on a Mars settlement. This is a novel method that couples waste management in space with the high cost of shipping materials to a Mars mission crew. Calgary iGEM has been working hard in engineering E. coli to produce bioplastics from human waste feedstock. The bioplastic of interest is poly(3-hydroxybutyrate) (PHB), which can be synthesized with their engineered bacteria using volatile fatty acids (VFA), compounds that are present in human waste. Calgary’s interest in our team stemmed from the need for a fast and effective E. coli detector to incorporate into their bioplastics synthesis system on a Mars colony. Our DNAzyme would serve as part of their containment system, and quickly alert crew to leakage from the synthesis system. Our team was very interested in this potential application of our DNAzyme and decided to mail a quantity of it over for pilot testing from Calgary iGEM.

Experiment

A small amount of DNAzyme was shipped to Calgary for testing its specificity in the presence of E. coli, and confirming lack of cleavage activity in the presence of other bacteria. The following outlines the procedure followed by Calgary iGEM and their results.
Overnight cultures of E. coli DH5α and Bacillus subtilis WB800 were prepared in liquid M9 media and diluted to a concentration of 106 cells/mL. Quadrants were drawn on agar plates with M9 media and labelled as either E. coli or B. subtilis, DNAzyme, and two negative controls (Figure 1).

On the quadrants labelled as bacteria, 30 µL of the overnight E. coli or B. subtilis were spread and left to grow for 36 hours. Calgary encountered a lack of bacterial growth following this incubation step. They decided to use a thicker agar plate and plate a higher concentration of cells (108 cells/mL), which resulted in colony growth (Figure 2).

Figure 2: Bacterial growth on M9 agar plates after 36 hours.

Next, 5 µL of 8µM DNAzyme was added to the quadrants containing bacteria. To check for background fluorescence, 1 µL of DNAzyme was added to the quadrant labelled as DNAzyme. The plates were left to incubate for 1 hour and viewed under UV light as an alternative to using a fluorescence gel imager.

Results

Unfortunately, the experiments conducted by Calgary iGEM proved unsuccessful. They were unable to detect fluorescence following the addition of DNAzyme under UV, let alone whether B. subtilis displayed a lack of fluorescence. Due to limited DNAzyme, repetition of the experiment was not possible.
There are a variety of improvements required if we are to continue with this collaboration. The first is checking the stability of the DNAzyme during the shipping process. The DNAzyme was pelleted when it was sent to Calgary, and they resuspended it before usage. Our team should check DNAzyme activity in our own lab using pelleted form to rule this out as a potential interferant. The second factor to consider is the use of UV vs fluorescence imaging. It would be optimal if Calgary gained access to a fluorescence gel imager (Typhoon Imager) to be consistent with our experiments and imaging.

Future Directions

Once the DNAzyme has been optimized, there is one big factor to take into consideration: use of the DNAzyme by non-specialists. If this collaboration were to proceed, our DNAzyme would need to be usable by Mars colonists and without the need for extensive bacterial culturing and plating. As well, a faster readout is required. The current 1 hour of incubation is quite long if the DNAzyme were to be used as part of a leakage monitoring system. The last aspect to consider is the activity of the DNAzyme in a variety of sample matrixes, without sample enrichment. Bacterial detection has to occur without pure bacteria, and in the bioplastics synthesis system there will a variety of molecules present that should not interfere with the DNAzyme. These are all considerations that would be taken into account when designing the DNAzyme for use in a Mars colony.

oGEM Manual of iGEM Teams

On July 17th, 2017, our annual oGEM meeting took place. Bringing together all of the post-secondary iGEM teams, the day allowed for discussion of our projects, our future, our strengths, and our weaknesses. While they were not at the center of the talk, surely but slowly common worries were brought forth, none of them relating to our projects at all but to our own selves: how do we even begin an iGEM team? How do we manage one that exists? How do we continue one that graduating students leave to us?

Throughout the years, this running theme of looking at our iGEM teams not through the lens of our projects and work but through the very essence of them, which is to say the people who make up iGEM in the first place, is something that has oft been pushed back and neglected. At the meeting that day, we realized that surprisingly, none of us were in the same stage of development in our growth as iGEM teams. Two teams just started that year; others had come back after some years of hiatus or had only been established for a number of years. Then, there were the teams that had been established as the forefront of oGEM, having been constants for years in this field. Yet, we all had our concerns about how to manage, grow, and develop our teams to be better. Despite our differences in experiences and years, or perhaps because of that, we came to realize that we could help each other build better, more solid foundations by exchanging the knowledge we had accumulated over the years. Thus, this manual came to be, out of a desire to be able to share and pass down that knowledge to all of us, and to you.

This manual will always remain open to edits, revisions, suggestions, comments, and questions. It is in our hopes that this manual grows as our teams will, and that what we have learned through this amazing journey in camaraderie and synthetic biology will always be available for those who need or want it.

Thank you, and good luck with your endeavours in iGEM. You’ll be amazing.

The Ontario iGEM chapters collaborating on this venture jointly by McMasterU, are: U of Guelph, Toronto, with entry contributions from Waterloo. The finished manual will then be distributed both in print and online to any chapters interested across Ontario and Canada.

Below is a list of the topics covered in the oGEM Manual, compiled by our Human Practices member Aline-Claire Huynh.

Section 1: The Process

Starting Your Team
What Do We Want Our Team to Look Like?
How Do We Register Our Team to iGEM?
How Many People Should We Aim to Recruit?
How Do We Hire the Right People?
Managing Your Current Team
Setting Team Expectations and Roles
Administration
Team Structures, Positions, and [Hierarchies]
Training New Members
You Said How Much Paperwork?
Communicating and Collaborating with Your Team
How Should Execs Keep in Touch?
How Should Each Team as a Whole Communicate?
How Should Each Subteam Communicate?
How Can All the Different Subteams Effectively and Efficiently Communicate with Each Other?
How Often Should We Communicate?
Developing Your Team
Mentorship
Transferring Knowledge
Team Bonding
Member Development
Growing Your Team
Marketing on Campus to Other Students
Keeping Your Social Media Updated and Shareable
Fundraisers and Awareness Campaigns
Troubleshooting Your Team
How to Prevent Setbacks as Much as Possible
How to Identify Minor Setbacks
How to Identify Major Setbacks
How to Handle Deadlines, Individually
How to Handle Deadlines, as a Team
How to Learn from Our Mistakes
Tips, Tricks, and Troubles for the Meta

Section 2: The Project

The Basics
What Are the Roles of Each Subteam?
What Do Our Timelines and Deadlines Look Like?
The Project
How Do We Decide on a Project?
How Do We Even Recruit a PI?
How Do We Get Lab Space?
Where Can We Find People to Teach Us How to Do Assays and Other Science?
How Can We Get Our PI to Believe in Us?
The Wet Wet Wet Wet Wet Lab
What are standard assays that we should use for ______?
The Mysterious Dry Lab
What Are the Most Important or Prominent Languages I Should Learn?
What Are Some Programs That Can Help for X?
Practices on Humans (or People, Policies, and Practices)
Understanding Synthetic Biology As Seen by the Public Eye
How Do We Explain SynBio to the Laymen?
Understanding the Science Behind SynBio Policies and Guidelines
Emailing Stakeholders and Interested Parties
Meeting with Stakeholders and Interested Parties
How to Reach Out to the Local Community
Organizing Workshops, Awareness Campaigns, and Fundraisers
Fundings, Sponsors, and Money Issues
How Do We Apply for/Get Funding and/or Grants?
Who Should We Look for as Sponsors/Partners?
Who Will Give Us Those Sweet Free Samples?
How Do We Capitalize on Resources?
The Wiki
What Is So Important About This Wiki, Anyway?
What Is the Wiki Freeze?
Why Are the Other Teams’ Wikis So Pretty?
The Competition
Medal Requirements
Collaborations
The Jamboree
Tips, Tricks, and Troubles for the Usual