Idea

Due to its nature as an information-encoding molecule, the use of DNA as an intercellular messaging molecule would enable more information-rich communication between populations of cells than traditional AHL-based messaging. The first demonstration of DNA messaging was published by the Endy group at Stanford University in late 2012, wherein DNA encoding instructions for expression of fluorescence and antibiotic resistance were transmitted from one bacterial population to another, carried by M13 bacteriophage particles.

Incorporation of well-established in vivo DNA modification techniques into DNA messaging will diversify and extend potential intercellular communication programs, and will enable the integration of recent developments in DNA digital logic with DNA messaging.

The goal of our project is to place on a DNA message a switch that can be flipped in receiver cells under inducible conditions, and whose state determines whether or not the DNA message is retransmitted from receiver cells to a population of secondary receiver cells. The switch consists of a promoter that can be inverted using a serine integrase, leading to transcription of different genes. It is directly inspired by the recombinase addressable data (RAD) module published by the Endy group in early 2012.

We have synthesized four such DNA switches and will soon test the ability of PhiC31 and Bxb1 serine integrases, along with the respective recombination directionality factors (RDFs), to control their states. We have also produced constructs that we will use to attempt to control the production of M13 viral particles containing a DNA message and we will test these soon. We will integrate these efforts to demonstrate our goal of incorporating digital DNA logic into DNA messaging. Through this work, we will broaden the horizons of engineered intercellular communication.

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Accomplishments

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Future Aspirations

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Results

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Conclusions

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BioBricks

• Hpdo with no gene 8
• Bxb1 rdf
• Bxb1 int
• Bxb1 switch
• BXB1 switch flipped
• ΦC31 rdf
• ΦC31 int
• ΦC31 switch
• ΦC31 switch flipped

Ottawa's Collaboration

Ottawa Ottawa Ottawa Ottawa Ottawa Ottawa Ottawa Ottawa Ottawa Ottawa Ottawa Ottawa Ottawa Ottawa

Notebook

Switch Modelling

x y z

Population & Infection Modelling

a b c

Phage Particle Production Modelling

a b c

Vlogs

Special Project

Intent to Invent

Laboratory

Safety

All experiments are carried out in a BL2 certified lab. Researcher safety when using E. coli, would not be compromised in safety issues due to use of M13. It poses no threat at all to humans. While, the E. coli strain used was relatively harmless, treatment of possible infections may potentially be affected by the antibiotic resistance. Furthermore, spontaneous mutations which result in increased infectivity may result. However, measures and precautions suggested by the Canadian biosafety guidelines were taken to minimize even the slight chance of infection. Additionally, the working conditions of the lab is already above the recommended safety level of BL1 for usage of M13 viruses. Every member of the team has been trained with safety modules and went through a week of lab training and continuous oversight from the Advisors and his graduate students in the lab. All lab members, including graduate students or other students that were working in the lab, wore appropriate PPEs and disposed all consumable in appropriate biological waste boxes. All surfaces were wiped down with ethanol after use and all glassware was washed immediately after their usage.

The design of the project does not call for release into the public. Additionally, the project design does not produce any harmful products. Through it is possible that the construct could get released to the general public accidentally. But, the product of the constructs only produce fluorescent proteins and it can only be used in a controlled setting with a certain type of chemical present in the environment, thus making it ineffective when released to the public. Because safety of the public and the lab members is our utmost concern, we have ensured that all wastes are thrown out appropriately and autoclaved so that accidentally release would never occur.

There are no additional risks posed by our projects compared to other general BL1 lab concerns. Our bacteria are not pathogenic and are unable to survive outside of the lab environment, because they are unable to effectively compete with other organisms in nature. As stated above, all wastes are discarded according to the Waterloo standards and autoclaved.

There is no potential for harm to human health through use of our constructs, as described above. There is therefore no risk of malicious use.

Our constructs pose no threats to human health, as described above, and scaling up would not change this. Our project is a "fundamental advance" that contributes to the coordination of population-level cellular behavior by allowing messages to be sent between populations of E. coli cells. However, many additional layers of complexity in engineering would be required to use our method to enable pathogenic or otherwise dangerous behaviors in populations of cells.

The cell to cell communication project does include packaging viral particles. Although there are only some proteins of the M13 virus that are packaged and are therefore not a safety risk. M13 is not a safety risk even if its whole genome is packaged. Our project poses no threat to safety and thus we haven't implemented any of these mechanisms.

All the lab and design team members successfully passed the following safety training: Employee Orientation Training Session: https://info.uwaterloo.ca/infohs/hse/online_training/employee-orientation/Staff%20Orientation.swf Workplace Violence and Harassment Training: https://info.uwaterloo.ca/infohs/hse/online_training/workplace_violence/workplace_violence.html General Laboratory Safety: https://info.uwaterloo.ca/infohs/hse/online_training/lab_safety/lab_safety_course.html WHMIS: http://www.safetyoffice.uwaterloo.ca/hse/lab_safety/index.html Laboratory BioSafety Training: https://info.uwaterloo.ca/infohs/hse/online_training/biosafety/biosafety.swf

The BioSafety Guidelines followed by uWaterloo iGEM team can be found here: http://www.safetyoffice.uwaterloo.ca/hse/bio_safety/legislation.html

University of Waterloo has a Biosafety Committee and can be found here: http://www.safetyoffice.uwaterloo.ca/hse/bio_safety/bsc.html. Although the project has not been discussed with the Biosafety Committee, it has been discussed with several faculty members and has been found to have no risks. Furthermore, the laboratories operating at the University of Waterloo have obtained permits from the Bio-Safety Committee in order to perform intended research. Since the Waterloo iGEM team performs all laboratory work in a parent lab under the guidance of the Masters and PhD students of that lab, the permits obtained by the parent lab cover the projects carried out in the lab.

Canada has very well established biosafety regulations and guidelines which can be found here: http://www.phac-aspc.gc.ca/lab-bio/

The laboratory we work on cell to cell communication project is rated level 1.

E.coli strains that Waterloo iGEM team works with falls within the risk level 1. Additionally the laboratory we operate in is certified for work with the above listed risk group of the E.coli.

Sponsors

Administrators

Lab & Design

M13 Group

BxB1 Group

Φ C31 Group

Mathematical Modelling

Human Practices

Advisors

Graduate Student Advisors