Team:Waterloo

From 2013.igem.org

Revision as of 04:13, 27 September 2013 by Nelk (Talk | contribs)

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.

Intercellular Communication and DNA Messaging

Intercellular communication between cells in nature allows for coordinated population-level behavior, enabling spatial and temporal organization and complex responses to environmental stimuli.

Synthetic biology is continually expanding the range of programmable cellular characteristics and behaviors, and incorporation of intercellular communication into engineered cell populations has extended programmable behavior to a population level.

Engineered AHL-based intercellular communication

Many bacteria naturally secrete acylated homoserine lactone molecules, or AHLs, which can be detected by other members of the bacterial population. Concentration-sensitive detection of AHL brings about significant qualitative changes in cell behavior via transcription regulation, including feedback on AHL production. Such natural quorum sensing systems are associated with coordinated behaviours such as biofilm formation [1] and bioluminescence [2].

These natural bacterial quorum-sensing systems have been successfully modulated to enable programmed intercellular communication in engineered bacterial populations. In this approach, genes associated with AHL production, detection, and response are “re-wired” such that they correspond to different input stimuli and output behaviors. Coordinated population-level behaviors including two-dimensional pattern formation [3], coordinated oscillations in gene expression [4], and even a system exhibiting predator-prey dynamics [5] have been demonstrated using this technique.

Why DNA messaging?

While AHL-based communication is a useful approach for engineering population-level coordination of bacterial cells, the quorum sensing messaging system has some inherent weaknesses that limit the diversity and information content of messages that can be communicated using this method

Ortiz and Endy [6] note that AHL communication acts only through regulation of transcription. In this way, the diversity of messages in AHL-based communication is restricted to regulation of genes present in the receiver.

They also note that the receptor or transcription factor affected by a particular AHL can only respond in one way, or perhaps a few ways if different concentrations correspond to different responses; that is, a single type of AHL signaling molecule cannot be used to communicate a great number of different messages within the same communication system. In order to diversify the number of potential messages, additional types AHL molecules must be used. In this way, the message and the molecule are coupled: “the message is the molecule”.

To improve in these areas, Ortiz and Endy designed and demonstrated a communication system where DNA is used as the messaging molecule used for information exchange between cells [6]. These “DNA messages” are carried between cells inside a hijacked M13 bacteriophage particle: through a cunning act of trickery, M13 viral proteins are deceived into packaging the non-viral DNA message inside viral particles instead of the viral genome itself. (Keep reading for details).

Baseballs and Bottles

An analogy is useful in appreciating the expansion of potential for communication afforded by DNA messaging over AHL based messaging. Consider the following absurd but illustrative situation:

Suppose you and I both natively speak, read, and write Italian, and I would like to communicate with you about how I am feeling. Suppose also that we are unfortunately too far away to speak directly and it is too foggy for us to see each other (no gestures), so we are forced to communicate by hurling baseballs over to each other. When I hurl lots of baseballs to you, you know I am in a particular mood, and when I hurl fewer you know I am in a different mood. Perhaps if I got very fancy I could devise a few patterns in my hurling that would add even one or two more expressible feelings to my repertoire. Or maybe if I had some tennis balls or golf balls I could hurl those as well, extending my expression a little further. While I would be profoundly grateful for this crude outlet for sharing my feelings through hurling baseballs, I would long to explain to you, in words, all the colorful flutterings of my heart.

Imagine, now, a slight change in the situation. Imagine that I have a pile of (unsmashable) bottles, a notepad, and a pen. Now instead of hurling baseballs, I can hurl bottles to you. But inside these bottles I can put a note, written in Italian! Provided we can write and read Italian, which we both can, I can send you an arbitrary range of messages expounding my full range of thought and emotion. I can philosophize, make jokes, and write you love letters, all in our native language of Italian.

The difference between these two scenarios is in the fundamental nature of our messaging tools. The problem with the first scenario is that baseballs, tennis balls, and golf balls are not able to carry much information! We can get some use out of them by setting up a system where you are able to detect how many balls I am throwing, but this could never compare to communication in our shared native language of Italian. Notepads and pens are tools that were specifically designed for communication in Italian, which allows for transmission of rich and densely encoded information.

As you’ve likely picked up, AHL here is analogous to baseballs, and DNA is analogous to a written note in Italian. An M13 viral particle is the bottle carrying the note. While AHL can be used by cells to communicate, it is not a particularly good information-encoding molecule. DNA is the master information molecule – it was specifically designed for this by nature – and all cells read and write the language of DNA. It is for this reason that DNA holds so much promise as an intercellular messaging molecule!

The Nuts and Bolts of DNA Messaging

Several key aspects of the M13 filamentous bacteriophage were harnessed by Ortiz and Endy [6] in their original design of the DNA messaging system.

Tricking M13 proteins into packaging heterologous DNA: In nature, M13 viral proteins package the M13 genome into viral particles. The M13 DNA is recognized through the M13 packaging sequence. Any DNA containing this packaging sequence will be packaged into viral particles. By removing the M13 packaging sequence from the M13 genome and placing it on a plasmid, we can trick the viral proteins produced from the M13 genome into packaging the plasmid DNA instead of the M13 DNA. A plasmid carrying the packaging sequence is called a phagemid, and a version of the M13 genome that does not efficiently package itself due to a reduced or removed packaging sequence is called a helper plasmid. A phagemid will be packaged into a viral particle in the presence of a helper plasmid.

DNA of arbitrary length can be packaged: M13 viral packaging occurs at the cell membrane, where the viral particle forms around the DNA as it is packaged and slowly secreted, forming a long filament. This allows DNA of arbitrary length to be packaged [7].

M13 is not lytic: Since M13 viral particles are secreted through the cell membrane, infected cells are able to continue living and dividing, albeit at ½ to ¾ their normal rate [7]. Because of this, cells sending a DNA message need not commit suicide to transmit their message!

Only F+ cells can be infected: The M13 bacteriophage must attach to the F pilus of an E. coli cell in order to infect it. Therefore, only E. coli cells carrying the F plasmid (F+ cells) are susceptible, while F- cells are not.

Sender cells contain a messaging phagemid and a helper plasmid, which allows them to secrete viral particles. Receiver cells must be F+. When the two cell populations are co-cultured, DNA messaging will take place. (Fig --)

Fig -- A sender population carrying a messaging phagemid and a helper plasmid can transmit a DNA message to an F+ receiver plasmid in co-culture.

DNA messaging was first established in 2012 when Ortiz and Endy demonstrated transmission and receipt of a DNA message encoding GFP and ampicillin resistance, as well as a separate message encoding T7 RNA polymerase [6].

This proof of principle demonstration indicates the viability of DNA messaging and suggests extension of the method to diversify potential communication programs.

References:

1. Lewis-Sauer K, Camper A, Ehrlich G, Costerton J, Davies D. Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. Journal of Bacteriology, 2002, 184 (4) pp 1140–54.

2. Nealson K, Platt T, Hastings JW. The cellular control of the synthesis and activity of the bacterial luminescent system. Journal of Bacteriology, 1970, 104 (1) pp 313–22.

3. Basu S, Gerchman Y, Collins CH, Arnold FH, Weiss R. A synthetic multicellular system for programmed pattern formation. Nature, 2005, 434 pp 1130–1134.

4. Danino T, Mondragón-Palomino O, Tsimring L, Hasty J. A synchronized quorum of genetic clocks. Nature, 2010, 463 pp 326–330.

5. Balagaddé FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Quake SR, You L. A synthetic Escherichia coli predator–prey ecosystem. Molecular Systems Biology, 2008, 4, pp 1–8.

6. Ortiz ME, Endy D. Engineered cell-cell communication via DNA messaging. Journal of Biological Engineering, 2012, 6:16.

7. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, NY. 2001.

Videos

Accomplishments

Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments Accomplishments

Future Aspirations

Future Aspirations Future Aspirations Future Aspirations Future Aspirations Future Aspirations Future Aspirations Future Aspirations Future Aspirations Future Aspirations Future Aspirations Future Aspirations Future Aspirations Future Aspirations

Results

Results Results Results Results Results Results Results Results Results Results Results

Conclusions

Conclusions Conclusions Conclusions Conclusions Conclusions Conclusions Conclusions Conclusions Conclusions Conclusions Conclusions

BioBricks

Ottawa's Collaboration

This year Waterloo iGEM collaborated with Ottawa iGEM. Ottawa team required help with Mathematical Modelling. In exchange Ottawa team was able to help us build the following constructs:

Promoter-LacI cassette-lock-Bxb1 integrase-Transcriptional Terminator-Promoter- Key-Transcriptional Terminator-Promoter- Lock- Bxb1 RDF

Promoter-LacI cassette-lock-PhiC31 integrase-Transcriptional Terminator-Promoter- Key-Transcriptional Terminator-Promoter- Lock- PhiC31 RDF

Promoter-LacI cassette-lock-Bxb1 integrase-Transcriptional Terminator-Promoter- Key-Transcriptional Terminator

Promoter-LacI cassette-lock-PhiC31 integrase-Transcriptional Terminator-Promoter- Key-Transcriptional Terminator

Notebook

Switch Modelling

x y z

Population & Infection Modelling

a b c

Phage Particle Production Modelling

a b c

The University of Waterloo’s iGEM – Human Practices team is a diverse team whose goal is to raise awareness on issues regarding synthetic biology. In addition, the team also provides the student community information about the latest in the research area of synthetic biology to help the community make informed, accurate and fact-based opinions. Our goal is to strengthen the bridge between the community and their knowledge of synthetic biology along with eliminating misconceptions regarding synthetic biology.

In the past year, this team gained valuable experience and information through the projects they worked on. Each project provided more insight on how informed the student community is on the topic of synthetic biology. This further helped us plan out activities that help us achieve our goal.

One of the main purposes behind the projects this year to enrich, educate and empower the student community. To achieve this goal, various activities were planned to inform the student community about the field of synthetic biology, it’s potentials and how it affects the world around us. These activities provide fundamental knowledge of synthetic biology and it’s uses, allowing the participants and the viewers to form an informed, accurate and fact-based opinion about the topic.

T.I.L.

iGEM is a community of people passionate about synthetic biology – how can we best convey this while reaching out to the public? Sometimes reading papers and textbooks doesn't quite do it for understanding an idea. As students, we know it can be difficult to grasp some concepts we’re not familiar with. So what’s a better way to communicate an idea? Could social media be the answer? That was the idea behind the VLOG series TIL: Syn Bio.

These series are a quick and effective way to convey the ideas and passion of synthetic biology. The series has many episodes that highlight various aspects of synthetic biology through a mixture of one-on-one videos and animated style videos. The series begin with episodes explaining “What is Synthetic Biology?, “Fundamental Advances” and “Cell-to-Cell Communication” (Waterloo iGEM’s 2013 project). This phase of the series is important to orient the viewers and provide some background information. During the second part of the series, the team takes a fun twist. Using the TIL: Synthetic Biology outreach event footage to compare the viewpoints of students and professors on various topics relating to synthetic biology. The footage from this event is used for addressing many factors associated with the idea of synthetic biology. These factors range from the background knowledge to stigma associated with synthetic biology and from the regulations needed to its future potential.

The series begins with these six videos, leaving the rest of the series to be shaped by viewers. Ultimately, viewers engage with the team about what they want to see in future videos, ask questions they want answered and connect with information from a variety of sources.

The TIL: Synthetic Biology outreach event (used as part of the video series) was well-received. The team came prepared with questions to ask passing students. Students were also given 4-5 days notice via Facebook. The idea behind this aspect of the video was to have it be a surprise. Questions like "do you support GMOs?", "would you eat modified fruit/meat?", "who should be able to practice synthetic biology/should it be open sourced?" and many more were asked. The team was in for some surprises with the diversity of knowledge on campus! We hope incorporating the footage into our series will give participants a fun look into their experience, which they can easily share with their friends and family. Overall, we hope that the team's work will inspire more leaders to take part and contribute to the advances in synthetic biology, regardless of their academic or professional background.

Intent to Invent

Intent to Invent was hosted on March 07, 2013 at the University of Waterloo’s Quantum Nano Center. The purpose of the event was to:

  1. Connect the students to experts in 3 key industries that use synthetic biology in their processes: Agriculture, Health and Pharmaceuticals.
  2. Bridge the level of discomfort a scientist has in regards to business.
  3. To encourage entrepreneurship within the scientific community by delivering resourceful content from industry experts.

The event promoted open panel discussions of emerging technologies in biotechnology and other advanced biological fields within the 3 industries. Students got a chance to see how synthetic biology is the connected to entrepreneurship, innovation and commercialization. They learned about the industry perspectives and barriers faced by biological companies at different stages in their business model. This talk also encouraged entrepreneurship within the scientific community by delivering resourceful content form industry experts. Each speaker gave a 20-minute mini lecture on topics including: Clinical Trial Drug Development, Commercialization of Biomass and Energy Products and Entrepreneurial Barriers for Biotechnology Companies.

Steve jobs once said, “I think the biggest innovation of 21st century will be the intersection of biology and technology. A new era is beginning, just like the digital one…”. Through sessions such as Intent to Invent, Waterloo iGEM hopes to enrich the experience of science enthusiasts as well as those just curious about synthetic biology and it’s potential. By connecting these students to industry experts, we were able to gage their interests in an innovative and entrepreneurial aspect of science. Many students showed interest in learning more about the bridge between science and business in the future. iGEM received good feedback regarding Intent to Invent, as many students felt that the information they learned was very valuable. Waterloo iGEM provided many students the appropriate connection and information they need to start connecting the scientist in them with the businessman/businesswoman in them.

VeloCity Science - For young people. By young people.

We want to inspire young people to eliminate the gap between science and business. The conventional education system does not provide for such activity. There is a job unemployment crisis throughout North America, and from the past experience, this is the perfect time to pursue this. It is time to do what we have done to the IT industry back in the 80s, but with synthetic biology this time.

Economic downturns have proven to be the best time for entrepreneurship. The perfect storm is brewing. The infrastructure is there to support initiative like this. We have bright young people hungry to make changes to the world.

What has University of Waterloo done to support this movement? We have created an entrepreneurship program that brings together the right business resources (networks, mentorship, legal and financial services, etc) and the right technical resources (wetlab space, consumables and equipment) to create kick-ass biotechnology start-ups.

But that’s not all. Above of all these resources, it is the sense of community that is crucial for the success of these entrepreneurs. The University of Waterloo has proven time and time over in providing a strong sense of community to our entrepreneurs through the programs like Accelerator Centre, Communitech Hub, and VeloCity.

That’s the story of VeloCity Science. And, we are just starting to write it.

Laboratory

Intent to Invent

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

Retrieved from "http://2013.igem.org/Team:Waterloo"