MC Interface Revealed!

Hi Everyone!

I am pleased to post (for the first time) screen shots of our Membrane Computing Agent Base Model. In this post i am focusing on interface mostly. Beside the fact that we want to have an accurate model, we also want a model that is easy to use. So we thought about design issues with our interface. How can we (computer scientists) hide as much code as possible so when a biologist uses our software, they don't freak out.

So lets take a look at our main screen:



If the size of the picture is small and you have a hard time making out the details, you can always click on the photo and see a full size version. Unfortunately this is the largest size that the blog allows for posting images online. Anyways going back to our interface, on the top you see "Quorum Sensing Model - Version 2.0". So this is our QS model and the version 2.0 is our latest release. We initially started with version 1.0 and gone through 1.1, 1.2, 1.3, ... , 1.7. With versions 1.x we were adding basic functionality to the system. Simply setting up a framework, introducing rules, figuring out how to define rules and so on. In version 2.0 we have build the interface on top of our framework so users can interact with the system without the need of knowing how the code works. You don't need to compile anything, or type a single line of code. This is the beautify of Mathematica. I don't want to get into Mathematica and our implementation; however I want to mentioned that all you need to know to run our program is a shift+enter and that will do the trick. We have also worked on improving run-time speed of our simulation and I am happy to say that version 2.0 is about 5x faster than version 1.0!

Going back to our interface, there are three different tabs on the top: Input, Visualization and About. Input tap includes 2 sub-tabs: Simulation parameters and Rule Constants. Under simulation parameters, we have different parameters to set before running the simulation. To get the user started, each parameter is set to a default value. User is able to modify each value before running the program. We will go over each parameter on our next post. For now lets focus on interface. We have different parameters for the simulation in general (population size and simulation step). Cytoplasm, Periplasmic space and environment also get their own list of parameters. At bottom of the interface we have a check box which allows user to introduce division into the mix. So there are two different way of running the simulation. When division check box is not selected, starting with 20 individual cells, the simulation ends with 20 cells and outputs a set of results. However when division checkbox is selected, user might start with 20 cells and based on how many steps is specified by the user, the system might end up with 40 or 80 cells. Results of the simulation also reflect the division. Next blow the checkbox we have a button for running the simulation and then the current status of simulation is shown below ("Ready!"). In order to give user an idea about current status of the simulation we have added a progress bar which only appears when the program is running. This progress bar gives the user a status update on how long will the simulation take before completion. Here is a screenshot of a simulation in progress:

In this example the simulation is 69% completed. Next is looking at rule constants. Each rule in our simulation has a constant associated with it. This constant is used for calculating probability of a reaction. The system is loaded with default values for each constant. However one can modify each value before running the simualtion. Here is an screen shot of rule constants:

Because it is hard to remember which constant belongs to which rule, we have enabled a tool-tip option so when a user moves the mouse pointer over each constant, its corresponding interaction rule is displayed (as shown above). This should help out the user in associating each constant value with an interaction rule in the system.

We are going to skip visualization section for now (still under development). Lets go to About tab.

Here under about tab we plan to include copy rights, information about developers and other related information. There is also another aspect of our modeling project which is on hold for now due to time constraints. That is, ability of exporting results. Lets take a look at what we are planning to have maybe by end of this year:

Yes, we are planning to integrate the ability of exporting results from the simulation to HTML (for posting online), SBML (Systems Biology Markup Language - for visualizing metabolic pathways and importing to popular biological modeling tools such as Cell Designer) and PDF (for sharing as well as printing). This is part of our future work for expanding the model and making it even more useful.

If you have been wondering about results, well our model is also able to produce nice and clean graph visualizations. Lets take a look at sample AI2 concentration over 1000 steps:

I am gong to present only this graph for our result section just to demonstrate that our model is able to not only simulate by also analyze the results and visualize them.

Ok. This should do it for now. Consider this post as a sneak preview of what hopefully will be a complete Quorum Sensing Model. We hope to have our completed model posted on UCalgary wiki by end of september.

Thanks for following our progress!

Afshin

Hiatus...Or not.

As summer winds to a close, and fall classes loom closer and closer, we recognize that it will be difficult for us to post by our regular schedule (as in, a greater deviation than usual).

However, rest assured that updates from all subteams will still be completed on a regular basis! Right, guys?

:D Enjoy the rest of the summer, and see you in September!

-Mandy

Labbing it up!

Hello virtual world of synthetic biologists, engineers and the rest of the cool kids floating around in cyber space!

Jamie is back with a short and sweet update from the lab side of things.

This past week Emily (with a little Vicki-probably about 0.1 of her to be exact) alongside with Kevin proceeded to test the reporter circuit with the mutant circuits. This involved a quick plasmid switch of the mutant Vicki, I mean OD47A into psB1AC3 simply due to antibiotic resistance and selection pressure. Overnights were grown and tested in the plate reader Synergy HT.

Jeremy and myself (Jeremy did most if not all of the pipetting; I might have plated one or two transformations here and there) just finished up the signalling pathway in pCS26. Sequencing results were analyzed with tools available over at ncbi.nlm.nih.gov (i.e. BLAST) and the signalling cascade is now officially completed with glycerol stocks, plasmid DNA and restreaks ready to go!

I (sort of) lied when I said the luxPQ-luxOU construct was done. Just a tad. Carol's project involving the synthetic sigma70 promoter is currently on hold as not only is Carol off in Vancouver on a well deserved holiday but new primers need to be made with the degenerate bases. We hope to resume progress on this after next week.

Meanwhile, I myself am fiddling around with mineral oil, plate reader, 96 well microplates,Vibrio harveyiSalmonella typhimurium, Escherichia coli, centrifuges in hopes of isolating AI-2. Please note this is all done without the handy dandiness of multi-channel pipettes or access to a VICTOR; nevertheless learning to pipette effectively and how to calibrate a different model of plate is useful and exciting!

Without further ado, on behalf of the lab team I will like to sign off (not permanently though ;) - can't get rid of us THAT easily) for the summer. Thank you everyone for following us throughout the sunny (and rainy) summer days and I look forward to blogging again once the school year rolls around. So until next next week a.k.a. September, see you later :D!

FM 707.1 Marketing Update for the Week of August 17th 2009

Hello everyone,
My name is Fahd Mirza and you are reading my FM 707.1 marketing update for the week of August 17th. This week was rather slow for marketing because our marketing team was either busy with lab or ethics work. However we still managed to get a lot of stuff done for marketing,
1) Last Thursday, we were interviewed by the CJSW 90.9 Radio station. CJSW is the University of Calgary Community Radio station whose aim is to promote the activities of the University of Calgary students in Calgary and surrounding areas. We were interviewed by Joe Burima, who is the current program director for CJSW. On behalf of the University of Calgary iGEM team, we would like to sincerely thank CJSW for their support to iGEM and hope that they would continue to support our team in the future.
2) This week, Prima and I continued to contact Syn-Bio and Oil & Gas Companies for potential sponsorship/partnership. We hope to wrap up the sponsorship agenda by the end of August.
3) The biggest news of this week is that we have surpassed the amount of funds that we needed for this year. All the funds generated from now on will be contributed towards our next year’s iGEM fund for recruitment and other purposes.
4) We have also been working on our August newsletter which will be out soon.

WHAT’S NEXT?
We have also been working on our August newsletter which will be out soon. Also Prima has contacted the company that will be responsible for making our University of Calgary 2009 iGEM team shirts. We will also be working on attracting the media’s attention for our aGEM and iGEM jamborees.

Letter from one island to another

Dear Mandy,

Due to finals early this week and you having left for Hawaii, second life team members have been sparse. I wanted to stay in touch and keep you up to date somehow.

As you are aware, since scripting and equipment in the lab has been completed, last week we began the base of the Biobrick spiral where some of the fundamentals of molecular biology, including the central dogma will be located. You will be happy to know I have completed the DNA replication animation that avatars will be able to click on in order to learn what is required to happen for successful replication as well as a initiation point/button to begin the activity as well as reset it. All that is left is make it user friendly since everything that can be touched will perform some action and these actions just need to be explained and secured so that order is important. This may also be done with the help of a notecard that has been made. The only reason this went so slowly is the fact that I am attempting to complete wiki notebook updates in parallel and believe me, it has been a painful experience decrypting the notes that I have. Now, transcription and translation will have to be added and will most likely be tackled by either you or myself in the near future. I have started the initial framework for transcription, but most likely will not be able to complete it before this week is finished.


As I understand it, the biobrick simulator is basically completed and now the levels have to be organized and the textures for the buttons of the biobrick simulator interface. Thank you for completing the buttons Patrick required for the HUD he has made. They look great and I’m sure he really appreciates it.


I see the area avatars first enter the island has been expanded by you and Stefan and now includes a map (including teleportation areas) and directive tubing into synthetic kingdom. To ignore the big red X’s and arrows would have to be intentional and pathway stones litter the landscape throughout. Stefan has been working on his eukaryotic cell and finishing up his disease hunting bacteria within it. Also, I would like to thank you for allocating an area for previous iGEM projects and I see that the logo now decorates the outer walls of the virtual lab. A section of the island will now be filled with worthy iGEM projects, which may be done in the fall with enough linden dollars.


Hoping you are able to locate a bobtail squid,

Katie

Sensitivity analysis: Models... have feelings too!

Vicki and Chinee has put quite an amout of time into tracking down those sensitive ones in our system. Vicki is currently working with the Sensitivity analysis tool in Matlab commandline in order to find the sensitive parameters that influence the GFP output the most. The following graph is the result her sensitivity analysis model has generated so far: (click to enlarge)



The closer the graph gets to 0, the lower the influence this parameter has on the GFP output, and vice versa. According to this graph, the kForward (reaction rates)values 1, 3, and 4 seems the most influential parameters relative to 2 and 5; thus these parameters will be looked at further.

From these results, Vicki performed parameter optimization on them. In parameter optimization function, she was able to play with the individual parameter values in order to come up with a best fit line model to the predicted levels of GFP output. The following is the graphical result of the above function.



Here, the circles represent the made up predicted data, and the colored lines represent each optimized parameters. The optimized rate constants for kForward1, 3, and 4 fit nicely to the pattern of the predicted data; however, the optimized rate constants for kForward 2 and 5 do not, meaning that these parameters do not play a big role in determining the GFP output values.

From these two functions, Vicki would be able to, once we get some lab results, optimize our significant rate constants to fit the behavior of our AI-2 system.

Along with Vicki, Chinee was also working on the parameter sensitivity; however, with a different tool. In Simbiology, Chinee was able to outline the key difference between the initial amount and the reaction rates of the parameter. The following is the graph when all the parameters have a reaction rate value of 1:



The following is the graph when all the parameters have a reaction rate value of 10:



The only difference between the above two graphs are that the lines in the second graph are much more steep than the lines in the first graph. This means that the reaction rate only influences how immediate the reactions happen.

And finally, the graph when all the parameters have an initial amount of 10:



The above graph is very different from the ones above. From this, one can see how initial amounts of parameter play a much bigger role than the rate constants of each parameters.

I, Kevin, and Carol attempted to fix some biological misunderstandings within the reactions, and produce a graph that displays the pattern of each parameters. The following is the graph:



The patterns observed are reasonable and match what was expected. The lab data is needed to get an accurate model of our system; however, from it we can still test the sensitivity of each parameters by changing them one by one and simulating the results.

We hope to get some lab data by this week.

FM 707.1 Marketing Update for the Weeks of August 3rd 2009 and August 10th 2009

Hi everyone,
My name is Fahd Mirza and you are reading the FM 707.1 Marketing Update for the week of August 3rd 2009 and August 10th 2009. These past two weeks have been intensely busy and highly productive for our marketing team.
Last week, we had organized a massive bake sale having more than 400 baked items to sell. This event managed to raise a net profit of $500.04 Cdn Dollars. All the funds would be directed towards our research project. We had also managed to get sponsorships from Corning Life Sciences (Thanks to Prima Moinul) and VWR Scientific (Courtesy Fahd Mirza) last week. Our July newsletter was also ready to be sent out to potential/current/future sponsors and to media organizations who gave us media coverage. I want to thank Jamie Feng and Prima Moinul for doing a marvelous job on the newsletter.
Nexen Inc. was declared our Sponsor of the Month for the month of August. It is with Nexen Inc.’s generous donation that iGEM Calgary would be able to achieve its goals.
This week, our iGEM Calgary team toured the Oil Sands in Fort McMurray courtesy of Andrew Hessel and OSLI (Oil Sands Leadership Initiative). The tour was a great learning experience and an exciting opportunity for iGEM Calgary team to work on future potential synthetic biology projects for iGEM. It was also an opportunity for our marketing team to form important partnerships with the Oil and Gas Development companies.
We are still working on getting more sponsorship for our research project and I hope that we are successful in this endeavor. Within the next couple of weeks our marketing team will be working on our August newsletter and getting media coverage for our project, the All Alberta iGEM Jamboree (also called aGEM) and the iGEM competition itself at MIT.

CLOSE UP of iGEM Calgary's LAB

Hey guys! Summer is coming to a close pretty quick here, and so is iGEM! We are working tremendously hard to try and bring our project to completion! Here’s where we stand as of today (August 12, 2009):

Signaling Circuit

LuxPQ-B0015-R0040-LuxOU-B0015 has been successfully verified in psB1AC3. But that’s old news. We have recently cloned this construct into the pCS26 vector, whose cloning site originally had LuxCDABE flanked by NotI sites. We cut our signaling construct with NotI enzyme, but this does not guarantee that our construct was cloned in the appropriate direction. We transformed the product into TOP10 and XL Gold competent cells, and grew them on Kanamycin plates (pCS26 has resistance for this antibiotic). We have isolated plasmid from eight colonies, and we will verify whether the direction of our construct is correct with a primer that anneals just outside the cloning site on the supposed LuxPQ side. This primer will be paired with a LuxPQ Reverse primer. If we see a PCR product of just over 4kb, we have cloned the construct in the right direction, if we get smearing, however, we have not cloned the product in the right direction.

Moving forward, we require the sigma 70 promoter library that will control the expression levels of LuxPQ in our system. We have had trouble cloning this into the pCS26 vector, and will be trying this again from the start.

Reporter Circuit and Mutants

Pqrr4-B0034-GFP has been successfully verified in psB2K3. Both mutants have been verified in the following constructs in psB1AC3: R0040-B0034-LuxO D47A-B0015 and R0040-B0034-LuxO D47E-B0015. The Pqrr4-B0034-GFP is currently in TOP10 cells, and being made competent once again in order to allow the subsequent transformation of the mutants into the cells. The purpose here is to test the mutant circuits to see if they are working.

Acquiring AI-2

The purpose of constructing all these circuits is to make an AI-2 signaling system. So what else do we need? AI-2! We are currently isolating AI-2 from a species of Salmonella by centrifuging and taking the supernatant that should contain the AI-2. Many controls will be used in order to test whether AI-2 is actually present in the supernatant, namely using Vibrio Harveyi as a response which should glow in the presence of AI-2 because it naturally has the AI-2 signaling system. Once verified that we have AI-2, it can then be used to see if our system responds to AI-2.

Crunch Time in Second Life

Hello again! This week has been another busy week for us Second Lifers. We are wrapping up a lot of our work for the summer. Patrick's update about the biobricker from last week was awesome, and this week I'll be describing what we've been up to in the Synthetic Kingdom and the Labs.





In the Synthetic Kingdom (where we show potential applications of synthetic biology), we are completing the pathway through the levels, and the descriptions and instructions for the stations. We will also be completing descriptions of free moving bacteria that can be interacted with, which visitors can try to look for them while following the path, creating a dynamic environment that still provides guidance for users. This area will also be expanded to include descriptions of past iGEM projects in an "iGEM Hall of Fame". The pathway through the kingdom is complete, as well as the individual stationed activities and the drop in point. Now we are just building on it to make it clear what sort of things we want users to learn as they travel through the kingdom.






All of the lab scripts are complete, and all of the lab activity parts have been duplicated and modified for the second lab. We are still working on the instructions and descriptions for each of these activities. Now that the lab missions are fully functional, we've also created prizes to be given for successful completion of the lab , as an incentive. As the lab missions get successively more difficult, the awesomeness of the prizes increases. :)

Since the lab component is mainly complete, we have begun working on base of the biobricker helix, where we will be putting up exhibits and other objects that will explain some basics: what are genes, how gene expression can be modified, replication, transcription, translation, and some important parts of iGEM (the registry, how biobricking works, etc.).

We're getting nearer to the end of summer, so now we're trying to complete as much as we can in hopes of having most (if not all) of the island ready for people to explore.

Modeling (Membrane Computing) Update

Hi Everyone!

This will be our first update on Membrane Computing (MC) and agent-based approach to model our complex biological system.

In regards to modeling of Autoinducer-II Quorum Signaling, we have been able to successfully complete our implementation of this signaling cascade. Monitoring each cell as an agent, we are able to monitor the system at both, cell level and population level.

Currently our simulation uses a well known algorithm called Gillespie's Algorithm. In simple words, the Gillespie algorithm keeps stochasticity (randomness) in our simulation. Why is it important to have this randomness in our model? Because this will push our model one step closer to actual biological system. Because cells don't interact with each other or their environment at discrete steps. As an example consider protein-protein interactions versus cell division. These two interactions have quite different time scales. Our implementation of Gillespie Algorithm takes care of accounting for such cases.

At this stage our model is able to produce concentration graphs and allows user to review how the system behaves at the cell level and also at colony level. We are working on implementing powerful visualizations and a user-friendly interface for our model.

We are also working on addition of new updating scheme for cells during simulation. Currently our algorithm updates state of each cell at single steps. It is worth mentioning that because of the way our Gillespie Algorithm is setup, time delays between each step can be as short as 0.0001 second (considered instantaneous) or a given step could take as long as one minute. This will compensate for different interactions and the time it takes for each interaction to complete. The second scheme which we are working now to complete hopefully before aGEM is where all cells in the system are updated at the same time. This allows cells to be in sync when applying interactions. With single-step updating scheme, as the number of cells increase over time, it takes longer and longer for cells to update their state based on changes in their environment. This side effect of non-synchronous updating scheme only kicks in when we have high population of cells. Since we have introduced cell division to our simulation where our system could start with one cell and end up with 300, a synchronous updating system could take us one step closer to how the actual biological system works.

We will post updates at least once a week on our current progress; We also hope to have some screen shots on our visualizations and interface in near future.

Thanks for following our progress :)

Second Life: Belated Update!

Hello again,

This will be a shorter update than the last few weeks, mostly because the SL team is hard at work. Our iGEM season ends alarmingly soon, on the 21st of August, so we're in crunch mode now to get the sim polished and ready for public consumption.

I will tell you what I've been up to toward the end of last week and the beginning of this one: the Biobrick builder (aka the Biobricker). This is one of the most important features of the Biobrick Simulator in SL, because it turns the very static devices seen in the video last week into plastic and redesignable systems. The interface for the Biobricker is finished, which enables the user to piece various promoters, coding sequences, terminators, and other Biobrick parts together. You can build a device of any length, insert and delete parts anywhere in the Biobrick too. The system isn't only critical for the final package, it will be very useful for testing the other new features that are yet to come. Clicking around the interface already feels nice and responsive, and very powerful!

The system is designed to be expandable, so while the initial selection of parts will be small it will be easy for myself or another intrepid SL coder to add additional elements. Did I mention that all of the components we're designing this summer will be made available for all to use in Second Life? We saw no point in restricting the use of the work we've done this summer, and I'd love to see what people can come up with.

The interface was finished up last week, but I have just finished the Linden Scripting Language code (that's Second Life's built in scripting language) to accomplish the construction of these Biobricks in world today. The Biobricker needs so pass a few more trace-throughs and a lot of testing before I certify this part of the project complete though.

A simplified view of what's going on behind the scenes, is that after using the Biobricker's interface to design the device you want, the interface object directs the assembly of a number of Biobrick objects in the real world. It creates each Biobrick part, gives it a name and position within the completed device, and orders all the DNA to link together into the final device. Guaranteeing that each part gets the information it needs is harder than it sounds though, especially given the nature of communications in Second Life! It's impossible to guarantee messages will arrive in the order they were sent, or that they will even arrive at all, so the construction system has a lot of fail-safes to ensure that your device comes out right every time.

That's all for this week, stay tuned!

Lab: Some Progress Finally!!!

Hi Everyone! Thanks for reading our lab blog once again! Just a quick lab update for all you readers this week. Emily has finally completed her mutant circuit for
luxOD47E. Congrats to her! Jeremy and Jamie are currently working on several things right now. First, they finally performed a plasmid switch from pSB1AK3 to pSB1AC3 for luxPQOU. Now that they have successfully switched plasmids, they will be constructing luxPQOU into pCS26 (surette vector) by cutting with NotI enzyme. Finally, they are working on verifying cllamda. Unfortunately, after many enzyme digestions, they are unable to verify that the sequence is in the vector. They are currently trying again and hopefully will get results later this week. Kevin is working on the reporter circuit and he is verifying that circuit today with restriction enzymes. He is going to look into how to test the mutant circuits this week as well. Finally I am still stuck with the sigma 70 promoter. Unfortunately, I am still unable to get any colonies. We are currently looking at other ways to optimize the results. Hopefully, I'll have better results to report next week! Thanks for reading!

Modelling: Collaborative Work

Hi Everyone! Not much to update for modelling this week. We had a big meeting with all the teams on friday, and the two modelling teams (Membrane Computing and Mathematical Modelling) will meet every tuesday and thursday for meetings. This will give us time to draw parallel between the two types of modelling. We have also discussed about how to seperate all the experiments to characterize our signalling system. All this detail will be touched tomorrow at the meeting. Other than that, not much to update. Hopefully next week, we have more to add! Peace :)

P.M back in da House!!

Hey guys,

I’m Prima and I’m back on the show again!! This has been a very successful week for iGEM Calgary. I’d like to specially thank VWR and Corning Life Sciences for sponsoring our project. Both of these generous contributors have been recognized under the Bronze category of our sponsors.

The marketing team also finished, edited and began to distribute the July newsletter to all companies. This newsletter highlights the achievements and progress of iGEM Calgary for the month of July. Aside from that, Jamie, Fahd, Jeremy and I are slaving away with our hard core marketing responsibilities! We followed up with numerous companies who are considering our sponsorship package, set up meetings and thanked all other companies for considering our project.

We have an exciting week coming up as the team prepares for its next ultimate fundraising project: iGEM Bake sale trinity! This time we’ll come prepared with double the treats, triple the advertisements and of course 15 talented iGEMers to make Wednesday August 5th a once in a life time event!! Come devour the ambrosia!!! We have high expectations for next week and if all else goes well, iGEM Calgary will celebrate with a end of the year team get together. That’s about it! Peace out!

Blogging about writing...in the lab part of the project

Welcome Jamie to the guest blogger position once again! Fun times.

So I plan on doing this post in 216 words or less because I need to save my daily typing quota for my paper ( =( sorry guys).

Anyways, Carol is still tanking along the path towards constructing the variable promoter library.

Vicki is chu chu-ing on the rails as she works hard on her paper. Emily too. They're both chu chu-ing. Except Emily might be going in the opposite direction as her spatial orientation seems a bit off (see non-existent post about B0015-J13002-luxOD47E construct). But nonetheless we are moving along! Just like Theodore.Thank you Emily for the correction. Vicki and Emily are like Thomas the Train, not Theodore the tugboat. My apologies for any confusion, pain and injuries resulting from this mistake.

Jeremy is errr... driving (?? I ran out of cool references to transportation) along, working on his paper and finishing up the signalling circuit.

Kevin is steaming away (he is a steamboat :O) as he figures out his reporter circuit and works with his rainbow colored assortment of fluorescent proteins.

Myself? I am sitting. At home. Writing my paper. First complete draft to be done in about 2 hours (yes it took me over 4 weeks to write it...but I was not working on it the ENTIRE time. Just 99.9% of it.)

In continuation of my last blog, I do not enjoy extracurriculars. Just being cool. It's really time consuming.

EDIT: Hi Jamie. I would just like to let you and our readers know that I finished my circuit today, my ethics paper draft, and my notebook updates. I hope everyone's had a productive time (in lab) today :)

<3 from Emily

Patrick and the Biobrick Simulator

Modeling: Learning how to write

Hi everyone, its Carol again!
This week for modeling, we focused mainly on writing up several ways to characterize the AI-2 signaling system. The characterization methods that we decided to explore are the following:
1. Static Performance
2. Dynamic Response
3. Response Time
4. Robustness
The robustness part of characterizing the system would be the most interesting. The degree of robustness depends on how sensitive the system is to fluctuations and changes. If this part of the signaling system can be explored in more detail, it will be an advantageous component to our project. We spent the past week summarizing this in paper format. I am currently working on a promoter library, which will play a huge role in exploring the expression of luxPQ. Once the circuits are done, some of these experiments can be done in a few days. We will hopefully by the end this summer have some characterization done. Stay tuned! Signing off now, peace!

SynthETHICS

Hello, Stefan here. Second Lifer extraordinaire and now official iGEM ethicist. You might be wondering what’s been going on in our Ethics department. Well, you’re in luck, because I’m about to tell you! Mandy, Emily, Fahd and I have been keeping busy by reading a few papers on synthetic biology. These include papers about the economics of synthetic biology, bioterrorism, and general ethical issues regarding the field. The ideas expressed in these papers will be used to elucidate both sides of ethical debates surrounding our project and to present evidence for our statements. For our project, the issues that we will look at will be: Environmental, Ethical, Economical, Legal, and Societal (E3LS). We will also take into account pre-cautionary perspectives (things are bad until considered safe) and pro-actionary perspectives (good until proven otherwise). Our paper will involve two (or more) applications of our signaling system that will be evaluated through E3LS. Can’t wait to get working on it!

In other news… We are thinking of holding an ethics conference in Second Life. We plan to invite many guests to share their thoughts as well as other people involved in iGEM. However, this probably be done in late September. Thanks for reading, and I’ll see you in future updates.

Jon: The Brave High School Kid Who Helped Big Kids Out

Update from Circuit World

Hi, this is Emily again! I'm part of the wetlab portion of our team and today I'm going to be updating you on the progress we've made over the past week or so. I'll start with the signalling circuit where Jeremy, Jamie and Carol have been concentrating their efforts.

This week, Carol has been working on our promoter library which Jeremy touched on last week. Essentially Carol is trying to find a promoter that provides the best expression of Luciferase so that we can find the optimal expression of LuxPQ. We don't know the optimal expression of PQ however as more epression of LuxPQ does not necessarily mean more expression of Lucierase. This is therfore the purpose of the Promoter Loibrary, to find the optimal expression of LuxPQ. To do this, Carol has set up PCR reactions with 16 forward primers and 16 reverse primers. She then digested the PCR products and the Surette vector with ZhoI and BamHI enzymes and is currently in the process of ligating them in order to trasnform them into competant cells. She has tried three different types of Ligation, QuikLigse, T4 NEB ligase and a week long ligase. The first two did not produce cells that glow (that express Luciferase) and there were not many cells that grew. The week long ligation will be done tomorrow and we are hoping that the results will be promising!

Last week Jamie and Jeremy finished the signalling circuit with LuxOU and LuxPQ. This week they have been working on a plasmid switch. Thw signalling circuit was contructed in an AK vector, however we need to now move it into a vector with K resitance (the surrette vector). Because moving something from AK to K does not offer any antibiotic selection pressure, we need to first do a plasmid switch to get it into an AC vector, and then we can move it into the Surrette vector.

Next we have the reporter circuit which Kevin has been working on. This week Kevin has been making a constrcut with pqrr4, RBS and GFP in order to test the functioning of the Signalling circuit. Kevin has been working on this construction this week and is waiting for

Finally we have our two Mutant circuits: LuxOD47E and LuxOD47A, which are being worked on by Emily and Vicki respectively. As of last week, Vicki had seccessfully completed and sequnced her circuit. This week she has been working on her pro paper that, with some additons and changes is well on its way to being done. Unfortunately, LuxOD47E and I are not having so much fun and I am pretty much at the same place that I was last week, trying to get the B0015 terminator in. This week's attempt seems much more promising as yesterday I did a verificatioon digest with goiod results and have since sent off a colony for sequncing. I have my fingers crossed that B0015 is in there and my circuit will finally be done.

Well that's about all for this week. We still have a fair ways to go in the lab, so hopefully we have more successes to report next week!

Scripting In the Virtual Lab

iGEM's next top model?

Hey there! So I’m a little late with this entry…hopefully none of you are too crushed by this. As a refresher, I’m Vicki, and we’ve already met repeatedly over lab editions. With my fabulous team of brilliance – Carol, Chinee, Kevin, Afshin and Iman – we’re going to give the term “America’s next top model” a whole new meaning.

It has been a very busy week of searching for reaction constants for our differential and membrane-computing efforts. Indeed, the literature is vast, but it sure isn’t easy to navigate. And it doesn’t help when published authors don’t really seem to make effective communication a priority. Regardless, we’re progressing, slowly but surely.

We have also been discussing how we’re going to approach the circuit characterisation matter. After a long and fruitful chat on what others have done previously to characterise their signalling circuits, we came up with a list of areas for which quantitative characterisation would be useful AND realistic to achieve. Because as much as we want to pay for sequencing every day to assess circuit stability, it doesn’t seem like the most effective use of our funds, especially with so other areas (read: ice cream parties) where the money would be better spent! We also outlined general procedure approaches that would enable us to collect the data to come up with and validate our models. Stay tuned!

FM707.1 July 16th 2009

Why Annotations are Useful

Under the Sea

NOW WITH WORKING SOUND!

Techy Biologist

iGEM Calgary on CTV News

P.M. In Da House

Carol Chan Battles LuxCDABE

Hi everyone, it’s Carol again! I won’t re-introduce myself again since I wrote the modelling blog a few days ago. I don’t have much to report since I’ve been having bad luck in the lab lately, actually from the start! I am working with Kevin (the nice individual) to construct the reporter circuit for the project; however, due to my lack of lab skills, I’m delaying the whole project and leaving Kevin with nothing to do! I’m just kidding. I spent a few weeks trying to concentrate DNA plasmids for LuxCDABE sequence in topo vector. After many failed maxi-prep and many mini-preps (with the help of my favourite lab equipment, the vacifuge), I was able to concentrate my sample. As well, before Biobrick construction, I was left with a difficult task of single site mutagenesis. For some reason (with my luck) after one trial I was able to mutate a specific site within the LuxCDABE gene. This past week, I was unable to successfully clone and transform the LuxCDABE into the Biobrick vector. The lab team are trying to think of other ‘innovative’ ways to get this large gene cloned into Biobrick vector. Hopefully, next week, I’ll be able to show some better results, as of now, only time will tell! Talk to you guys on Monday!

Kevin Loves Rainbows

I, Kevin Shin, being a nice individual, am also involved in wet lab part of our team. Carol and I are responsible for our Reporter circuit, which involves Qrr4 promoter and LuxCDABE gene. As of now, I have completed verifying and glycerol stocking last year’s Pqrr4 part, and am waiting for LuxCDABE part (which is much longer, meaning harder to work with) to be done.

While waiting, I am conducting an exciting side project involving a variety of FLUORESCENT proteins!!(and dry ice...) Woot! Although I am really disappointed at not being able to gain access to purple, orange, and blue fluorescent proteins, we have secured a supply of red, green, yellow, and cyan for me to have fun with. Today, I will be transforming plasmids with these genes in them and by tomorrow, I should be able to draw some bacterial pictures! We will keep you posted on those glowing masterpieces.

How Negative Controls Became Positive

Hi, I'm Emily and I’m going into second year Biomedical Sciences. When I’m not fighting with my LuxOD47E gene, I’m usually highland dancing or playing the Oboe. In the lab, I’m in charge of the mutant LuxOD47E circuit This week I am excited to say that sequencing has confirmed that I've finally biobricked my gene of interest, LuxOD47E. Yay! This has taken a lot longer than anticipated due to several negative control contamination issues (negative controls are not my friends) and a battle with strange reappearing bands in restriction digest and PCR products. Nevertheless, this gene is now biobricked and it is on to the construction of my circuit! This week I will be trying to get the J13002 promoter in front of LuxOD47E as well as the BOO15 terminator behind. I’ll be doing this through restriction digest with EcoRI, XbaI and SpeI followed by an awful lot of verification. Maybe, just maybe I’ll see some clean negative controls! If this is successful, my circuit should be completed and sequenced by Monday!

Jeremy Kubik Wrestles With LuxPQ LIVE!

My name is Jeremy Kubik and I am part of iGEM Calgary’s wet lab and marketing team. This is my first time participating in iGEM and I am having a great time working with the other students on this awesome team. Outside of the lab, I enjoy competitive sports, all the way from swimming with the Varsity team at the U of C to ripping it up on the streets of Calgary for a little hockey action. I also enjoy long walks on the beach, sunrises and fine wines.

Within the lab, I am taking care of part of the signaling circuit. Our overall project is to create a Quorum Sensing (QS) system with the signaling molecule autoinducer-II (AI-2) as the input to the system. My collaboration with Jamie will eventually lead to the construction of a circuit with the genes LuxPQ and LuxOU, all of which code for important proteins with respect to the transmitting the AI-2 to induce a specified response (which was recently decided to be a protein output that degrades biofilms!). So where am I as of today? Stuck. Well, not really stuck, but I have had some difficulties with LuxPQ. The part is verified to be in the TOPO vector and to have a length of 3.8kb, and I have had quite some trouble simply cloning LuxPQ into a BioBrick Vector (psB1AC3). I performed a construction last week and finally got some colonies, of which I ran a colony PCR, isolated plasmid, and have sent plasmids of two colonies down to sequencing. I will find out in a few hours whether LuxPQ has been successfully cloned into the BioBrick vector. If it is, I can begin construction with Jamie’s circuit (LuxOU with promoter and terminators). If it has not been successfully cloned, it is back to the drawing board: trying different conditions to transfer LuxPQ from TOPO vector into a BioBrick vector.

Jamie + Synthetic Biology + Blogging = Jamie on Youtube

So I decided to be lazy and outdo everyone on my team by doing some snazzy Web 2.0 related thing: VIDEO BLOGGING!!!!!

Amazing stuff.

One downfall of video blogging is it is HARD to edit stuff out. :( And after reading everyone else's blog post and realizing I forgot to introduce myself OUTSIDE of iGEM (do I even exist???) and being to lazy to re-record anything I will include the following disclosure:

Jamie enjoys doing iGEM from 9-5 Monday to Fridays. Sometime 10-6, dependent on how late I sleep in :D

I will include my extracurriculars next week (I will have had enough time by then to think of at least two extracurriculars so I do not look like a complete loser).

Anyways, enjoy the video.

Building Circuits: A Few Successful Moves to the End, but too many Failures...

Hi! You heard from me last week in my third-person commentary and now I’m back in the first-person flesh. My name is Vicki and I recently graduated from the Engineering Science program at the University of Toronto, with a major in biomedical engineering. I grew up in Calgary and am very excited to help my hometown kick some synthetic biology butt. When I am not indulging in the satisfaction that only a properly-sequenced synthetic BioBricked circuit can provide, I am usually either swimming, skating, biking, learning German or a combination of any of the above (ever try riding a bicycle whilst on skates? It’s as perilous as it sounds!).

Over the last few weeks, I have been deeply entrenched in converting a mutant protein sequence in a TOPO plasmid into a fully functional and biobricked sequence in a pSB1AK3 plasmid, complete with the appropriate and properly-integrated promoter, RBS and terminator sequences. Indeed, it has been a most gruelling month of gradient PCRs, colony PCRs, plasmid isolations, restriction digests, ligations and – craziest of all – understanding what I’m doing and explaining it to the lab group in coherent sentences! Because even though the principles of synthetic biology and biobricking are supposed to make genetic engineering so easy that even an engineer like me can work with it, it’s really quite challenging when you cannot see what is really happening in terms of molecular interactions on the nano-scale and smaller. Although many parts of the project have flummoxed me, I am developing a better appreciation of what I am doing as I gain more experience in the lab.

So, here is my work to date.

Battle 1: Vicki vs gradient PCR of LuxOD47A in TOPO. The purpose of this step was to use biobrick gene-specific primers to make copies of a biobricked version of LuxOD47A.

Winner: Vicki

Battle 2: Vicki vs ligation of LuxOD47A into the pSB1AC3 plasmid. This is so that the gene sequence can be integrated into competent TOP 10 bacteria. Prefix-end cuts were made with EcoRI and XbaI (in separate samples).

Winner (VK vs EcoRI cuts): EcoRI cuts L. The restriction digests of these look like someone painted white-out in the 12 kb range just to spite me.

Winner (VK vs XbaI cuts): Vicki. The EcoRI enzyme sample that wasn’t up to par is spending its days in solitary confinement at room temperature. Indeed, things are so much cleaner when enzymes decide to cooperate.

Battle 3: Vicki vs the sequencing machine, part I. After a successful restriction digest of the XbaI-cut samples, I sent them down for confirmation that my PCR and restriction digest results weren’t just lying to me.

Winner: Vicki

Battle 4: Vicki vs integration of promoter and terminator sequences, attempt I. I tried both to see which would work best.

Winner (VK vs J13002 promoter): J13002 promoter L. That one didn’t integrate in any of the colonies that I PCR’d and RD’d

Winner (VK vs B0015 terminator): Vicki. We’ll move forward with a newly-made LuxOD47A-B0015 construct, while the attempted J13002-LuxOD47A samples can go roast in the autoclave.

Battle 5: Vicki vs the sequencing machine, part II. Yea, I know I skipped a lot of steps here. It wouldn’t tell you anything interesting beyond what you’ve already read. After good restriction digests and colony PCR results that would have been fine if not for a superfluous negative control band that didn’t show up anywhere else, we sequenced anyway. We had a scare when a simple [Crtl+F _ (B0015 sequence)] didn’t yield any results on the text file, but were reassured when we blasted the sequences against each other as that algorithm accounts for the reverse sequence given by the reverse primers that I used.

Winner: Vicki, with credit to BLAST

Battle 6: Vicki vs the integration of the nefarious promoter sequence. Lest the J13002 samples go the same way as our uncooperative EcoRI enzyme, we decided to attempt this integration thing one last time. Two colonies behaved appropriately, and the J-part lived to see another day.

Winner: Vicki

Battle 7: Vicki vs the sequencing machine, part III. Although I had to wait four days for my results, they were what we expected.

Winner: Vicki

This takes us to now. I need to make more of the biobricked sequence in question by letting it grow in bacteria, which I’ll do later today so that it can grow overnight. Results to come!

School of Video Games

Hello, my name is Patrick King, and I am one of four students at iGEM Calgary working on a very unique subproject this year: creating an educational environment for synthetic biology in Second Life.

Second Life is an online, virtual world, where every object, costume, creature, and patch of land is designed by the world's inhabitants. It is a 3D chat engine, a videogame platform, a virtual reality, and an online classroom all rolled into one. It's a fascinating place, an internet search or two will reveal the variety of creations and experiences to be had there. Access to Second Life is free, create a free account at their website, download the client, and you're ready to go.

iGEM is the International Genetically Engineered Machines competition. It is an annual contest of scientific skill between teams of undergraduates at universities around the world, to engineer the most useful and most interesting organisms around. I can't sum up all of iGEM in this space, but for the unfamiliar, the official introduction is a great place to start.

So what does iGEM have to do with Second Life? Well, the problem with doing an iGEM project at university is this: sooner or later all of the students graduate. Even those who decide to participate in an iGEM team several years in a row (those poor souls!) have to get on with their lives sooner or later. The result is that a huge amount of time is spent educating the new year of students, and this problem can be especially acute for teams of entirely undergraduates like ours. Many of our students are in their first or second year, many have never been in a molecular biology lab before, and they've got to learn all the ropes if they want to have a hope of competing at iGEM level.

Ultimately, all this time spent training the next generation will pay off (we hope!) with a generation of new scientists, the synthetic biologists. But for right now, we really just need to get all the cloning and biobrick assembly and system tests and a dozen other kinds of work done by the end of summer.

An educational area in Second Life offered by Nature Publishing Group

Enter Second Life. SL has gained a lot of attention for its potential as an educational platform. The most often touted feature is that SL can offer a classroom-like environment for people at any distance from one another. While the iGEM Calgary island will make an excellent hangout for idle igemmers the world over, our focus is less on creating a classroom, and more on presenting concepts directly. We want to make it easier for new students to grasp the basics of synthetic biology by making it accessible and interactive. This is where SL's object creation and scripting facilities come into play: we can create anything we want, from molecules to cells to lab equipment, and then make it behave like the real thing.

For example, take one of the Second Life subprojects: the Biobrick Simulator (provisional title). The goal of the simulator is to represent a number of commonly used biobrick types by simulating the actions of single molecules. In Second Life, you won't read about RNA polymerase in your textbook, or hear about it in lecture, or click buttons on a flash animation. You will grab an RNAP molecule floating in front of you, and bring it to the promoter region on the DNA also floating before you, and watch it create an mRNA and pop off at the terminator. Unless you drag the RNAP to some DNA that doesn't happen to be a promoter, or move it perhaps to a promoter with a repressor complex attached. Then it won't do anything at all. Simple!

The Biobrick Simulator is my pet project, I will be posting more about it in the future. I plan to have examples for the visitor drawn from the real world, including classical systems like the Lac operon and the Tetracycline resistance operon, and synthetic systems like the repressilator, and the bistable switch (and many, many other systems). The main event will be the ability to assemble your own genetic circuit from scratch, and see how it works!

You've already heard from Mandy a little about the virtual lab that is also under construction, next week we'll give an introduction to the third and final subproject: the Synthetic Domain (name also tentative!). It seems quite unlikely that SL will replace traditional instruction, but the power of learning by doing is enormous. Putting some smart students together in a lab with a few biological reagents is the basis of the iGEM competition, after all.

My number one goal for this project is for it to be useful to others, especially early university or high school students just beginning with iGEM, but also biology students in general, and the public. For it to be useful, it must be used; feedback on the accuracy of our work is essential! I hope that Lindsay Island will be open to the public near the end of the summer, but the real test will not come until iGEM
2010, when we will meet our first batch of fresh students.

Modelling: How Engineers Understand the Social Life of Bacteria...

Hi! My name is Carol and I’m heading into my third year of biomedical and chemical engineering in the fall, and I have completed a degree in biomedical sciences. I love watching sports (especially football and hockey) and I enjoy cooking and reading. I’m part of the laboratory and modelling team, so you’ll be hearing from me lots this summer!

In the past few weeks, the modelling team focused on familiarizing with Matlab and Simbiology. Before I move on, I should introduce you to my team mates that make this team possible! Vicki is a recent graduate from Engineering from the University of Toronto and she has worked with Matlab and its applications extensively throughout her studies. Chinee is also a third year chemical engineering and has some experience with Matlab as well. Finally, Kevin who is going into his second year of Kinesiology is just helping out because he is just a nice individual! With all the skills from each team member, we believe that we can produce a great model for our project. Back to modelling, one of our facilitators (Dr. Nygren) gave us an assignment that helped us understand the program more. We had to model the three gene repressilator in Matlab via two different methods, stochastic and deterministic. I’m going to spend some time now to explain the two methods that we will be using to model our quorum sensing model.

Differential (Deterministic) Model

This model uses equations that involve derivatives to illustrate concentrations of different molecules within a network. In our case, we will be using equations to describe the concentrations of different molecules within the Autoinducer-II (AI-2) cascade. With the ability to solve the differential equations, we can investigate how the concentration of different molecules within the cascade modifies compared with initial conditions. However, this method of modelling is often used with systems with high concentrations of chemicals and we expect that the importance of rare events is low. Furthermore, this type of modelling is often used in smaller networks.

Stochastic Models

The other type of model that can be used to describe our model is through probabilistic equations that can describe the probability that a certain chemical reaction will occur between certain types of molecules at any instant. Furthermore, these equations can also be used to calculate the quantities of all species at the end of a small time step. Therefore, it is plausible to evaluate how the molecules within the cascade change over time by repeating this process over many steps. Since random variable input is involved with this type of modelling, each simulation run can produce varying results. By using averages through numerous simulation runs, a trend can be predicted. This type of modelling is used for small numbers of molecules because they take more computing power. This is because of the random nature of molecular interaction and it accounts for the probability of rare events occurring.

Our goal for this week is to successfully build the AI-2 system in Simbiology. We’ve been busy the last few weeks reading up on literature and we were able to successfully find some phosphorylation rates that can be incorporated into our model. Next week, we will show you what we’ve built in Simbiology and give you a tour of Simbiology and Matlab! Stay Tuned!

Baking Alberta

Some of the culinary delights offered at our bakesale

Some musings from mutant-circuit-world

The last few days have been flavoured by a plethora of successes, not-quite-successes, and other incidences of interest and amusement.

THE GOOD: Jamie has usurped the throne of AI-2 Circuitland. With a complete and properly-sequenced LuxOU component of the circuit, complete with the necessary promoter and terminator sequences, Jamie gets to sit back, chillax and admire his work as he helps the rest of the lab group complete their work. Congrats, Jamie, and B0015-R0040-LuxOU-B0015 FTW!

THE BAD: Negative controls FTL! Emily and Vicki have both been plagued by unsightly bands in the negative control lane, despite using new and purportedly uncontaminated equipment in their PCRs and restriction digests every time. Of particular bamboozlement, Vicki’s negative control in her latest colony PCR had a band at 1kb that did not appear anywhere else in the gel. Nevertheless, the two will continue to move forward with their experiments. The sequencing results of Vicki’s LuxOD47A BBk circuit showed that she does indeed have the proper product present. And Emily is set to conquer the colony PCR, so hopefully we’ll see the awesome results of that at the end of the day.

THE UGLY: Vicki spat into a K-laced plate and placed it in the incubator to see what would happen. She was most disappointed to see that the plate was cleaner when she returned than when she first left it there. She’ll try again with a plate free of antibiotics next week. Results to come!

Lab Tours Express

New Beginnings

Welcome to Synthetic BLOGology!
This is the blog for the University of Calgary's iGEM Team. You can find us, and info about our project at:
iGEM Calgary

Second Life: The first day on our virtual island, May 27th 2009

Here, we will be posting updates for Modeling, Second Life, Wet Lab, Marketing, & Ethics/Outreach, so stay tuned!