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!