Team:Calgary/31 July 2009

From 2009.igem.org

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Transformation of R0040 (promoter) +pCS26 (vector) into XL Gold cells
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WIKI CODING HERE
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* From the overnight digest of R0040 with XhoI and BamHI, the promoter was ligated with vector, pCS26.
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* These plasmids were transformed into XL Gold cells and was plated on Kanamycin plates overnight.
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Team meeting:
 +
* Lab:discussed about promoter library and the failure to see the expression of luciferase. Also, discussed about the primer design of R0040, so that we can clone the constitutively ON promoter in front of luciferase. This will show us what the luciferase expression will look like.
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* Modelling: Discussed about Robustness. The three things that we decided to look at for robustness is: 1. PQ expression (the selection of the 'best' sigma 70 promoter) 2. Temperature Alterations (to see if changes in temperature (from 37<sup>o</sup>C to another temperature would affect expression) 3. Type of media (LB vs. LM)
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Sequencing of J13002-LuxOD47E-B0015 Construct in psB1AC3
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*Sent Colony 3 of my J13002-LuxOD47E-B0015 construct down for sequencing, results will come in on Monday hopefully.
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*Explored Secondlife and met with Rob from UChicago.
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*Prepared the Human Practices project write-up for the Wiki.
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*Team Meeting- Talked about the exploration of seminars in Second Life and what ideas that has given us.  Talked about the pending completion of the J13002-LuxOD47E-B0015 curcuit, results will come in on Monday.
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Marketing and Ethics for July 31st 2009
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WIKI CODING HERE
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Today I worked more on the ethcial aspect of our project. I read more papers on the environmental and economical issues of our team calgary project.
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I also made follow up calls to a couple of comapnies.
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More Educational Aspects: Rules Animations
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The next step to enrich the educational aspects of our model is to animate each rule. With doing so, biologists can make connection with the rules as well as the notations used to implement these rules. It is really important for us that make our model convenient to understand for biologists as they are the main users of it. that is why we have designed these animation so that biology and computer science could be connected.
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[[Image:Rules2.png]]
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That is what I have been working on this week.
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In addition we had our meeting, and here is a summary for modeling team:
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The team members involved in modelling need to meet more and learn more about what the others are doing. We will address this by meeting on Tuesdays and Thursdays at 10h00 am.
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On the differential side, we have a lot of breadth to our approach but it's still fairly superficial. We have established a differential reaction-based model on SimBiology, but finding values for phosphorylation constants has been a much greater challenge than we had anticipated. We will need to set up a model anyway and collect some input/output data, and from that we can approximate a set of parameters. It's highly unlikely that they'll be unique, but they will give us something to work with and help us evaluate which ones are limiting steps and would be worth tweaking in a real system
 +
The point of standardisation and characterisation is to have a set of parts to work with and choose from, based on characteristics that can be compared among different parts. Endy's 2006 paper is interesting, but it only presents characterisation for one part. It would be useful to have more parts characterised, especially if they actually work. We are submitting a few parts to the Registry: a signalling circuit, a reporter, mutants - all with various combinations of promoter and terminator sequences. It would be useful to characterise as many of them as possible, even if it's just for static and dynamic respones, so that future users have a quantifiable way of comparing performance.
 +
We are testing a signalling-reporter combination, but we don't know how much the reporter will affect our model of the signalling circuit. For example, if we consider response time, we don't know if our circuit response is limited by the signalling circuit, the reporter circuit, or a combination of both. If the circuit response is slowed mostly by the reporter circuit, it would cast doubt on the validity of the model as soon as a different reporter or sexy circuit were implemented. If there is a way to characterise the reporter by itself along with the signalling + reporter (and if we could do the same with the mutants, that would be interesting too), it would help us assess how good our model is for the signalling circuit by itself, and how it will perform when used with a different reporter or sexy circuit at the end.
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Verification of cl lambda inverter in psB2K3
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WIKI CODING HERE
 
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<ul><li>Plasmid miniprep of overnight cultures with Sigma Genelute Miniprep kit. Standard manufacturer protocol used; elution in 40&#181;L of ddH<sub>2</sub>O.</li>
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<li>Vacufuge to concentrate.</li>
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<li> Verification digest with <i>Xba</i>I and <i>Pst</i>I. Control: cl lambda part from the Registry distribution plate.</li>
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<li>Results: Faint bands were present.  Looked into vector characteristics and it turns out it is high-copy number with IPTG induction (Thanks Thane!)
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Isolation of cl lambda; Sequencing for LuxPQ-LuxOU construct
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Six overnight cultures of cl lambda colony 1 were prepared with LB-Kanamycin broth. Plasmid was then isolated using the Sigma GenElute MiniPrep kit. Two plasmids were not successfully isolated, but the other four tubes were pooled into two tubes and vacufuged to increase their concentration. A restriction digest with XbaI and PstI was then set up for 2 hours at 37ºC.
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The LuxPQ-B-R-LuxOU-B construct in psB1AC3 was sent for sequencing today based on verification techniques performed over the past few days. Primers used include BBK CP F/R and R0040 Reverse.
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Modifications to DNA Extraction, Restriction Digest, Instructions and Write-ups
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The movement of the restriction digest tubes have been modified so they are more interactive so the user is asked what piece of equipment is required once all required items have been added in the correct order to each tube. The three pieces of equipment to touch are the water bath, heating block and incubator (for the insert) respectively. I still am required to place my restriction digest instructions into a notecard on the lab table, which I will do next week.
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I have been able to modify the first five steps in DNA extraction so that the order in which a avatar proceeds is important. Now if the initial step of resuspension has not been accomplished, if you touch the lysis activity you will not be allowed to perform the activity. However, once a step has been unlocked an avatar may return to it at any time if they believe they missed something or just wish to redo a step. Once an avatar reaches the end of the activity a message is sent to all objects, which resets the entire activity. Now I will have to begin incorporating the containers that store: Wash Solution, High Salt Buffer, Isopropanol, and one containing a DNA buffer as well as one more centrifuge step.
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Some more instruction were added to bacterial transformation so there is a response for everything an avatar does in attempt to complete the activity. For example, if a user drops an item into the quiz’s inventory that is of the same type that has been submitted before it will alert them to the fact that they have answered that set of questions before and will not ask them again. I have been given the task of creating more products for this activity as well so I will have to add to my transformation script so that the plates with growth will give you the proper object to be used within the lab elsewhere.
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I also wrote an introduction to the virtual lab for our project description for the wiki, which generally explains the lab’s content as well as its purpose for the second life project.
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cPCR of the reporter circuit
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In order to verify cPCR, I have once again ran a cPCR of the reporter circuit, <i>Pqrr4</i>+B0034(RBS)+K082003(GFP+LVA) with <i>Pqrr4</i> Forward and Biobrick CP Reverse primers. Of course, pTaq was used to amplify the DNA. The product was then ran on 2% agarose gel at 90V. The following is a picture of the gel.<br><br>
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[[Image:Calgary_2009.07.31.reporter_cPCR.png|700px]]
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<br><br>
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As you can see, the bands in most of the colony lanes match the positive control of <i>Pqrr4</i>+B0034, which means that the cells contain those pieces, not the one of interest. It seems that only the colony 9 is between 1000bp and 1500bp, which is what was expected. However, even this band is suspicious. The size of the circuit is about 1043bp, and with about 125bp addition due to Biobrick CP reverse primer annealing outside of the circuit; however, the band on colony 9 lane is at about 1250bp. Another problem can be noticed in the negative control lane, which is another contamination. It is very disappointing to see a whole day of work gone to waste :(! Yet, it is too early to give up hope. I can try to isolate the plasmid of the Colony 9 and perform a Restriction digest on it to see if I still do not see the expected band, which is at this point unlikely.
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<b>Distribution of our baking sale posters</b><br><br>
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Fahd left a stack of Baking sale posters for us to post throughout the building, and they were posted before 11am today.
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Automagic Biobricks
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WIKI CODING HERE
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I spent most of today being very productive with the Biobricker portion of the Biobrick Simulator. This is a little user interface upgrade that will permit you to build any biobrick device you please, from a small library of parts implemented in Second Life. In SL terms, the UI is an object owned by the user's avatar, that can be attached to your display. The simulator's UI will have quite a few features to make your digital bioengineering easier, of which the Biobricker is an important one.
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I finished working on all of the controls for the biobricker, including some scroll buttons for the parts in the device under construction, and the ability to add parts from the UI's inventory, and delete them too. The Biobricker is all done except for one last control... the build button! This single button will be as much work as the rest of the Biobricker put together, though. The task is to create a copy of each individual Biobrick in world, inform each one where it belongs in the completed device, and link them all together. This will involve a lot of communication back and forth between the user interface object and the biobrick objects out in the world.
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 +
The Biobricker is in a very primitive state right now, with letters standing in for where all the pretty icons will be in the final version. So instead here's a screenshot of the Main Menu, with the link to the Biobricker visible.
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<center>[[Image:Calgary Biobrick Simulator UI Preview.png|400px]]</center>
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The UI fits in at the top left corner of the screen. The red letters are buttons, that cause the UI to be redrawn with a new menu for whichever module you've chosen. I hope this gives you an idea of how the UI fits into the overall simulator.
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Continuation of last day's work
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WIKI CODING HERE
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Today, I edited the high school presentation proposition again and sent it out to Mrs. Ellechuk. I filled in the lab notebook and filled out the daily updates on the wiki.
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I finished typing out the acknowledgement for each mentor/advisor of iGEM Calgary. I also helped to write references for Sonja. Then I finished off with mailing out more newsletters. We also had a very productive team meeting today.
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On Monday I have to follow up with the companies which I have been contacting for the past few days. I will also call our hotel in Boston to change a few rooms around.
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Continuing along the path
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WIKI CODING HERE
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Construction of the path is almost complete and finals plans were mapped out but are subject to change to make room for future virtual demonstrations. In order to complete each station I've decided that a Hawaiian Bobtail Squid will be added to each that will tell visitors about what they see. It will preferably be very interactive (ie. move around and talk to you rather than give a notecard) so as to be fun and educational for the junior high and high school audience we are targeting. The squid that was already in the synthetic kingdom was slightly modified in order to match the physical characteristics of the Hawaiian Bobtail Squid. 
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Discussion write-up, referencing and wiki posting
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(This isn't terribly exciting. I continued with the discussion section of the technical report, I learned all about APA referencing, and I posted part of my notebook on the wiki).
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Lab meeting
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We discussed characterisation elements for our signalling circuit, as described in Carol's section. I focussed on dynamic performance and response time, or, in other words, what happens when you add a lot of AI-2 into a system where it wasn't there previously.
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We also discussed how we could consolidate our modelling efforts and come up with something with a little depth to it in time for the competition. Some of the main points that came out of that are:
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*The team members involved in modelling need to meet more and learn more about what the others are doing. We will address this by meeting on Tuesdays and Thursdays at 10h00 am.
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*On the differential side, we have a lot of breadth to our approach but it's still fairly superficial. We have established a differential reaction-based model on SimBiology, but finding values for phosphorylation constants has been a much greater challenge than we had anticipated. We will need to set up a model anyway and collect some input/output data, and from that we can approximate a set of parameters. It's highly unlikely that they'll be unique, but they will give us something to work with and help us evaluate which ones are limiting steps and would be worth tweaking in a real system
 +
*The point of standardisation and characterisation is to have a set of parts to work with and choose from, based on characteristics that can be compared among different parts. Endy's 2006 paper is interesting, but it only presents characterisation for one part. It would be useful to have more parts characterised, especially if they actually work. We are submitting a few parts to the Registry: a signalling circuit, a reporter, mutants - all with various combinations of promoter and terminator sequences. It would be useful to characterise as many of them as possible, even if it's just for static and dynamic respones, so that future users have a quantifiable way of comparing performance.
 +
*We are testing a signalling-reporter combination, but we don't know how much the reporter will affect our model of the signalling circuit. For example, if we consider response time, we don't know if our circuit response is limited by the signalling circuit, the reporter circuit, or a combination of both. If the circuit response is slowed mostly by the reporter circuit, it would cast doubt on the validity of the model as soon as a different reporter or sexy circuit were implemented. If there is a way to characterise the reporter by itself along with the signalling + reporter (and if we could do the same with the mutants, that would be interesting too), it would help us assess how good our model is for the signalling circuit by itself, and how it will perform when used with a different reporter or sexy circuit at the end.
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Latest revision as of 19:16, 1 October 2009

University of Calgary

UNIVERSITY OF CALGARY



THIS MONTH

July
MTWTFSS
    [http://2009.igem.org/Team:Calgary/1_July_2009 1] [http://2009.igem.org/Team:Calgary/2_July_2009 2] [http://2009.igem.org/Team:Calgary/3_July_2009 3] [http://2009.igem.org/Team:Calgary/4_July_2009 4] [http://2009.igem.org/Team:Calgary/5_July_2009 5]
[http://2009.igem.org/Team:Calgary/6_July_2009 6] [http://2009.igem.org/Team:Calgary/7_July_2009 7] [http://2009.igem.org/Team:Calgary/8_July_2009 8] [http://2009.igem.org/Team:Calgary/9_July_2009 9] [http://2009.igem.org/Team:Calgary/10_July_2009 10] [http://2009.igem.org/Team:Calgary/11_July_2009 11] [http://2009.igem.org/Team:Calgary/12_July_2009 12]
[http://2009.igem.org/Team:Calgary/13_July_2009 13] [http://2009.igem.org/Team:Calgary/14_July_2009 14] [http://2009.igem.org/Team:Calgary/15_July_2009 15] [http://2009.igem.org/Team:Calgary/16_July_2009 16] [http://2009.igem.org/Team:Calgary/17_July_2009 17] [http://2009.igem.org/Team:Calgary/18_July_2009 18] [http://2009.igem.org/Team:Calgary/19_July_2009 19]
[http://2009.igem.org/Team:Calgary/20_July_2009 20] [http://2009.igem.org/Team:Calgary/21_July_2009 21] [http://2009.igem.org/Team:Calgary/22_July_2009 22] [http://2009.igem.org/Team:Calgary/23_July_2009 23] [http://2009.igem.org/Team:Calgary/24_July_2009 24] [http://2009.igem.org/Team:Calgary/25_July_2009 25] [http://2009.igem.org/Team:Calgary/26_July_2009 26]
[http://2009.igem.org/Team:Calgary/27_July_2009 27] [http://2009.igem.org/Team:Calgary/28_July_2009 28] [http://2009.igem.org/Team:Calgary/29_July_2009 29] [http://2009.igem.org/Team:Calgary/30_July_2009 30] [http://2009.igem.org/Team:Calgary/31_July_2009 31]


NOTEBOOK PAGE INDEX



CALENDAR

May
MTWTFSS
        [http://2009.igem.org/Team:Calgary/1_May_2009 1] [http://2009.igem.org/Team:Calgary/2_May_2009 2] [http://2009.igem.org/Team:Calgary/3_May_2009 3]
[http://2009.igem.org/Team:Calgary/4_May_2009 4] [http://2009.igem.org/Team:Calgary/5_May_2009 5] [http://2009.igem.org/Team:Calgary/6_May_2009 6] [http://2009.igem.org/Team:Calgary/7_May_2009 7] [http://2009.igem.org/Team:Calgary/8_May_2009 8] [http://2009.igem.org/Team:Calgary/9_May_2009 9] [http://2009.igem.org/Team:Calgary/10_May_2009 10]
[http://2009.igem.org/Team:Calgary/11_May_2009 11] [http://2009.igem.org/Team:Calgary/12_May_2009 12] [http://2009.igem.org/Team:Calgary/13_May_2009 13] [http://2009.igem.org/Team:Calgary/14_May_2009 14] [http://2009.igem.org/Team:Calgary/15_May_2009 15] [http://2009.igem.org/Team:Calgary/16_May_2009 16] [http://2009.igem.org/Team:Calgary/17_May_2009 17]
[http://2009.igem.org/Team:Calgary/18_May_2009 18] [http://2009.igem.org/Team:Calgary/19_May_2009 19] [http://2009.igem.org/Team:Calgary/20_May_2009 20] [http://2009.igem.org/Team:Calgary/21_May_2009 21] [http://2009.igem.org/Team:Calgary/22_May_2009 22] [http://2009.igem.org/Team:Calgary/23_May_2009 23] [http://2009.igem.org/Team:Calgary/24_May_2009 24]
[http://2009.igem.org/Team:Calgary/25_May_2009 25] [http://2009.igem.org/Team:Calgary/26_May_2009 26] [http://2009.igem.org/Team:Calgary/27_May_2009 27] [http://2009.igem.org/Team:Calgary/28_May_2009 28] [http://2009.igem.org/Team:Calgary/29_May_2009 29] [http://2009.igem.org/Team:Calgary/30_May_2009 30] [http://2009.igem.org/Team:Calgary/31_May_2009 31]


June
MTWTFSS
[http://2009.igem.org/Team:Calgary/1_June_2009 1] [http://2009.igem.org/Team:Calgary/2_June_2009 2] [http://2009.igem.org/Team:Calgary/3_June_2009 3] [http://2009.igem.org/Team:Calgary/4_June_2009 4] [http://2009.igem.org/Team:Calgary/5_June_2009 5] [http://2009.igem.org/Team:Calgary/6_June_2009 6] [http://2009.igem.org/Team:Calgary/7_June_2009 7]
[http://2009.igem.org/Team:Calgary/8_June_2009 8] [http://2009.igem.org/Team:Calgary/9_June_2009 9] [http://2009.igem.org/Team:Calgary/10_June_2009 10] [http://2009.igem.org/Team:Calgary/11_June_2009 11] [http://2009.igem.org/Team:Calgary/12_June_2009 12] [http://2009.igem.org/Team:Calgary/13_June_2009 13] [http://2009.igem.org/Team:Calgary/14_June_2009 14]
[http://2009.igem.org/Team:Calgary/15_June_2009 15] [http://2009.igem.org/Team:Calgary/16_June_2009 16] [http://2009.igem.org/Team:Calgary/17_June_2009 17] [http://2009.igem.org/Team:Calgary/18_June_2009 18] [http://2009.igem.org/Team:Calgary/19_June_2009 19] [http://2009.igem.org/Team:Calgary/20_June_2009 20] [http://2009.igem.org/Team:Calgary/21_June_2009 21]
[http://2009.igem.org/Team:Calgary/22_June_2009 22] [http://2009.igem.org/Team:Calgary/23_June_2009 23] [http://2009.igem.org/Team:Calgary/24_June_2009 24] [http://2009.igem.org/Team:Calgary/25_June_2009 25] [http://2009.igem.org/Team:Calgary/26_June_2009 26] [http://2009.igem.org/Team:Calgary/27_June_2009 27] [http://2009.igem.org/Team:Calgary/28_June_2009 28]
[http://2009.igem.org/Team:Calgary/29_June_2009 29] [http://2009.igem.org/Team:Calgary/30_June_2009 30]


July
MTWTFSS
    [http://2009.igem.org/Team:Calgary/1_July_2009 1] [http://2009.igem.org/Team:Calgary/2_July_2009 2] [http://2009.igem.org/Team:Calgary/3_July_2009 3] [http://2009.igem.org/Team:Calgary/4_July_2009 4] [http://2009.igem.org/Team:Calgary/5_July_2009 5]
[http://2009.igem.org/Team:Calgary/6_July_2009 6] [http://2009.igem.org/Team:Calgary/7_July_2009 7] [http://2009.igem.org/Team:Calgary/8_July_2009 8] [http://2009.igem.org/Team:Calgary/9_July_2009 9] [http://2009.igem.org/Team:Calgary/10_July_2009 10] [http://2009.igem.org/Team:Calgary/11_July_2009 11] [http://2009.igem.org/Team:Calgary/12_July_2009 12]
[http://2009.igem.org/Team:Calgary/13_July_2009 13] [http://2009.igem.org/Team:Calgary/14_July_2009 14] [http://2009.igem.org/Team:Calgary/15_July_2009 15] [http://2009.igem.org/Team:Calgary/16_July_2009 16] [http://2009.igem.org/Team:Calgary/17_July_2009 17] [http://2009.igem.org/Team:Calgary/18_July_2009 18] [http://2009.igem.org/Team:Calgary/19_July_2009 19]
[http://2009.igem.org/Team:Calgary/20_July_2009 20] [http://2009.igem.org/Team:Calgary/21_July_2009 21] [http://2009.igem.org/Team:Calgary/22_July_2009 22] [http://2009.igem.org/Team:Calgary/23_July_2009 23] [http://2009.igem.org/Team:Calgary/24_July_2009 24] [http://2009.igem.org/Team:Calgary/25_July_2009 25] [http://2009.igem.org/Team:Calgary/26_July_2009 26]
[http://2009.igem.org/Team:Calgary/27_July_2009 27] [http://2009.igem.org/Team:Calgary/28_July_2009 28] [http://2009.igem.org/Team:Calgary/29_July_2009 29] [http://2009.igem.org/Team:Calgary/30_July_2009 30] [http://2009.igem.org/Team:Calgary/31_July_2009 31]


August
MTWTFSS
          [http://2009.igem.org/Team:Calgary/1_August_2009 1] [http://2009.igem.org/Team:Calgary/2_August_2009 2]
[http://2009.igem.org/Team:Calgary/3_August_2009 3] [http://2009.igem.org/Team:Calgary/4_August_2009 4] [http://2009.igem.org/Team:Calgary/5_August_2009 5] [http://2009.igem.org/Team:Calgary/6_August_2009 6] [http://2009.igem.org/Team:Calgary/7_August_2009 7] [http://2009.igem.org/Team:Calgary/8_August_2009 8] [http://2009.igem.org/Team:Calgary/9_August_2009 9]
[http://2009.igem.org/Team:Calgary/10_August_2009 10] [http://2009.igem.org/Team:Calgary/11_August_2009 11] [http://2009.igem.org/Team:Calgary/12_August_2009 12] [http://2009.igem.org/Team:Calgary/13_August_2009 13] [http://2009.igem.org/Team:Calgary/14_August_2009 14] [http://2009.igem.org/Team:Calgary/15_August_2009 15] [http://2009.igem.org/Team:Calgary/16_August_2009 16]
[http://2009.igem.org/Team:Calgary/17_August_2009 17] [http://2009.igem.org/Team:Calgary/18_August_2009 18] [http://2009.igem.org/Team:Calgary/19_August_2009 19] [http://2009.igem.org/Team:Calgary/20_August_2009 20] [http://2009.igem.org/Team:Calgary/21_August_2009 21] [http://2009.igem.org/Team:Calgary/22_August_2009 22] [http://2009.igem.org/Team:Calgary/23_August_2009 23]
[http://2009.igem.org/Team:Calgary/24_August_2009 24] [http://2009.igem.org/Team:Calgary/25_August_2009 25] [http://2009.igem.org/Team:Calgary/26_August_2009 26] [http://2009.igem.org/Team:Calgary/27_August_2009 27] [http://2009.igem.org/Team:Calgary/28_August_2009 28] [http://2009.igem.org/Team:Calgary/29_August_2009 29] [http://2009.igem.org/Team:Calgary/30_August_2009 30]
[http://2009.igem.org/Team:Calgary/31_August_2009 31]


September
MTWTFSS
  [http://2009.igem.org/Team:Calgary/1_September_2009 1] [http://2009.igem.org/Team:Calgary/2_September_2009 2] [http://2009.igem.org/Team:Calgary/3_September_2009 3] [http://2009.igem.org/Team:Calgary/4_September_2009 4] [http://2009.igem.org/Team:Calgary/5_September_2009 5] [http://2009.igem.org/Team:Calgary/6_September_2009 6]
[http://2009.igem.org/Team:Calgary/7_September_2009 7] [http://2009.igem.org/Team:Calgary/8_September_2009 8] [http://2009.igem.org/Team:Calgary/9_September_2009 9] [http://2009.igem.org/Team:Calgary/10_September_2009 10] [http://2009.igem.org/Team:Calgary/11_September_2009 11] [http://2009.igem.org/Team:Calgary/12_September_2009 12] [http://2009.igem.org/Team:Calgary/13_September_2009 13]
[http://2009.igem.org/Team:Calgary/14_September_2009 14] [http://2009.igem.org/Team:Calgary/15_September_2009 15] [http://2009.igem.org/Team:Calgary/16_September_2009 16] [http://2009.igem.org/Team:Calgary/17_September_2009 17] [http://2009.igem.org/Team:Calgary/18_September_2009 18] [http://2009.igem.org/Team:Calgary/19_September_2009 19] [http://2009.igem.org/Team:Calgary/20_September_2009 20]
[http://2009.igem.org/Team:Calgary/21_September_2009 21] [http://2009.igem.org/Team:Calgary/22_September_2009 22] [http://2009.igem.org/Team:Calgary/23_September_2009 23] [http://2009.igem.org/Team:Calgary/24_September_2009 24] [http://2009.igem.org/Team:Calgary/25_September_2009 25] [http://2009.igem.org/Team:Calgary/26_September_2009 26] [http://2009.igem.org/Team:Calgary/27_September_2009 27]
[http://2009.igem.org/Team:Calgary/28_September_2009 28] [http://2009.igem.org/Team:Calgary/29_September_2009 29] [http://2009.igem.org/Team:Calgary/30_September_2009 30]


October
MTWTFSS
      [http://2009.igem.org/Team:Calgary/1_October_2009 1] [http://2009.igem.org/Team:Calgary/2_October_2009 2] [http://2009.igem.org/Team:Calgary/3_October_2009 3] [http://2009.igem.org/Team:Calgary/4_October_2009 4]
[http://2009.igem.org/Team:Calgary/5_October_2009 5] [http://2009.igem.org/Team:Calgary/6_October_2009 6] [http://2009.igem.org/Team:Calgary/7_October_2009 7] [http://2009.igem.org/Team:Calgary/8_October_2009 8] [http://2009.igem.org/Team:Calgary/9_October_2009 9] [http://2009.igem.org/Team:Calgary/10_October_2009 10] [http://2009.igem.org/Team:Calgary/11_October_2009 11]
[http://2009.igem.org/Team:Calgary/12_October_2009 12] [http://2009.igem.org/Team:Calgary/13_October_2009 13] [http://2009.igem.org/Team:Calgary/14_October_2009 14] [http://2009.igem.org/Team:Calgary/15_October_2009 15] [http://2009.igem.org/Team:Calgary/16_October_2009 16] [http://2009.igem.org/Team:Calgary/17_October_2009 17] [http://2009.igem.org/Team:Calgary/18_October_2009 18]
[http://2009.igem.org/wiki/index.php?title=Team:Calgary/19_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/20_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/21_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/22_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/23_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/24_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/25_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 25]
[http://2009.igem.org/wiki/index.php?title=Team:Calgary/26_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/27_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/28_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/29_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/30_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/31_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 31]



JULY 31, 2009


CAROL

Transformation of R0040 (promoter) +pCS26 (vector) into XL Gold cells

  • From the overnight digest of R0040 with XhoI and BamHI, the promoter was ligated with vector, pCS26.
  • These plasmids were transformed into XL Gold cells and was plated on Kanamycin plates overnight.

Team meeting:

  • Lab:discussed about promoter library and the failure to see the expression of luciferase. Also, discussed about the primer design of R0040, so that we can clone the constitutively ON promoter in front of luciferase. This will show us what the luciferase expression will look like.
  • Modelling: Discussed about Robustness. The three things that we decided to look at for robustness is: 1. PQ expression (the selection of the 'best' sigma 70 promoter) 2. Temperature Alterations (to see if changes in temperature (from 37oC to another temperature would affect expression) 3. Type of media (LB vs. LM)


CHINMOYEE

Descriptive Title of What You're Doing

WIKI CODING HERE


EMILY

Sequencing of J13002-LuxOD47E-B0015 Construct in psB1AC3

  • Sent Colony 3 of my J13002-LuxOD47E-B0015 construct down for sequencing, results will come in on Monday hopefully.
  • Explored Secondlife and met with Rob from UChicago.
  • Prepared the Human Practices project write-up for the Wiki.
  • Team Meeting- Talked about the exploration of seminars in Second Life and what ideas that has given us. Talked about the pending completion of the J13002-LuxOD47E-B0015 curcuit, results will come in on Monday.


FAHD

Marketing and Ethics for July 31st 2009

Today I worked more on the ethcial aspect of our project. I read more papers on the environmental and economical issues of our team calgary project.

I also made follow up calls to a couple of comapnies.


IMAN

More Educational Aspects: Rules Animations

The next step to enrich the educational aspects of our model is to animate each rule. With doing so, biologists can make connection with the rules as well as the notations used to implement these rules. It is really important for us that make our model convenient to understand for biologists as they are the main users of it. that is why we have designed these animation so that biology and computer science could be connected.

Rules2.png

That is what I have been working on this week.

In addition we had our meeting, and here is a summary for modeling team:

The team members involved in modelling need to meet more and learn more about what the others are doing. We will address this by meeting on Tuesdays and Thursdays at 10h00 am. On the differential side, we have a lot of breadth to our approach but it's still fairly superficial. We have established a differential reaction-based model on SimBiology, but finding values for phosphorylation constants has been a much greater challenge than we had anticipated. We will need to set up a model anyway and collect some input/output data, and from that we can approximate a set of parameters. It's highly unlikely that they'll be unique, but they will give us something to work with and help us evaluate which ones are limiting steps and would be worth tweaking in a real system The point of standardisation and characterisation is to have a set of parts to work with and choose from, based on characteristics that can be compared among different parts. Endy's 2006 paper is interesting, but it only presents characterisation for one part. It would be useful to have more parts characterised, especially if they actually work. We are submitting a few parts to the Registry: a signalling circuit, a reporter, mutants - all with various combinations of promoter and terminator sequences. It would be useful to characterise as many of them as possible, even if it's just for static and dynamic respones, so that future users have a quantifiable way of comparing performance. We are testing a signalling-reporter combination, but we don't know how much the reporter will affect our model of the signalling circuit. For example, if we consider response time, we don't know if our circuit response is limited by the signalling circuit, the reporter circuit, or a combination of both. If the circuit response is slowed mostly by the reporter circuit, it would cast doubt on the validity of the model as soon as a different reporter or sexy circuit were implemented. If there is a way to characterise the reporter by itself along with the signalling + reporter (and if we could do the same with the mutants, that would be interesting too), it would help us assess how good our model is for the signalling circuit by itself, and how it will perform when used with a different reporter or sexy circuit at the end.


JAMIE

Verification of cl lambda inverter in psB2K3

  • Plasmid miniprep of overnight cultures with Sigma Genelute Miniprep kit. Standard manufacturer protocol used; elution in 40µL of ddH2O.
  • Vacufuge to concentrate.
  • Verification digest with XbaI and PstI. Control: cl lambda part from the Registry distribution plate.
  • Results: Faint bands were present. Looked into vector characteristics and it turns out it is high-copy number with IPTG induction (Thanks Thane!)

JEREMY

Isolation of cl lambda; Sequencing for LuxPQ-LuxOU construct

Six overnight cultures of cl lambda colony 1 were prepared with LB-Kanamycin broth. Plasmid was then isolated using the Sigma GenElute MiniPrep kit. Two plasmids were not successfully isolated, but the other four tubes were pooled into two tubes and vacufuged to increase their concentration. A restriction digest with XbaI and PstI was then set up for 2 hours at 37ºC.

The LuxPQ-B-R-LuxOU-B construct in psB1AC3 was sent for sequencing today based on verification techniques performed over the past few days. Primers used include BBK CP F/R and R0040 Reverse.


KATIE

Modifications to DNA Extraction, Restriction Digest, Instructions and Write-ups

The movement of the restriction digest tubes have been modified so they are more interactive so the user is asked what piece of equipment is required once all required items have been added in the correct order to each tube. The three pieces of equipment to touch are the water bath, heating block and incubator (for the insert) respectively. I still am required to place my restriction digest instructions into a notecard on the lab table, which I will do next week.

I have been able to modify the first five steps in DNA extraction so that the order in which a avatar proceeds is important. Now if the initial step of resuspension has not been accomplished, if you touch the lysis activity you will not be allowed to perform the activity. However, once a step has been unlocked an avatar may return to it at any time if they believe they missed something or just wish to redo a step. Once an avatar reaches the end of the activity a message is sent to all objects, which resets the entire activity. Now I will have to begin incorporating the containers that store: Wash Solution, High Salt Buffer, Isopropanol, and one containing a DNA buffer as well as one more centrifuge step.

Some more instruction were added to bacterial transformation so there is a response for everything an avatar does in attempt to complete the activity. For example, if a user drops an item into the quiz’s inventory that is of the same type that has been submitted before it will alert them to the fact that they have answered that set of questions before and will not ask them again. I have been given the task of creating more products for this activity as well so I will have to add to my transformation script so that the plates with growth will give you the proper object to be used within the lab elsewhere.

I also wrote an introduction to the virtual lab for our project description for the wiki, which generally explains the lab’s content as well as its purpose for the second life project.


KEVIN

cPCR of the reporter circuit

In order to verify cPCR, I have once again ran a cPCR of the reporter circuit, Pqrr4+B0034(RBS)+K082003(GFP+LVA) with Pqrr4 Forward and Biobrick CP Reverse primers. Of course, pTaq was used to amplify the DNA. The product was then ran on 2% agarose gel at 90V. The following is a picture of the gel.

Calgary 2009.07.31.reporter cPCR.png

As you can see, the bands in most of the colony lanes match the positive control of Pqrr4+B0034, which means that the cells contain those pieces, not the one of interest. It seems that only the colony 9 is between 1000bp and 1500bp, which is what was expected. However, even this band is suspicious. The size of the circuit is about 1043bp, and with about 125bp addition due to Biobrick CP reverse primer annealing outside of the circuit; however, the band on colony 9 lane is at about 1250bp. Another problem can be noticed in the negative control lane, which is another contamination. It is very disappointing to see a whole day of work gone to waste :(! Yet, it is too early to give up hope. I can try to isolate the plasmid of the Colony 9 and perform a Restriction digest on it to see if I still do not see the expected band, which is at this point unlikely.

Distribution of our baking sale posters

Fahd left a stack of Baking sale posters for us to post throughout the building, and they were posted before 11am today.

MANDY

Descriptive Title of What You're Doing

WIKI CODING HERE


PATRICK

Automagic Biobricks

I spent most of today being very productive with the Biobricker portion of the Biobrick Simulator. This is a little user interface upgrade that will permit you to build any biobrick device you please, from a small library of parts implemented in Second Life. In SL terms, the UI is an object owned by the user's avatar, that can be attached to your display. The simulator's UI will have quite a few features to make your digital bioengineering easier, of which the Biobricker is an important one.

I finished working on all of the controls for the biobricker, including some scroll buttons for the parts in the device under construction, and the ability to add parts from the UI's inventory, and delete them too. The Biobricker is all done except for one last control... the build button! This single button will be as much work as the rest of the Biobricker put together, though. The task is to create a copy of each individual Biobrick in world, inform each one where it belongs in the completed device, and link them all together. This will involve a lot of communication back and forth between the user interface object and the biobrick objects out in the world.

The Biobricker is in a very primitive state right now, with letters standing in for where all the pretty icons will be in the final version. So instead here's a screenshot of the Main Menu, with the link to the Biobricker visible.

Calgary Biobrick Simulator UI Preview.png

The UI fits in at the top left corner of the screen. The red letters are buttons, that cause the UI to be redrawn with a new menu for whichever module you've chosen. I hope this gives you an idea of how the UI fits into the overall simulator.


PRIMA

Continuation of last day's work

Today, I edited the high school presentation proposition again and sent it out to Mrs. Ellechuk. I filled in the lab notebook and filled out the daily updates on the wiki.

I finished typing out the acknowledgement for each mentor/advisor of iGEM Calgary. I also helped to write references for Sonja. Then I finished off with mailing out more newsletters. We also had a very productive team meeting today.

On Monday I have to follow up with the companies which I have been contacting for the past few days. I will also call our hotel in Boston to change a few rooms around.



STEFAN

Continuing along the path

Construction of the path is almost complete and finals plans were mapped out but are subject to change to make room for future virtual demonstrations. In order to complete each station I've decided that a Hawaiian Bobtail Squid will be added to each that will tell visitors about what they see. It will preferably be very interactive (ie. move around and talk to you rather than give a notecard) so as to be fun and educational for the junior high and high school audience we are targeting. The squid that was already in the synthetic kingdom was slightly modified in order to match the physical characteristics of the Hawaiian Bobtail Squid.


VICKI

Discussion write-up, referencing and wiki posting

(This isn't terribly exciting. I continued with the discussion section of the technical report, I learned all about APA referencing, and I posted part of my notebook on the wiki).

Lab meeting

We discussed characterisation elements for our signalling circuit, as described in Carol's section. I focussed on dynamic performance and response time, or, in other words, what happens when you add a lot of AI-2 into a system where it wasn't there previously.

We also discussed how we could consolidate our modelling efforts and come up with something with a little depth to it in time for the competition. Some of the main points that came out of that are:

  • The team members involved in modelling need to meet more and learn more about what the others are doing. We will address this by meeting on Tuesdays and Thursdays at 10h00 am.
  • On the differential side, we have a lot of breadth to our approach but it's still fairly superficial. We have established a differential reaction-based model on SimBiology, but finding values for phosphorylation constants has been a much greater challenge than we had anticipated. We will need to set up a model anyway and collect some input/output data, and from that we can approximate a set of parameters. It's highly unlikely that they'll be unique, but they will give us something to work with and help us evaluate which ones are limiting steps and would be worth tweaking in a real system
  • The point of standardisation and characterisation is to have a set of parts to work with and choose from, based on characteristics that can be compared among different parts. Endy's 2006 paper is interesting, but it only presents characterisation for one part. It would be useful to have more parts characterised, especially if they actually work. We are submitting a few parts to the Registry: a signalling circuit, a reporter, mutants - all with various combinations of promoter and terminator sequences. It would be useful to characterise as many of them as possible, even if it's just for static and dynamic respones, so that future users have a quantifiable way of comparing performance.
  • We are testing a signalling-reporter combination, but we don't know how much the reporter will affect our model of the signalling circuit. For example, if we consider response time, we don't know if our circuit response is limited by the signalling circuit, the reporter circuit, or a combination of both. If the circuit response is slowed mostly by the reporter circuit, it would cast doubt on the validity of the model as soon as a different reporter or sexy circuit were implemented. If there is a way to characterise the reporter by itself along with the signalling + reporter (and if we could do the same with the mutants, that would be interesting too), it would help us assess how good our model is for the signalling circuit by itself, and how it will perform when used with a different reporter or sexy circuit at the end.