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Team:Calgary/17 August 2009
From 2009.igem.org
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| - | + | Transformation of J13002-LuxOD47A-B0015 into TOP10 cells, restreaks and overnight cultures of J13002-LuxOD47E-B0015 in KT1144 cells | |
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| - | + | *This morning I made a detailed lab plan of what I need to do for the week. I also spent some time updating my lab notebook as well as the Wiki Notebook. | |
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| + | *This afternoon I ligated J13002-LuxOD47A-B0015 and the psB1AC3 vector. I then transformed the ligated product into TOP10 cells and plated on KC plates and left for overnight growth. | ||
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| + | *I also did restreaks of my construct (J13002-LuxOD47E-B0015) in KT1144 cells on KC plates. I also prepared overnight cultrues of selected colonies in KC broth. Tomorrow Kevin and I will be using the plate reader to test these cultures for glow. | ||
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| - | + | Ethics and Marketing for August 17th 2009 | |
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<div class="desc"> | <div class="desc"> | ||
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| - | + | Today, I concentrated my energies on working on the ethical aspect of our project and did some marketing for our project. I started my day by working on the ethics essay that we will be writing for our project. I edited the essay and added some more information to elaborate on some topics. It is still not complete but I will be working on it for the whole week. | |
| + | <br> | ||
| + | <br> | ||
| + | For marketing, I contacted my team-mates who are working on 2009 iGEM Calgary Shirts and asked them if they needed any help. I will be contacting the contact for the shirts company and ask them for a better deal on the shirts or a possible partnership. I also worked on the August Newsletter; I started to write a couple of write-ups for some sections that we will be adding to the newsletter. | ||
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| - | + | Sequencing Analysis of luxPQ-luxOU construct in pCS26 and Analyzing Results from the V. harveyi AI-2 Activity Assay #1 | |
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<div class="desc"> | <div class="desc"> | ||
| - | </ | + | Sequencing results indicated successful cloning of the signalling cascade into the Surette vector after many BLAST alignments and highlighting different coding sequences. <br /> <br /> |
| - | + | More time was spent with the plate reader Synergy HT figuring out not only how to export data into an excel CSV format so analysis of the data could occur. Preliminary analysis presented several anomalies (i.e. DH5a producing AI-2 and 14028 not causing glowing <i>V. harveyi</i>). | |
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| - | + | PQ-OU verified by sequencing in pCS26 + OUTREACH | |
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| - | + | Sequencing results came back from the LuxPQ-OU construct in pCS26 for | |
| + | colony 8, revealing a successful cloning. | ||
| + | <br> | ||
| + | <br> | ||
| + | Thank you to students Nicole (RICE Univeristy), Julia (Dalhousie) and Mike | ||
| + | (UBC) for coming into the labs today to see what iGEM is all about. They | ||
| + | had a great time learning about synthetic biology and quorum sensing and | ||
| + | how these principles fall within the scope of our project. This outreach | ||
| + | activity was a great learning experience, and spiked their interest in | ||
| + | iGEM for future years. | ||
| + | |||
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| - | + | Completion of DNA Replication and More Writing | |
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<div class="desc"> | <div class="desc"> | ||
</html> | </html> | ||
| - | + | I was able to complete a blog update that will be posted sometime on Tuesday and DNA replication was completed except for the user prompts that will be included with the notecard. I had to make single stranded binding proteins that attach close to where the helicase is and the primase will only rez one set of primers, which is the same for DNA polymerase until it has been reset. Other things that I made today were: | |
| + | <br> | ||
| + | <br> | ||
| + | * A button has been made for intro to the activity that acts as a reset point so that everything is returned to their original position. | ||
| + | * While the gyrase is active it will glow and when it is not the rest of the activity will not work | ||
| - | + | I also posted wiki updates for the majority of May and also made a couple for July. I have now begun on my wiki notebook for June, which I would like to complete by Wednesday at the latest. | |
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| - | + | Next, I would like to begin working on either transcription or translation, which I will decide on tomorrow. I will also link the whole replication activity together so it can be moved to the base of the spiral on the island. | |
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| - | <a name=" | + | <a name="Kevin"></a> |
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| - | + | KEVIN | |
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<div class="heading"> | <div class="heading"> | ||
| - | + | Construction of <i>Pqrr4</i>+B0034+K082003 | |
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<br> | <br> | ||
<div class="desc"> | <div class="desc"> | ||
</html> | </html> | ||
| - | + | Once again, the <i>Pqrr4</i>+B0034+K082003 circuit was constructed. This time, the vector was <i>Pqrr4</i>+B0034 Colony 1. The vector was cut with SpeI and PstI, and the insert, K082003, was cut with XbaI and PstI. The vector was phosphotase treated, and then they were ligated together. Next, this constructed circuit was transformed into TOP10 cells and are now growing on Kanamycin + Chloramphenicol plates. | |
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| - | + | Modelling Blog | |
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| - | + | ||
</div> | </div> | ||
<br> | <br> | ||
<div class="desc"> | <div class="desc"> | ||
</html> | </html> | ||
| - | + | Another Modelling blog was written today. Most of the modelling work last week was done by Vicki and Chinee, working hard to track down those sensitive parameters. Here's the link to the blog: | |
| - | + | ||
<html> | <html> | ||
| + | <a href='http://igemcalgary.blogspot.com/2009/08/sensitivity-analysis-models-have.html'>Sensitivity analysis: Models... have feelings too!</a> | ||
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| - | + | Colony PCR and Marketing | |
</div> | </div> | ||
<br> | <br> | ||
<div class="desc"> | <div class="desc"> | ||
</html> | </html> | ||
| - | + | It turns out that I had used the Low-melt agarose on Friday to make my 1% gel. Unfortunately, the gel didn't turn out well and I had to postpone my work to Monday. Today, I made a 1% gel with the normal agarose powder and remade my stock (3 microL PCR product, 2 microL dye & 15 microL ddH2O) and ran my gel at 100V. | |
| + | The results showed exactly what I was expecting. Since I had used regular BBK CP primers and length of my construct was 885bp, the total length of the bands should've been 1135bp. My bands were just above the 1kb ladder band! I let the students visiting our lab make my overnight cultures. Tomorrow, I'll do a mini-prep and a restriction digest to verify the size of my construct. If all else is good, I'll send it to sequencing. | ||
| + | |||
| + | I followed up with the T-shirt company but unfortunately I couldn't get a hold of her. I'll follow up tomorrow. I also called numerous new companies and sent our sponsorship package to one of them. I left a voice mail for the rest of the companies. I'll follow up with them tomorrow. I also began write-ups for the August newsletter. | ||
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| - | + | Disease Hunting | |
</div> | </div> | ||
<br> | <br> | ||
<div class="desc"> | <div class="desc"> | ||
</html> | </html> | ||
| - | + | I was working on completing the disease hunting station today. I used the book "Scripting your world: the official guide to Second Life scripting" by Dana Moore, Michael Thome, and Dr. Karen Zita Haigh for help in making things work. Once I wrote this script I went to another sim (in case things went awry) to experiment by modifying it to suit the disease hunting. I accomplished making the following: | |
| + | <br> | ||
| + | <br> | ||
| + | *A nucleus that turns red when in contact with "Bad Guy" bacteria and turns back after a few seconds if bacteria is gone | ||
| + | *"Bad Guy" bacteria that senses the nucleus and moves towards it as well as changes color and subsequently dies if touched by "Champion" | ||
| + | *"Champion" bacteria that sense the "Bad Guys" and move towards them | ||
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| - | + | Sensitivity analysis and parameter optimisation | |
</div> | </div> | ||
<br> | <br> | ||
<div class="desc"> | <div class="desc"> | ||
</html> | </html> | ||
| - | + | My primary goals for today were: | |
| + | * to learn how to perform a sensitivity analysis for a given system | ||
| + | * to use that information to make the parameter estimation more useful and efficient | ||
| + | |||
| + | SENSITIVITY ANALYSIS | ||
| + | |||
| + | I followed the SimBiology example and applied that to the WithoutAI-2 system. While Chinee collected information on what affects the sensitivity of the outputs with respect to different parameters, I worked out how to automate that at the command line level. I explored GFP output specifically, with respect to 5 different parameters. Of those, my results showed GFP displays high sensitivity to parameters 1, 3 and 4; and very low sensitivity to parameters 2 and 5. | ||
| + | |||
| + | PARAMETER ESTIMATION | ||
| + | |||
| + | To support the sensitivity analysis, I performed the parameter optimisation on each of the 5 different parameters to try to fit the curve to output data that I made up. The idea is that if you can optimise just one of the parameters and fit the curve to the data, it means that the system is highly responsive to tweaking of that one parameter. The results of this were encouraging: by modifying either parameters 1, 3 or 4, I was able to achieve a 92% fit. However, even when I optimised parameters 2 or 5 as much as I possibly could, I could only achieve a 40% correlation. This supports the results of the sensitivity analysis, by showing that the system is most responsive to parameters 1, 3 and 4. | ||
| + | |||
| + | I then conducted the optimisation on parameters 1, 3 and 4 all at once. The results were surprising: the R-squared fit was .92 - slightly higher than by just tweaking parameters 3 or 4, but slightly lower than when I only tweaked parameter 1. I will explore this further to see what is going on. | ||
| + | |||
| + | If anyone would like a demonstration, these results can be seen by using the command "do_work" on my computer. | ||
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Latest revision as of 03:41, 22 October 2009
UNIVERSITY OF CALGARY
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CHINMOYEE
Working with MATLAB Day1
Researched Sensitivity Analysis:
Intro into sensitivity Analysis , by Lucia Breierova and Mark Choudhari... Content: Examples / Tutorial on how to do sensitivity analysis on data ( No examples related to our work) Learned: Sensitivity investigates the robustness of a study when the study includes some form of math modelling. Sensitivity allows the modeller to determine what level of accuracy is needed for a parameter to make the model sufficiently useful and valid. When the parameter is insensitive in the model , making an estimation is a good idea. With Systems Dynamic models the k parameter doesn't affect the behaviour of the graph . The shape of the graph stays the same . Matlab Programing: Looked into the effect of changing the kinetic constants on the graph. This resulted in no change in the behavior of the graph as predicted by the paper. The max/min/ steepness of the graphs changed as a result of changing k values. The change of some k values effected the steepness of the lines very much while changing the value of other k values didn't change the steepeness all that much. Also looked into the effect of changing the initial concentration of the species . This completely changed the behavior of the graphs. Looked into sensitivity analysis with simbiology interface . Still trying to work out some kinks.
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EMILY
Transformation of J13002-LuxOD47A-B0015 into TOP10 cells, restreaks and overnight cultures of J13002-LuxOD47E-B0015 in KT1144 cells
|
|
FAHD
Ethics and Marketing for August 17th 2009
Today, I concentrated my energies on working on the ethical aspect of our project and did some marketing for our project. I started my day by working on the ethics essay that we will be writing for our project. I edited the essay and added some more information to elaborate on some topics. It is still not complete but I will be working on it for the whole week.
For marketing, I contacted my team-mates who are working on 2009 iGEM Calgary Shirts and asked them if they needed any help. I will be contacting the contact for the shirts company and ask them for a better deal on the shirts or a possible partnership. I also worked on the August Newsletter; I started to write a couple of write-ups for some sections that we will be adding to the newsletter.
|
|
IMAN
Working on Paper
I am gathering information needed to write down the paper we have been asked to write. I have written some sections so far.
|
|
JAMIE
Sequencing Analysis of luxPQ-luxOU construct in pCS26 and Analyzing Results from the V. harveyi AI-2 Activity Assay #1
Sequencing results indicated successful cloning of the signalling cascade into the Surette vector after many BLAST alignments and highlighting different coding sequences.
More time was spent with the plate reader Synergy HT figuring out not only how to export data into an excel CSV format so analysis of the data could occur. Preliminary analysis presented several anomalies (i.e. DH5a producing AI-2 and 14028 not causing glowing V. harveyi). |
|
JEREMY
PQ-OU verified by sequencing in pCS26 + OUTREACH
Sequencing results came back from the LuxPQ-OU construct in pCS26 for
colony 8, revealing a successful cloning.
Thank you to students Nicole (RICE Univeristy), Julia (Dalhousie) and Mike (UBC) for coming into the labs today to see what iGEM is all about. They had a great time learning about synthetic biology and quorum sensing and how these principles fall within the scope of our project. This outreach activity was a great learning experience, and spiked their interest in iGEM for future years.
|
|
KATIE
Completion of DNA Replication and More Writing
I was able to complete a blog update that will be posted sometime on Tuesday and DNA replication was completed except for the user prompts that will be included with the notecard. I had to make single stranded binding proteins that attach close to where the helicase is and the primase will only rez one set of primers, which is the same for DNA polymerase until it has been reset. Other things that I made today were:
I also posted wiki updates for the majority of May and also made a couple for July. I have now begun on my wiki notebook for June, which I would like to complete by Wednesday at the latest.
|
|
KEVIN
Construction of Pqrr4+B0034+K082003
Once again, the Pqrr4+B0034+K082003 circuit was constructed. This time, the vector was Pqrr4+B0034 Colony 1. The vector was cut with SpeI and PstI, and the insert, K082003, was cut with XbaI and PstI. The vector was phosphotase treated, and then they were ligated together. Next, this constructed circuit was transformed into TOP10 cells and are now growing on Kanamycin + Chloramphenicol plates.
Modelling Blog
Another Modelling blog was written today. Most of the modelling work last week was done by Vicki and Chinee, working hard to track down those sensitive parameters. Here's the link to the blog:
Sensitivity analysis: Models... have feelings too!
|
|
PRIMA
Colony PCR and Marketing
It turns out that I had used the Low-melt agarose on Friday to make my 1% gel. Unfortunately, the gel didn't turn out well and I had to postpone my work to Monday. Today, I made a 1% gel with the normal agarose powder and remade my stock (3 microL PCR product, 2 microL dye & 15 microL ddH2O) and ran my gel at 100V.
The results showed exactly what I was expecting. Since I had used regular BBK CP primers and length of my construct was 885bp, the total length of the bands should've been 1135bp. My bands were just above the 1kb ladder band! I let the students visiting our lab make my overnight cultures. Tomorrow, I'll do a mini-prep and a restriction digest to verify the size of my construct. If all else is good, I'll send it to sequencing.
I followed up with the T-shirt company but unfortunately I couldn't get a hold of her. I'll follow up tomorrow. I also called numerous new companies and sent our sponsorship package to one of them. I left a voice mail for the rest of the companies. I'll follow up with them tomorrow. I also began write-ups for the August newsletter.
|
|
STEFAN
Disease Hunting
I was working on completing the disease hunting station today. I used the book "Scripting your world: the official guide to Second Life scripting" by Dana Moore, Michael Thome, and Dr. Karen Zita Haigh for help in making things work. Once I wrote this script I went to another sim (in case things went awry) to experiment by modifying it to suit the disease hunting. I accomplished making the following:
|
|
VICKI
Sensitivity analysis and parameter optimisation
My primary goals for today were:
SENSITIVITY ANALYSIS I followed the SimBiology example and applied that to the WithoutAI-2 system. While Chinee collected information on what affects the sensitivity of the outputs with respect to different parameters, I worked out how to automate that at the command line level. I explored GFP output specifically, with respect to 5 different parameters. Of those, my results showed GFP displays high sensitivity to parameters 1, 3 and 4; and very low sensitivity to parameters 2 and 5. PARAMETER ESTIMATION To support the sensitivity analysis, I performed the parameter optimisation on each of the 5 different parameters to try to fit the curve to output data that I made up. The idea is that if you can optimise just one of the parameters and fit the curve to the data, it means that the system is highly responsive to tweaking of that one parameter. The results of this were encouraging: by modifying either parameters 1, 3 or 4, I was able to achieve a 92% fit. However, even when I optimised parameters 2 or 5 as much as I possibly could, I could only achieve a 40% correlation. This supports the results of the sensitivity analysis, by showing that the system is most responsive to parameters 1, 3 and 4. I then conducted the optimisation on parameters 1, 3 and 4 all at once. The results were surprising: the R-squared fit was .92 - slightly higher than by just tweaking parameters 3 or 4, but slightly lower than when I only tweaked parameter 1. I will explore this further to see what is going on. If anyone would like a demonstration, these results can be seen by using the command "do_work" on my computer.
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