Team:Calgary/4 June 2009

From 2009.igem.org

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<div class="heading">NOTEBOOK PAGE INDEX</div>
 
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<a href="#Carol">Carol</a><br>
 
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<a href="#Chinmoyee">Chinmoyee</a><br>
 
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<a href="#Emily">Emily</a><br>
 
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<a href="#Fahd">Fahd</a><br>
 
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<a href="#Iman">Iman</a><br>
 
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<a href="#Jamie">Jamie</a><br>
 
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<a href="#Jeremy">Jeremy</a><br>
 
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<a href="#Katie">Katie</a><br>
 
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<a href="#Kevin">Kevin</a><br>
 
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<a href="#Mandy">Mandy</a><br>
 
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<a href="#Patrick">Patrick</a><br>
 
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<a href="#Prima">Prima</a><br>
 
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<a href="#Stefan">Stefan</a><br>
 
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<a href="#Vicki">Vicki</a><br>
 
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June 4, 2009
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JUNE 4, 2009
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Modelling Papers
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WIKI CODING HERE
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The modelling team continued exploring Simbiology and looking at several papers in how genetic modelling can work and what areas should our iGEM team should explore.
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CHINMOYEE
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EMILY
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Biobricking LuxOD47A
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WIKI CODING HERE
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*Objective: To form linear copies of LuxOD47A (currently inside TOPO T/A) with BioBrick ends.
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Materials and methods:
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* Template: LuxOD47E C4-8 in TOPO T/A [40 ng/uL]- original concedntration = [409.5 ng/uL]
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* Forward primer: Lux0-F-RS star(Tm = 60 degrees C)
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* Reverse primer: LuxO-R-RS primer (Tm = 60 degrees C)
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Master Mix contents (for 15 tubes)
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* 10X Pfx Amplification buffer (75 uL)
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* 10mM dNTPs mixture (15 uL)
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* 50mM MgSO4 (22.5 uL)
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* Forward primer [10 uM] (15 uL)
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* Reverse primer [10 uM] (15 uL)
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* Platinum Pfx DNA polymerase [2.5 units] (10 uL)
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* Autoclaved distilled HOH (577.5 uL)
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* TOTAL: 735 uL
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* PCR reaction volume for Master Mix: 49 uL
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PCR steps:
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* Denaturation: 94 degrees C, 5 minutes
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EMILY
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* Amplification: 36 cycles of {
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** Denaturation (94 degrees C, 15 seconds);
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** Annealing (58-66 degrees C, 30 seconds);
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** Extension (68 degrees C, 1.5 minutes)}
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Descriptive Title of What You're Doing
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* Final extension: 68 degrees C, 15 minutes
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* Hold temperature: 4 degrees C
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12 tubes were prepared to span the gradient, each of which contained 1 uL of template DNA and 49 uL master mix. A negative control with ddH2O in lieu of template DNA was also prepared.
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WIKI CODING HERE
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Results:
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The gradient PCR products were visualised on a 1% agarose gel, as shown below.  Lanes 1-12 contain template and labe 13 is a negative control with ddH20 which shows some contamination.  The bands are at the right size (~1.4 kb) to suggest that LuxOD47E is in fact in the vector.  We will proceed by isolating plasmid and sending it down for DNA sequencing.
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[[Image:2009.06.04.LuxOD47E_BBKAmf+PCR2.jpg|350px]]
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Marketing and Outreach for June 4th 2009
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WIKI CODING HERE
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Today, I continued filling out funding applications for some pharmaceutical companies. Some of these companies include Pfizer Canada, Merck Frosst Canada and Glaxosmithkline Canada. I almost got them completed and once they are done, I will run them through
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IMAN
 
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I continued working on the Campus Fair agenda as the Campus Fair Day way coming close.
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WIKI CODING HERE
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Gradient PCR amplification <i>luxOU</i> with Biobrick suffix/prefix from TOPO vector
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pPFX (Invitrogen, CA) was used to amplify <i>luxOU</i> from the TOPO BL plasmid with luxOU gene-specific primers with the appropriate suffix or prefix (T<sub>m</sub>: 68<sup>o</sup>C).  Denaturation at 94<sup>o</sup>C for 5 minutes followed by 36 cycles of 94<sup>o</sup>C for 15sec, 58-66<sup>o</sup>C for 30sec , 68<sup>o</sup>C for 2 minutes.  Final extension at 68<sup>o</sup>C for 15 minutes.  Visualization of 3uL of product on 1% agarose gel, 120V.
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WIKI CODING HERE
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<center>[[Image:Calgary_luxOU_GradPCR.jpg | 500px]]</center>
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Figure. 1% agarose gel (100V) for gradient PCR amplification of luxOU with Biobrick suffix and prefix.  pPFX amplified 40ng of TOPO BL with luxOU.  Gene specific primers with flanking Biobrick restriction sites were used at annealing temperatures ranging from 58oC (left) to 66oC (right).  Lanes 2-13 are samples from each reaction carried out at different temperatures.  Lane 14 is a negative control.  Expected band size is 2kb. 5μL of GeneRuler 1kb Plus DNA Ladder was loaded.
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Preparation of TOP10 Competent Cells
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WIKI CODING HERE
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In order to replicate plasmid and ensure successful transformation in the summer, today’s work was devoted to making TOP10 competent cells.
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To test whether the cells were competent, pBluescript was used as a positive control, and grown on LB-Ampicillin plates. Colonies were seen the next day, verifying that the cells were made competent.
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Improving Usability
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WIKI CODING HERE
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I have found a decent way to ask questions and allow users to answer in a better way then entering things into chat. By using the llDialog function we can also communicate on negative channels, which is not possible in the chat window and helps to divide communication happening between people and between people and objects or objects and other objects.
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So, I will be making everything entered in chat for the PCR machine into dialog prompt where avatars may pick from specific choices. This also gets rid of the risk of spelling errors and increases usability.
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Modelling papers
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WIKI CODING HERE
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In order to characterize our system to the best of our ability, we have chosen the part F2620 as our role model for characterization. I have looked into and read papers regarding the rate of reactions, such as the rate of the de-phosphorylation of LuxO.
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MANDY
 
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WIKI CODING HERE
 
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Early Development
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WIKI CODING HERE
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Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day.
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First began charting out the differences between the types of biobrick part that I wanted to support. Initially thought each type would get its own type of (ie a Promoter Script existing only in promoters), but later resolved to create a single script for to run all of the types for maintainability; and to specify the part's type in the same way internal name and next part in the sequence's names are specified.
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Ran some more tests of how the messaging system works in SL, and created six different versions of the device over the course of the day. I began using version numbers to track how my scripts had changed, and was already up to versions 4 and 3 for some scripts, things were changing very quickly early on. 
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Marketing and Outreach
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WIKI CODING HERE
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Today, Fahd and I were planning our work for the Campus Fair this Saturday. We decided that we wanted to do the following:
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Pipetting competition - for children aged 8 - 13 using colored dye - gloves, lab coats and goggles were to be used.
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Bacteria crafts - design your own bacteria using foam and pipe cleaners and glitter - for children aged 3 - 8
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Draw - pretending tube caps were bacteria and people had to guess how many bacteria are in the bag. The answer was 500!
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I followed up and emailed almost all the companies that I was in charge of contacting. I haven't heard back from most of them but I'll continue to contact them.
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WIKI CODING HERE
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Gradient PCR for biobrick cloning of LuxOD47A pieces
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WIKI CODING HERE
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Note: I have switched with Jeremy: I will now be dealing with LuxOD47A and he will be handling LuxPQ
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Purpose: To form linear copies of LuxOD47A (currently inside TOPO T/A) with BioBrick ends.
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 +
Materials and methods:
 +
 +
* Template: LuxOD47A in TOPO T/A [40 ng/uL]
 +
* Forward primer: LuxOD47A BBk forward primer (Tm = 60 degrees C)
 +
* Reverse primer: LuxOD47A BBk reverse primer (Tm = 60 degrees C)
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Master Mix contents (for 15 tubes)
 +
* 10X Pfx Amplification buffer (75 uL)
 +
* 10mM dNTPs mixture (15 uL)
 +
* 50mM MgSO4 (22.5 uL)
 +
* Forward primer [10 uM] (15 uL)
 +
* Reverse primer [10 uM] (15 uL)
 +
* Platinum Pfx DNA polymerase [2.5 units] (10 uL)
 +
* Autoclaved distilled HOH (577.5 uL)
 +
 +
* TOTAL: 735 uL
 +
* PCR reaction volume for Master Mix: 49 uL
 +
 +
PCR steps:
 +
* Denaturation: 94 degrees C, 5 minutes
 +
* Amplification: 36 cycles of {
 +
** Denaturation (94 degrees C, 15 seconds);
 +
** Annealing (58-66 degrees C, 30 seconds);
 +
** Extension (68 degrees C, 1.5 minutes)}
 +
* Final extension: 68 degrees C, 15 minutes
 +
* Hold temperature: 4 degrees C
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 +
12 tubes were prepared to span the gradient, each of which contained 1 uL of template DNA and 49 uL master mix. A negative control with ddHOH in lieu of template DNA was also prepared.
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 +
Results:
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The gradient PCR products were visualised on a 1% agarose gel, as shown below. The right-most lane is the negative control, although it can’t really be distinguished in the results. This doesn’t seem to have worked, so it will be repeated later.
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[[Image:June4.png|700px]]
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Latest revision as of 04:01, 19 October 2009

University of Calgary

UNIVERSITY OF CALGARY



THIS MONTH

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]


NOTEBOOK PAGE INDEX
Carol
Chinmoyee
Emily
Fahd
Iman
Jamie
Jeremy
Katie
Kevin
Mandy
Patrick
Prima
Stefan
Vicki



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]



JUNE 4, 2009


CAROL

Modelling Papers

The modelling team continued exploring Simbiology and looking at several papers in how genetic modelling can work and what areas should our iGEM team should explore.


EMILY

Biobricking LuxOD47A

  • Objective: To form linear copies of LuxOD47A (currently inside TOPO T/A) with BioBrick ends.

Materials and methods:

  • Template: LuxOD47E C4-8 in TOPO T/A [40 ng/uL]- original concedntration = [409.5 ng/uL]
  • Forward primer: Lux0-F-RS star(Tm = 60 degrees C)
  • Reverse primer: LuxO-R-RS primer (Tm = 60 degrees C)

Master Mix contents (for 15 tubes)

  • 10X Pfx Amplification buffer (75 uL)
  • 10mM dNTPs mixture (15 uL)
  • 50mM MgSO4 (22.5 uL)
  • Forward primer [10 uM] (15 uL)
  • Reverse primer [10 uM] (15 uL)
  • Platinum Pfx DNA polymerase [2.5 units] (10 uL)
  • Autoclaved distilled HOH (577.5 uL)
  • TOTAL: 735 uL
  • PCR reaction volume for Master Mix: 49 uL

PCR steps:

  • Denaturation: 94 degrees C, 5 minutes
  • Amplification: 36 cycles of {
    • Denaturation (94 degrees C, 15 seconds);
    • Annealing (58-66 degrees C, 30 seconds);
    • Extension (68 degrees C, 1.5 minutes)}
  • Final extension: 68 degrees C, 15 minutes
  • Hold temperature: 4 degrees C

12 tubes were prepared to span the gradient, each of which contained 1 uL of template DNA and 49 uL master mix. A negative control with ddH2O in lieu of template DNA was also prepared.

Results:

The gradient PCR products were visualised on a 1% agarose gel, as shown below. Lanes 1-12 contain template and labe 13 is a negative control with ddH20 which shows some contamination. The bands are at the right size (~1.4 kb) to suggest that LuxOD47E is in fact in the vector. We will proceed by isolating plasmid and sending it down for DNA sequencing.

2009.06.04.LuxOD47E BBKAmf+PCR2.jpg


FAHD

Marketing and Outreach for June 4th 2009

Today, I continued filling out funding applications for some pharmaceutical companies. Some of these companies include Pfizer Canada, Merck Frosst Canada and Glaxosmithkline Canada. I almost got them completed and once they are done, I will run them through

I continued working on the Campus Fair agenda as the Campus Fair Day way coming close.


JAMIE

Gradient PCR amplification luxOU with Biobrick suffix/prefix from TOPO vector

pPFX (Invitrogen, CA) was used to amplify luxOU from the TOPO BL plasmid with luxOU gene-specific primers with the appropriate suffix or prefix (Tm: 68oC). Denaturation at 94oC for 5 minutes followed by 36 cycles of 94oC for 15sec, 58-66oC for 30sec , 68oC for 2 minutes. Final extension at 68oC for 15 minutes. Visualization of 3uL of product on 1% agarose gel, 120V.

Calgary luxOU GradPCR.jpg


Figure. 1% agarose gel (100V) for gradient PCR amplification of luxOU with Biobrick suffix and prefix. pPFX amplified 40ng of TOPO BL with luxOU. Gene specific primers with flanking Biobrick restriction sites were used at annealing temperatures ranging from 58oC (left) to 66oC (right). Lanes 2-13 are samples from each reaction carried out at different temperatures. Lane 14 is a negative control. Expected band size is 2kb. 5μL of GeneRuler 1kb Plus DNA Ladder was loaded.



JEREMY

Preparation of TOP10 Competent Cells

In order to replicate plasmid and ensure successful transformation in the summer, today’s work was devoted to making TOP10 competent cells.
To test whether the cells were competent, pBluescript was used as a positive control, and grown on LB-Ampicillin plates. Colonies were seen the next day, verifying that the cells were made competent.


KATIE

Improving Usability

I have found a decent way to ask questions and allow users to answer in a better way then entering things into chat. By using the llDialog function we can also communicate on negative channels, which is not possible in the chat window and helps to divide communication happening between people and between people and objects or objects and other objects.

So, I will be making everything entered in chat for the PCR machine into dialog prompt where avatars may pick from specific choices. This also gets rid of the risk of spelling errors and increases usability.


KEVIN

Modelling papers

In order to characterize our system to the best of our ability, we have chosen the part F2620 as our role model for characterization. I have looked into and read papers regarding the rate of reactions, such as the rate of the de-phosphorylation of LuxO.


PATRICK

Early Development

Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day.

First began charting out the differences between the types of biobrick part that I wanted to support. Initially thought each type would get its own type of (ie a Promoter Script existing only in promoters), but later resolved to create a single script for to run all of the types for maintainability; and to specify the part's type in the same way internal name and next part in the sequence's names are specified.

Ran some more tests of how the messaging system works in SL, and created six different versions of the device over the course of the day. I began using version numbers to track how my scripts had changed, and was already up to versions 4 and 3 for some scripts, things were changing very quickly early on.


PRIMA

Marketing and Outreach

Today, Fahd and I were planning our work for the Campus Fair this Saturday. We decided that we wanted to do the following:

Pipetting competition - for children aged 8 - 13 using colored dye - gloves, lab coats and goggles were to be used. Bacteria crafts - design your own bacteria using foam and pipe cleaners and glitter - for children aged 3 - 8 Draw - pretending tube caps were bacteria and people had to guess how many bacteria are in the bag. The answer was 500!

I followed up and emailed almost all the companies that I was in charge of contacting. I haven't heard back from most of them but I'll continue to contact them.


VICKI

Gradient PCR for biobrick cloning of LuxOD47A pieces

Note: I have switched with Jeremy: I will now be dealing with LuxOD47A and he will be handling LuxPQ

Purpose: To form linear copies of LuxOD47A (currently inside TOPO T/A) with BioBrick ends.

Materials and methods:

  • Template: LuxOD47A in TOPO T/A [40 ng/uL]
  • Forward primer: LuxOD47A BBk forward primer (Tm = 60 degrees C)
  • Reverse primer: LuxOD47A BBk reverse primer (Tm = 60 degrees C)

Master Mix contents (for 15 tubes)

  • 10X Pfx Amplification buffer (75 uL)
  • 10mM dNTPs mixture (15 uL)
  • 50mM MgSO4 (22.5 uL)
  • Forward primer [10 uM] (15 uL)
  • Reverse primer [10 uM] (15 uL)
  • Platinum Pfx DNA polymerase [2.5 units] (10 uL)
  • Autoclaved distilled HOH (577.5 uL)
  • TOTAL: 735 uL
  • PCR reaction volume for Master Mix: 49 uL

PCR steps:

  • Denaturation: 94 degrees C, 5 minutes
  • Amplification: 36 cycles of {
    • Denaturation (94 degrees C, 15 seconds);
    • Annealing (58-66 degrees C, 30 seconds);
    • Extension (68 degrees C, 1.5 minutes)}
  • Final extension: 68 degrees C, 15 minutes
  • Hold temperature: 4 degrees C

12 tubes were prepared to span the gradient, each of which contained 1 uL of template DNA and 49 uL master mix. A negative control with ddHOH in lieu of template DNA was also prepared.

Results:

The gradient PCR products were visualised on a 1% agarose gel, as shown below. The right-most lane is the negative control, although it can’t really be distinguished in the results. This doesn’t seem to have worked, so it will be repeated later.



June4.png

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