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| | * After completion of making competent cells, transformed ligated products (pCS26+promoters ligated using NEB Quick Ligase) into XL Gold Ultracompetent cells. These cells were plated on LB+Kan agar plates and was incubated overnight at 37<sup>o</sup>C. | | * After completion of making competent cells, transformed ligated products (pCS26+promoters ligated using NEB Quick Ligase) into XL Gold Ultracompetent cells. These cells were plated on LB+Kan agar plates and was incubated overnight at 37<sup>o</sup>C. |
| | * As well, as a control, we transformed pBluescript into both XL Gold Ultracompetent cells and Top 10 Cells and plated cells on LB plates to incubate overnight at 37<sup>o</sup>C. | | * As well, as a control, we transformed pBluescript into both XL Gold Ultracompetent cells and Top 10 Cells and plated cells on LB plates to incubate overnight at 37<sup>o</sup>C. |
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| | *Did a miniprep of overnight cultures and took concentrations. | | *Did a miniprep of overnight cultures and took concentrations. |
| - | *Set up a Restriction Digest for verification (that the B0015 terminator is present in my contruct) with XbaI and PstI. If successful, we expected to see bands at about 1.7 kb and 3 kb for the contruct (J13002-LuxOD47E-B0015) and the psb1ac3 vector respectively. | + | *Set up a Restriction Digest for verification (that the B0015 terminator is present in my contruct) with XbaI and PstI. If successful, we expected to see bands at about 1.7 kb and 3 kb for the contruct (J13002-LuxOD47E-B0015) and the psb1ac3 vector respectively. Ran RD products on a 1% agarose gel at 120V with 1.0kb+ GeneRuler DNA Ladder. See gel ohoto below. |
| | <Br> | | <Br> |
| | Lane 1- J13002-LuxOD47E-B0015 C5, cut with XbaI and PstI | | Lane 1- J13002-LuxOD47E-B0015 C5, cut with XbaI and PstI |
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| | Lane 10- Size control- BBK LuxOD47E | | Lane 10- Size control- BBK LuxOD47E |
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| | *Analysis: From this gel it looks like colonies 5, 6 and 7 may have worked (contain the B0015 terminator) as we see two bands at the expected sizes. Colony 8 has some random bands, so I will ignore this one. Colony 6 looks the most promising as it is the cleanest (no random bands). I will proceed by sending this colony down for sequencing. | | *Analysis: From this gel it looks like colonies 5, 6 and 7 may have worked (contain the B0015 terminator) as we see two bands at the expected sizes. Colony 8 has some random bands, so I will ignore this one. Colony 6 looks the most promising as it is the cleanest (no random bands). I will proceed by sending this colony down for sequencing. |
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| | *Wrote out a lab blog for this week that I shall post tomorrow morning. | | *Wrote out a lab blog for this week that I shall post tomorrow morning. |
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| - | WIKI CODING HERE
| + | Was one of a handful of people who were in and out of the process for making competent cells (following Sambrook et al., 2001 protocol). |
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| + | Plasmid PCR redo from July 21 to verify signaling circuit in AC plasmid |
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| - | WIKI CODING HERE
| + | Purpose: use PCR to verify the presence of PQ-B-R-OU-B in the psB1AC3 vector for colonies 6 and 7, and to have a clean negative control lane. A pTaq PCR was set up using BBK CP F/R and LuxPQ F and LuxOU R primers using the following conditions: 94ºC for 3 minutes; 36X (94ºC for 30 seconds; 55ºC for 45 seconds; 72ºC for 6 min 30 sec); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel. The only bands seen on the gel were those of the positive control (PQ-B-R-OU-B in AK), and we must therefore either repeat this PCR, or use a different verification technique (i.e. Restriction Digest). |
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| | * What is a gene? | | * What is a gene? |
| | * What is DNA? etc. | | * What is DNA? etc. |
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| - | Two types of competent cells were made today, and they were TOP10 and XL Gold. Top 10 cells were made because we were running out on them, and XL gold was made because Top10 seemed to be not competent enough for Carol’s luciferase. | + | Two types of competent cells were made today, and they were TOP10 and XL Gold. Top 10 cells were made because we were running out on them, and XL gold was made because Top10 seemed to be not competent enough for Carol’s luciferase. |
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| + | Wiki Notebook Page Construction & Housekeeping |
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| - | WIKI CODING HERE
| + | Updates sections for all teams are up. Lab section has both a notebook that goes by day (calendar) and a section for each part (so, like the reports. Carol wrote up modelling, lab, and overall project descriptions. |
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| | + | FINALLY figured out how to insert wiki code into HTML tables. I had no idea you could just end <html> tags in the MIDDLE of tables, put in wiki code, and then continue with the <html> again afterwards. This is extremely useful for the main sections, as wikicode allows us to have a mini table of contents at the top of the page, so I can just put navigational/and mini introductions in the left hand bar. |
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| | + | Jen Hill sent us a few photos from dragon’s den and presentation workshops, so they have been added to the Alberta Events page. |
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| - | WIKI CODING HERE
| + | Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day. |
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| | + | Spent much of today doing research, about potential systems to incorporate into the simulator levels, and of other elements of the cell that might be worth representing. Still trying to wrap my head around how to do something like the arabinose operon, which requires folding DNA, or how to do the Lux operon, which requires distinct compartments. |
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| | + | Came up with two potential systems for the levels: a positively autoregulated system (ie, permanent switch for cell memory), and an [http://www.nature.com/nature/journal/v456/n7221/full/nature07389.html] interesting oscillator. This inspired me to investigate regulation motifs known to systems biology in greater detail. |
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| | + | My work in Second Life was stopped cold though, when the TetR object that I had been working on got knocked away from my working space accidentally, and I wasn't able to chase after it fast enough. The TetR object simply vanished, and the script inside had seen many changes but hadn't been backed up in more than a week! |
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| | + | I searched the internet for solutions to the problem, and after all those suggestions failed to find my TetR I resorted to using a server administration tool to return *all* of my object across the entire island to my inventory. It still didn't show up, which indicated to me that my TetR might be well and truly gone. |
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| | + | In how many other development environments do you lose work because a representation of a physical object containing your code gets lost? |
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| | Company 6 | | Company 6 |
| - | -called the lady for the 20th time in the last 10 days, she’s not in her office and there’s APPRENTLY no one else who handles sponsorship. GRRRR. But I’m gonna keep harassing her. Customer service APPRENTLY has no way to get a hold of her but they saw coming in and out of her office so she’s not on vacation. | + | -called the lady for the 20th time in the last 10 days, she’s not in her office and there’s APPARENTLY no one else who handles sponsorship. GRRRR. But I’m gonna keep harassing her. Customer service APPARENTLY has no way to get a hold of her but they saw coming in and out of her office so she’s not on vacation. |
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| | GlaxoSmithKline | | GlaxoSmithKline |
| | - this is the new company that I found yesterday. Called, left a voice mail. I need her email address so as soon as I get a hold of her I’ll talk to her and send pkg | | - this is the new company that I found yesterday. Called, left a voice mail. I need her email address so as soon as I get a hold of her I’ll talk to her and send pkg |
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| - | I also edited the newsletter that Jamie sent to the marketing team last night. I’ll discuss the corrections tomorrow. Then I checked over a couple of marketing emails that Jeremy and Fahd were sending out companies. Then Fahd and I wrote up an email toVWR and Mandel Scientific requesting specifically the reagents and pipets that Thane wanted us to order. We checked the email over with Jeremy before sending. | + | I also edited the newsletter that Jamie sent to the marketing team last night. I’ll discuss the corrections tomorrow. Then I checked over a couple of marketing emails that Jeremy and Fahd were sending out companies. Then Fahd and I wrote up an email toVWR and Mandel Scientific requesting specifically the reagents and pipettes that Thane wanted us to order. We checked the email over with Jeremy before sending. |
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| | For about 20 mins, I was pointing out a few things/suggestions for the wiki with Mandy. | | For about 20 mins, I was pointing out a few things/suggestions for the wiki with Mandy. |
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| - | I was working in Second Life for most of today, specifically the | + | I was working in Second Life for most of today, specifically the eukaryotic cell. I scrapped the previous endoplasmic reticulum and installed a new one and it looks much better. Scripts were added to make it "sparkle" in order to give it that ribosomal look. Smooth ER was also created today. Experimentation with particle systems proved to be a worthwhile endeavor. I had trouble with changing the settings, but I eventually figured out how. the result was vesicles coming out of the Golgi body periodically (you should check it out!). I have also chosen a location for the cell and thus, was able to script some of the organelles to move and expand. Tomorrow there will be a meeting with Gregor about ethics at 10 so that should be interesting. |
| - | eukaryotic cell. I scrapped the previous endoplasmic reticulum and | + | |
| - | installed a new one and it looks much better. Scripts were added to | + | |
| - | make it "sparkle" in order to give it that ribosomal look. Smooth ER | + | |
| - | was also created today. Experimentation with particle systems proved | + | |
| - | to be a worthwhile endeavor. I had trouble with changing the settings, | + | |
| - | but I eventually figured out how. the result was vesicles coming out | + | |
| - | of the Golgi body periodically (you should check it out!). I have also | + | |
| - | chosen a location for the cell and thus, was able to script some of | + | |
| - | the organelles to move and expand. Tomorrow there will be a meeting | + | |
| - | with Gregor about ethics at 10 so that should be interesting. | + | |
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CAROL
Making Competent Cells
- Prepared sterilized LB broth for growing cells. Followed procedure found in the protocol page for making competent cells.
- After completion of making competent cells, transformed ligated products (pCS26+promoters ligated using NEB Quick Ligase) into XL Gold Ultracompetent cells. These cells were plated on LB+Kan agar plates and was incubated overnight at 37oC.
- As well, as a control, we transformed pBluescript into both XL Gold Ultracompetent cells and Top 10 Cells and plated cells on LB plates to incubate overnight at 37oC.
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EMILY
Verification Digest of J13002-LuxOD47E-B0015
- Did a miniprep of overnight cultures and took concentrations.
- Set up a Restriction Digest for verification (that the B0015 terminator is present in my contruct) with XbaI and PstI. If successful, we expected to see bands at about 1.7 kb and 3 kb for the contruct (J13002-LuxOD47E-B0015) and the psb1ac3 vector respectively. Ran RD products on a 1% agarose gel at 120V with 1.0kb+ GeneRuler DNA Ladder. See gel ohoto below.
Lane 1- J13002-LuxOD47E-B0015 C5, cut with XbaI and PstI
Lane 2- C5 uncut
Lane 3- C6 cut
Lane 4- C6 uncut
Lane 5- C7 cut
Lane 6- C7 uncut
Lane 7- C8 cut
Lane 8- C8 uncut
Lane 9- Size control- J13002-LuxOD47E
Lane 10- Size control- BBK LuxOD47E
- Analysis: From this gel it looks like colonies 5, 6 and 7 may have worked (contain the B0015 terminator) as we see two bands at the expected sizes. Colony 8 has some random bands, so I will ignore this one. Colony 6 looks the most promising as it is the cleanest (no random bands). I will proceed by sending this colony down for sequencing.
- Wrote out a lab blog for this week that I shall post tomorrow morning.
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FAHD
Marketing for July 22nd 2009
Today, I concentrated my energies at marketing our iGEM project and looking at the ethical aspect of our project. Here is what I did today:
1) I was very happy when i read NEXEN INC.’s e-mail that they would give us 3000 dollars. Nexen Inc. would be our first silver sponsor.
2) I e-mailed a thank you letter to Nexen Inc. and also sent them PDF copies of our Sponsor webpage. I will also mail in a thank you letter.
3) Helped approve/edit the July's newletter.
4) E-mailed the lab equipment and reagents list to VWR
5) Called Life Technologies and left her a voicemail.
6) Called EMD biochemicals and left a voicemail for us.
7) Called Bietz Resources Ltd. He will not be able to help us but he has offered his services(contacts) to our team.
8) Called Albian Sands Energy Ltd. and left a voicemail for him
9) Called Alberta Pacific Forest Industries Inc. and left a voicemail for him.
10) Helped out in the lab: I was putting tubes in the racks and closing the lids.
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JAMIE
Competent cells
Was one of a handful of people who were in and out of the process for making competent cells (following Sambrook et al., 2001 protocol).
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JEREMY
Plasmid PCR redo from July 21 to verify signaling circuit in AC plasmid
Purpose: use PCR to verify the presence of PQ-B-R-OU-B in the psB1AC3 vector for colonies 6 and 7, and to have a clean negative control lane. A pTaq PCR was set up using BBK CP F/R and LuxPQ F and LuxOU R primers using the following conditions: 94ºC for 3 minutes; 36X (94ºC for 30 seconds; 55ºC for 45 seconds; 72ºC for 6 min 30 sec); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel. The only bands seen on the gel were those of the positive control (PQ-B-R-OU-B in AK), and we must therefore either repeat this PCR, or use a different verification technique (i.e. Restriction Digest).
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KATIE
Implementing the Notecard Reader
The bacterial transformation activity was fixed in the morning and I added to the conditionals that tell you if the question has already been answered so once a question has been answered it cannot be answered again unless the quiz has been completed or reset at any time. I also added a further instructions option to the PCR machine, which gives a notecard for now, but I am interested in trying the instructions as audio. The instructions are for those who throw themselves into the lab first thing or those who need a reminder of how to operate their tools in second life.
The first and second construction site now use the notecard reader and I added more options to the Sequencing station so more can be sequenced. Now I just need to know the sequence of base pairs for each notecard I will be using. At the moment the notecards I am testing with contain random data. Ligating two genes together may be something I will be adding to the restriction digest activity once I can get it all working together since it is just adding more conditions. However, I would have to redesign how you could put things together so I believe I will hold off coding anymore of this until the activity has been finished. Tomorrow morning I can quickly complete the third construction site script and test with generic notecards.
One thing I'm not really comfortable with the dataserver event yet, which is very important to reading the notecard so I think it would be useful for me to trace the code within this event and determine exactly what it does just so I completely understand how notecard reading works.
Since we should probably get started on the base of the spiral that will contain levels I have started to write up some more notecards that are even more basic than the ones I have been writing (transcription, translation etc.) like:
- What is a gene?
- What is DNA? etc.
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KEVIN
1. Sequencing
I have sequenced by Colony 1 and 9 of my Pqrr4+Boo34 (RBS) because they were the closest to the positive controls and the cleanest lanes. For this, I have only used Biobrick CP forward sequencing primers, and no reverse. I only used forward and not reverse because the piece is quite short, and forward primer is enough to read every base pair. These are hopefully being sequenced right now and I am waiting for the result to be in.
2. Competent cells.
Two types of competent cells were made today, and they were TOP10 and XL Gold. Top 10 cells were made because we were running out on them, and XL gold was made because Top10 seemed to be not competent enough for Carol’s luciferase.
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MANDY
Wiki Notebook Page Construction & Housekeeping
Updates sections for all teams are up. Lab section has both a notebook that goes by day (calendar) and a section for each part (so, like the reports. Carol wrote up modelling, lab, and overall project descriptions.
FINALLY figured out how to insert wiki code into HTML tables. I had no idea you could just end tags in the MIDDLE of tables, put in wiki code, and then continue with the again afterwards. This is extremely useful for the main sections, as wikicode allows us to have a mini table of contents at the top of the page, so I can just put navigational/and mini introductions in the left hand bar.
Jen Hill sent us a few photos from dragon’s den and presentation workshops, so they have been added to the Alberta Events page.
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PATRICK
Research
Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day.
Spent much of today doing research, about potential systems to incorporate into the simulator levels, and of other elements of the cell that might be worth representing. Still trying to wrap my head around how to do something like the arabinose operon, which requires folding DNA, or how to do the Lux operon, which requires distinct compartments.
Came up with two potential systems for the levels: a positively autoregulated system (ie, permanent switch for cell memory), and an [http://www.nature.com/nature/journal/v456/n7221/full/nature07389.html] interesting oscillator. This inspired me to investigate regulation motifs known to systems biology in greater detail.
My work in Second Life was stopped cold though, when the TetR object that I had been working on got knocked away from my working space accidentally, and I wasn't able to chase after it fast enough. The TetR object simply vanished, and the script inside had seen many changes but hadn't been backed up in more than a week!
I searched the internet for solutions to the problem, and after all those suggestions failed to find my TetR I resorted to using a server administration tool to return *all* of my object across the entire island to my inventory. It still didn't show up, which indicated to me that my TetR might be well and truly gone.
In how many other development environments do you lose work because a representation of a physical object containing your code gets lost?
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PRIMA
Marketing Continuation
Today:
I followed up with:
Company 1: called Dr. Maloney’s asst. and she said he’s unable to meet with us this month but she’ll discuss our sponsorship pkg with him. I was thinking of calling his assistant again tomorrow. Then I’ll follow up on the sponsorship pkg on July 208h
Company 2
Currently looking over our sponsorship pkg. I called and the Marketing In-charge was not in the office so I left a msg: follow up July 23 (tomorrow). Everytime I leave a, I’m saying that we’re not asking for large grants and that our donations have ranged from 200 to 6000 dollars so any contribution will be greatly appreciated by the team. I told the rest of the marketing team to say the same thing.
Company 3
-called to follow up- the Service Desk lady said she forwarded our pkg to the right person and since they didn’t reply back it probably means they’re not interested. I asked for that person’s contact info, but she said it’s against their policy to give out staff contact info.
Company 4
-currently looking at our sponsorship pkg – called but she wasn’t in the office, left a msg so follow up July 23
Company 5
-called, not in the office, so I left a msg. Follow up July 23.
Company 6
-called the lady for the 20th time in the last 10 days, she’s not in her office and there’s APPARENTLY no one else who handles sponsorship. GRRRR. But I’m gonna keep harassing her. Customer service APPARENTLY has no way to get a hold of her but they saw coming in and out of her office so she’s not on vacation.
GlaxoSmithKline
- this is the new company that I found yesterday. Called, left a voice mail. I need her email address so as soon as I get a hold of her I’ll talk to her and send pkg
I also edited the newsletter that Jamie sent to the marketing team last night. I’ll discuss the corrections tomorrow. Then I checked over a couple of marketing emails that Jeremy and Fahd were sending out companies. Then Fahd and I wrote up an email toVWR and Mandel Scientific requesting specifically the reagents and pipettes that Thane wanted us to order. We checked the email over with Jeremy before sending.
For about 20 mins, I was pointing out a few things/suggestions for the wiki with Mandy.
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STEFAN
Building inside the eukaryotic cell
I was working in Second Life for most of today, specifically the eukaryotic cell. I scrapped the previous endoplasmic reticulum and installed a new one and it looks much better. Scripts were added to make it "sparkle" in order to give it that ribosomal look. Smooth ER was also created today. Experimentation with particle systems proved to be a worthwhile endeavor. I had trouble with changing the settings, but I eventually figured out how. the result was vesicles coming out of the Golgi body periodically (you should check it out!). I have also chosen a location for the cell and thus, was able to script some of the organelles to move and expand. Tomorrow there will be a meeting with Gregor about ethics at 10 so that should be interesting.
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VICKI
More report writing
I wrote a preliminary introduction and most of the materials and methods section for the LuxO D47A mutant.
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