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| - | Descriptive Title of What You're Doing
| + | Colony PCR to verify LuxPQ plasmid switch (from AC to AK – see July 8) and to verify PQ-B-R-OU-B (July 9) construct |
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| - | WIKI CODING HERE
| + | Purpose: To perform a colony PCR to verify the presence of the above constructs. Each Colony pTaq PCR reaction used the BBK CP F/R primers, each with the following similar conditions: 94ºC for 6 minutes; 36X (94ºC for 30 seconds; 55ºC for 45 seconds; 72ºC for 4 minutes for plasmid switch (or 6 min 10 sec for PQ-B-R-OU-B construct); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel (see below). For the plasmid switch, we can see that colonies 2, 5, 6, and 8 gave the desired size of LuxPQ (3.8kb), whereas in the signaling construct verification, we see desired sizes (~6.0kb) for colonies 11, 12 and 13. Next steps will be to isolate plasmid and proceed with further verification techniques on the colonies that appear to be what we want. |
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CAROL
Descriptive Title of What You’re Doing
WIKI CODING HERE
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CHINMOYEE
Looked at Markov Models ( Part of Optimization)
The Matlab Help files were helpful in providing basic background about Markov Models and their uses . I also tried to recall my MATH211 notes where we had briefly studied Markov Models.
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EMILY
Colony PCR for Verification of J13002-LuxOD47E-B0015 Construction
- Performed a colony PCR of J13002-LuxOD47E-B0015 to see if the B0015 terminator was seuccessfully cloned in, using BBK-CP forward and reverse primers and p Taq. Cycling conditions were the same as used for the last Colony PCR (see July 9th 2009 for details). PCR products were visualized on a 1% agarose gel and run with Generuler 1 kb+ DNA ladder along with J13002-LuxOD47E and LuxOD47E as size controls. See photo of gel below.
- Analysis: Lanes 1-4 are J13002-LuxOD47E-B0015 trial 1 (treating B0015 as the Insert). Lanes 5-8 are trial 2 (treating J13002-LuxOD47E as the Insert). Lane 9 is J13002-LuxOD47E (a size control), Lane 10 is LuxOD47E BBK (another size control) and Lane 11 is a negative control.
- This gel seems ot be kind of slanted, which isn't very good for size comparison. It looks like some of the first lanes of Trial 1 may have worked (contain the B0015 terminator) as they appear slighlty higher than other lanes, however we will run a new gel with the PCR product to be sure.
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FAHD
Marketing for July 10th 2009
TODAY, I concentrated my energies on marketing our iGEM project. The following are the companies I had contacted:
1) Contacted Sigma Aldrich
2) Contacted Nexen
3) Contacted Neuroimage Inc.
I will make follow up phone calls next week.
I also worked on my US visa application and attended our weekly meetings with my supervisors.
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IMAN
Descriptive Title of What You're Doing
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JAMIE
Descriptive Title of What You're Doing
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JEREMY
Colony PCR to verify LuxPQ plasmid switch (from AC to AK – see July 8) and to verify PQ-B-R-OU-B (July 9) construct
Purpose: To perform a colony PCR to verify the presence of the above constructs. Each Colony pTaq PCR reaction used the BBK CP F/R primers, each with the following similar conditions: 94ºC for 6 minutes; 36X (94ºC for 30 seconds; 55ºC for 45 seconds; 72ºC for 4 minutes for plasmid switch (or 6 min 10 sec for PQ-B-R-OU-B construct); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel (see below). For the plasmid switch, we can see that colonies 2, 5, 6, and 8 gave the desired size of LuxPQ (3.8kb), whereas in the signaling construct verification, we see desired sizes (~6.0kb) for colonies 11, 12 and 13. Next steps will be to isolate plasmid and proceed with further verification techniques on the colonies that appear to be what we want.
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KATIE
Descriptive Title of What You're Doing
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KEVIN
Weekly meeting: Second life
Each sectional members are now put on the spot for weekly updates, so we get more in-depth look at each accomplishments.
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MANDY
Descriptive Title of What You're Doing
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PATRICK
Reorganization and Planning
Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day.
Had a meeting today, got lots more feedback for the whole Second Life project from the point of view of an educator, extremely useful!
Produced a tentative list of levels for the simulator today:
- 1 my first transcription + biobrick parts
- 2 repressor
- 3 inducible repressor
- 4 repressor w. alosteric inhibition / lacI
- 5 activator
- 6 inducible activator
- 7 inhibitable activator
- 8 2 circuits. one constituitive, represses the other
- 9 bistable switch
- 10 repressilator
Began reorganizing the HUD's screens to be easier to navigate and understand. Came up with the idea of a Home screen, which is a base camp for each level, and links to all of the other features of the simulator HUD.
Also established the progression for visitors to the island, and filled in a few gaps:
Progression
- -basic controls
- -synthetic kingdonm
- -molecular bio basics
- --dna
- --central dogma
- --big picture: synthetic biology
- -biobrick tower (aka Biobrick Simulator)
- -lab (aka Virtual Lab)
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PRIMA
Mini-prep Lux OD47A from original plates and liquid cultures
Purpose: purify upto 20 (microg) of high-copy plasmid DNA from 1.5ml overnight cultures of E.coli in LB medium.
Procedures: followed the Qiagen mini-prep procedures.
I shadowed Vicky today.
Colonies 1 & 2 - Lux OD47A BBK from original plates
Colonies 3 & 4 - Lux OD47A BBK from liquid cultures
We used 2 samples of original Colony 6 and 2 samples of plate cultures.
The following chart is showing the Nano-drop results (concentration of DNA) obtained after isolating the plasmids.
| Plasmid | 260/280 | 260/230 | Concentration [ng/μL]
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| Lux OD47A -plate | 1.88 | 2.25 | 92.7
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| Lux OD47A -plate | 1.84 | 1.84 | 84.8
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| Lux OD47A - liquid culture | 1.91 | 2.20 | 69.6
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| Lux OD47A - liquid culture | 1.98 | 2.37 | 56.6
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STEFAN
Robots!
Yesterday, time was spent on building a robot (Biobert) that will act
as a guide to our lab and also the rest of the island. It had to be
built from scratch because the first one wasn't very good and creeped
people out because it transformed from a pipette and had an empty
soulless expression. Once it was built, Mandy added the script so it
could talk to people (including several secret phrases!). I designed
it to move its arms and mouth when touched and optimized to use the
least prims possible in order to make sure we have enough left for
other things on the island. Today, a second robot was also made in the
same fashion (Synthia) because Biobert was lonely and she needed a
friend. We also had an important meeting deciding on the structure of the Second Life areas.
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VICKI
Plasmid isolation
…was also done exactly as I described in my July 8 update. One of my tubes struggled through the lyse stage but the rest seemed to work better. The plasmid yield was not as lucrative as last time – around 70 ng/uL (100 uL sample), but it’s an improvement on the 7 ng/uL present the first time I conducted this procedure with these cells.
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