Team:Calgary/27 July 2009

• Finished writing modeling paper that summarizes what we will be doing once the circuit is completed. The experiments should not take long to accomplished and these results can be tabulated in Matlab quickly.
• Plated the week-long ligation of the promoter library in both XL Gold Ultra competent cells and Top 10 cells, but there is no growth.
• A reason that we thought of that might have caused minimal colonies is the phosphatase step. The 32 primers that we have synthesized does not have 5' phosphates. If we phosphatase the vector, that might result in no ligation.
• Re-started restriction enzyme digestion on vector pCS26 with XhoI and BamHI at 37^oC for 4 hours. Then I ligated the promoter with the vector with Quick Ligase for 5 minutes. Then I transformed the plasmids into both XL Gold Ultracompetent cells and Top 10 cells. Incubate at 37^oC for 16 hours.

• Did a construction digest of the J13002-LuxOD47E construct and B0015 terminator with XbaI/ PstI/ SpeI enzymes.
• Transformation into TOP10 cells and plated on AC plates.
• Hopefully things will go smoothly from here and my circuit will be fully-contructed by Thursday!

• Today I talked with Fahd, Stefan and Mandy about our Ethics paper as well as our conference. Mandy has a location in mind and she want Fahd and I to log into Second Life and explore the ethics area a bit. Mandy had a chance to look at our paper first thing this morning and she made some good suggestions for areas to add on to the introduction, bringing in some of the stuff that we learned about in the webinar. I spent a large part of today re-writing that part and I will have a copy of the rough draft of our paper by tonight!

-Fraiser Milner Casgrain LLP

-Graymont Western Canada Inc. 

7) I wrote a rejection thank you letter for APotex Canada and asked them if i could have some contacts who would be able to sponsor us.

Positive control = LuxPQ-B0015-R0040-LuxOU-B0015 in AK3

PCR conditions

Result: the only bands that appeared on the gel were those of the ladders. Therefore, start new construction.

Colony PCR of ∆LuxPQ in psB1AC3 using BBK CP F/R and LuxPQ F/R primers
Purpose: To verify the presence of ∆LuxPQ in psB1AC3 (to verify a successful plasmid switch)
Protocol

Isolating plasmid from LuxPQ-B0015-R0040-LuxOU-B0015 (AC) and LuxPQ (in AC)
Purpose: isolate and measure concentrations of pure plasmids.

Construction of LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)

Purpose: To construct LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)
Protocol
Recipient

Query: Pqrr4+I13500 Forward
Subject: Pqrr4+I13500 Reverse

>lcl|789 
Length=1034

Score = 1663 bits (900),  Expect = 0.0
Identities = 916/923 (99%), Gaps = 6/923 (0%)
Strand=Plus/Minus

Query  128   CATGAAACTAGAGCCCCGCACACTTGCGGGGCTTTTTAATTTTGAATTTCTTTCTTATTA  187
             ||||||||||||||||||||||| |||||||| ||||||||||||||||||||  |||||
Sbjct  1034  CATGAAACTAGAGCCCCGCACAC-TGCGGGGC-TTTTAATTTTGAATTTCTTT-NTATTA  978

Query  188   AAACGCCATTTTTCTGATAAATGTATTAGTAGCAATGCGCATGGTGGCATATTTGCATCA  247
             |||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  977   AAACGCCA-TTTTCTGATAAATGTATTAGTAGCAATGCGCATGGTGGCATATTTGCATCA  919

Query  248   TTTTGCATTTTGCAAATGCGATTTGCAAAATGCGTGCTCAATAAAGCACCAATATGCATC  307
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  918   TTTTGCATTTTGCAAATGCGATTTGCAAAATGCGTGCTCAATAAAGCACCAATATGCATC  859

Query  308   AGGATCGAAGAAAAAAGGCGTTTTTAAAAGTTGGCACGCATCGTGCTTTATACAGATACT  367
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  858   AGGATCGAAGAAAAAAGGCGTTTTTAAAAGTTGGCACGCATCGTGCTTTATACAGATACT  799

Query  368   AGAGAAAGAGGAGAAATACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCC  427
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  798   AGAGAAAGAGGAGAAATACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCC  739

Query  428   AATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGG  487
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  738   AATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGG  679

Query  488   TGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACT  547
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  678   TGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACT  619

Query  548   ACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAG  607
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  618   ACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAG  559

Query  608   ATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGT  667
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  558   ATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGT  499

Query  668   ACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAA  727
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  498   ACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAA  439

Query  728   GTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGA  787
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  438   GTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGA  379

Query  788   TGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCAT  847
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  378   TGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCAT  319

Query  848   GGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGA  907
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  318   GGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGA  259

Query  908   TGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGT  967
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  258   TGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGT  199

Query  968   CCTTTTACCAGACAACCATTACCTGTCCACACA-TCTGCCCTTTCGAA-GATCCCAACGA  1025
             ||||||||||||||||||||||||||||||||| |||||||||||||| |||||||||||
Sbjct  198   CCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGA  139

Query  1026  AAAGAGAGACCACATGGTCCTTC  1048 
             |||||||||||||||||||||||
Sbjct  138   AAAGAGAGACCACATGGTCCTTC  116

From it, we can see that the Forward sequence has mismatches towards the bottom, and Reverse sequences has mismatches near the beginning, which makes sense. Because the two sequences match perfectly in the middle, I can assume that I have the right plasmid in these cells.

Next step for this would be to make these cells competent again for Emily and Vicki to put their mutant circuits into it.

Seeing the biobrick simulator systems in action in the [http://igemcalgary.blogspot.com/2009/07/patrick-and-biobrick-simulator.html blog video] I put together was an excellent way to point out its flaws!

Made one simple but huge upgrade to all of the proteins that bind DNA: caused them to glow briefly when the binding event is accepted by the DNA. No more banging that RNAP on the DNA wondering whether it will take, or whether it wasn't close enough yet! Now it lets you know, as soon as you see your RNAP light up you can stop dragging it and let it get to business!

Also, decided today to scrap multimeric proteins, despite all the work I'd done on them. Putting your TetR together before you can use it is just a pain, transcribing/translating it twice before you can use it is a pain, representing larger protein complexes accurately in SL is nearly impossible, and it all adds very little of value to the simulation. Removing this functionality was surprisingly easy to do without introducing any new bugs, and the unbind screen in the HUD still remains useful for getting your now single object proteins to detach from DNA.

The only reason that I might resuse the type of binding I had before, is if I have time to introduce cofactors to the sim. It only makes sense for IPTG to associate with LacI, for example, and this would add quite a lot of power to the simulation.

Emailed John Winter (Account Manager) for Corning Life Sciences and he’s giving us filtered pipet tips and conical tubes (total of approx $1300)……so they are our bronze sponsors

Called Company 2 – left a message  follow up tomorrow

Called Company 3– apparently my last message was sent to the right person but since he never got back to me I got his personal email address and sent him (marketing VP) the sponsorship pkg  follow up July 29

Emailed Canadian society of Life Sciences for sponsorship …follow up July 29

Called Company 4 – left a msg  follow up tomorrow

Research and emailed the following oil & gas industries: - Prairie Mines and Royalty Ltd – related to iGEM through pipelines and biofilms - North American Construction Group – related to iGEM through pipelines and biofims - Northwest Hydraulics Consultants – cleaning water… Wrote a thank you letter to Jon’s dad and got the rest of the marketing team to look it over and gave it to Jon. Then I looked up some T-shirts companies and looked up deals.

Tomorrow - Follow-up with Sigma Aldrich tomorrow- currently looking over sponsorship pkg, - Call some of the companies I found earlier about T-shirts and try to get discounts/quotes - Talk about next mass bake sale w/ team - Talk about other fundraising activities w/ team

What is it? Extracting GFP from a jellyfish and putting it in a bacteria, which then glows green.

How do you do it? Simply click the jellyfish to receive the protein. Then, open the inventory of the bacteria, put the GFP inside, click on it and watch it glow!

Why would I do this? This will help people get used to using the SL inventory system because it will be required in the lab portion of our island. At the same time, it will be a visual representation of how cool it is to extract this stuff from animals and put it to use in bacteria.

Problems: Once I put the GFP inside the glowing bacteria stays like that. I've been using a sleep script for it to turn off but that doesn't eliminate the original GFP in the inventory so if I left it like this the GFP would build up inside. This would not be optimal, so I've obtained a script from Katie (she also helped me with the inventory script) to delete the protein inside. I hope to get this working by tomorrow.

In other news, I brainstormed a few pathways for the Synthetic Kingdom so people don't get lost and wonder around like Brownian motion. I also visited other islands in order to get an idea of how to structure the Help Island the people who come to our sim teleport on. Soon, I will have a rough draft of the narrative and path people follow (the only thing I have decided on is glowing platforms. They are ESSENTIAL).