Team:Calgary/27 July 2009

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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 Positive control = ∆LuxPQ in psB1AK3 PCR conditions 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). This Restriction digest was set up overnight with the following sets of enzymes: XbaI and PstI (insert) and SpeI/PstI (recipient).



 KATIE Continuing to Progress in the Lab I had to change the order script within the restriction digest tubes since everything needed to be added backward, which turned out to be a problem with my inventory function, which I now have adding inventory names to a list in the opposite order so that it is now correct. I completed the movement scripts I have been working on within the restriction digest tubes so they will move to the water bath, the heating block and then the insert tube moves to the incubator and I think that maybe I will make it wait there for a while until the avatar goes to get it as well as placing it on a timer so if someone does not go to retrieve it will return to its original position anyway and just not give anything and reset the whole script. Bacteria colony note cards have been made to give when the texture has been changed on the plates, when they are empty, they are inactive, just needed to determine what permission I will need to set on these note cards so that they cannot be copied to inventory, cannot be modified but are still able to be read although not when contained within an object. I am changing names within construction sites, but decided to keep some of the ones I was going to change general instead of part information, which will now be within the note cards, which people can look at after it is given. I would really like to add more genes to the biobrick construction activity so then I may ask what biobricked part they would like to perform restriction digest on, which provides more of a variety of genes that could be used (I think we will stick with the same promoter/rbs and terminator since it lessens time spent performing restriction digest on similar parts, but people may create circuits that serve different functions). I have planned out how to begin the lagging strand simulation, which will have to use detection or collision in order to be successful since it will detect (DNA polymerase that is) when it comes across a RNA primer it will begin adding nucleotides. Once the polymerase has traveled down the strand it will signal to I will try to get the replication out into the open at the base of the levels since I have just been using the lab and then taking everything into my inventory. I will also be changing the PCR machine a bit for the activities so that it may use specific primer you will apparently be given in a starter folder as well as adding a product you may receive.



 KEVIN Sequencing result of Pqrr4+I13500 (GFP+RBS) Purpose of this sequencing was to verify the presence of both Pqrr4 and I13500 for Emily to test her mutant circuit. I have received the sequencing result today. At first, I was disappointed because the sequencing that was done with Forward primer had a perfect match with expected Pqrr4 sequence, but only matched 96% of I13500, and as for the sequencing done with Reverse primer, it had perfect match with the expected I13500 sequence, but only matched 99% of Pqrr4. However, because polymerases make mistakes after about 1000bp, this often occurs. So I decided to align the Forward sequence and the reverse sequence. The following is the result:

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.



 MANDY Changing Up the Wiki Did regular update of blog onto different sections of the page, so that will keep being updated and it’s great that everybody has been keeping up with posting on the required days. I fixed up the notebook template so it can be accessed easily from the header menu, and it now applies to all team members (not just the lab people). I’ve also edited the instructions for posting in it to include some more fundamental formatting requirements (with many helpful suggestions from Jamie). TO DO: Completion of DNA Extraction Activity in Second Life The DNA extraction activity is finally done, and it works! (I think). This officially makes me the slowest scripter ever D:<. I am still thinking of a way to make it easier to do. It is intuitive in that it gives you direct instructions, but right now, people can kind of jump in to the middle of it without doing the first bit (which I’m not sure is a good thing – it IS a long activity after all). Following the DNA extraction activity, I’ve completed most of the instructions/description for the third lab mission. Tomorrow, I will be checking with Katie to make sure the alternative components fit in both the PCR and restriction digest activities to give the desired products (eg. PCR to make a linearized biobricked part). We will also be changing notecards and mission instructions to reflect the changes to these activities. Speaking of notecards, I’m editing the one I wrote for DNA extraction. I’ve started writing up the instructions for the first lab mission (bacterial transformation) – it’s a ridiculous task (INVISIBLE BACTERIA :D), but I thought it would be fun. This was inspired by seeing Katie make a thing you can wear on your avatar to make you mostly invisible- we might be able to give something like that as a prize?
 * more blog update inclusion
 * Edit Fahd’s news story about NUTV
 * Post video of NUTV
 * Edit Sponsor page
 * Teach Prima how to edit Acknowledgement page
 * edit content already there
 * add more to intros for human practices and second life



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

Seeing the biobrick simulator systems in action in the 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.



 PRIMA Sponsorship deals continued and hotel searches in Boston Today: Called Company 1- said they’re not sponsoring any outside projects this year. But they’ll keep us in mind for next year. Wrote a thank you letter and asked for contacts.

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

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</a> STEFAN GFP zone Today I added a new "exhibit" to the underwater kingdom!

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).

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</a> VICKI Materials&methods and results section work Both are complete (save the culture conditions for the bacteria in the Materials/Methods), although I think that they’ll require substantial editing and reorganisation. I had to take extra time to prepare my sequence results in nice form with the biobrick sites highlighted, which took longer than it was supposed to. I’m sending you the PDF now and I’ll convert it to Word later tonight.