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Team:Calgary/29 July 2009
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| - | + | Wiki Updates | |
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| - | + | * Updated the wiki pages today, so there is full descriptions for all the parts of our project. As well, started working on the protocols for our wiki today. | |
| + | * The transformation with pCS26 vector and the sigma 70 promoters ligated by T4 Invitrogen Ligase showed small colonies on plates late in the afternoon, left overnight in the incubator. | ||
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| - | + | Verifictaion Digest of J13002-LuxOD47E-B0015 | |
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| - | + | *Today I did a minprep of my overnight cultures of J13002-LuxOD47E-B0015. I took the concentration and did a restriction digest with XbaI and PstI. I ran this on a gel this afternoon with uncut plasmids of each colony as a control as well as LuxOD47E BBK (uncut) and J13002-LuxOD47E (uncut) as size controls. See gel photo below. | |
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| + | [[Image:2009.07.29J13002-LUuxOD47E-B0015VerRD.jpg|350px]] | ||
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| + | Lane 1- J13002-LuxOD47E-B0015 Trial 2, C1 cut with XbaI and PstI | ||
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| + | Lane 1- C 1, uncut | ||
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| + | Lane 3- C2, Cut | ||
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| + | Lane 4- C2, Uncut | ||
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| + | Lane5- C3, Cut | ||
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| + | Lane 6- C3, Uncut | ||
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| + | Lane 7-LuxOD47E BBK (Uncut) | ||
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| + | Lane 8- J13002-LuxOD47E (Uncut) | ||
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| + | Outreach/ Ethics | ||
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| + | *Stefan, Fahd and I met to talk about Ethics this morning to make a plan of what needs to be done for the Erhics conference. We're planning the conference for some time in late September/ early October, but we want to start inviting people now. So today we made a list of possible speakers as well as people that we will invite just to come and listen. | ||
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| + | *We decided that the first step would be to familiarize ourselves with Second Life. So, this afternoon I made an account on SecondLife and explored a little. I walked through the boards with instructions on them to learn how to talk, move, ect. Mandy then gave me access to our Island, so I got a chance to walk around and look at some of the stuff that our team has been looking on. Tomorrow I'm going to explore an ethics area that Mandy knows about as well as try to find some conferences to sit in on to get a better idea of how they are run. Fahd and I need to get planning our Ethics conference so I think it's a really good idea to become familiar with what we can do in Second Life so that we can decide where we want to have it, what we need to build for it, how we're going to advertise it within Second Life, etc. | ||
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| - | + | Second Life Conference, July Newsletter, Education and Outreach: | |
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| - | + | Today I sent out July letters to the Following comapnies: | |
| + | |||
| + | 1)Nexen Inc. | ||
| + | <br> | ||
| + | 2)New England Biolabs | ||
| + | <br> | ||
| + | 3)Alberta Ingenuity Fund | ||
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| + | 4)Boheringer Ingleheim Canada | ||
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| + | 5)Apotex Canada | ||
| + | |||
| + | My Ethics teammates and I today discussed some ways to conduct the Ethics conference on synthetic biology in the online virtual world called Second Life. | ||
| + | |||
| + | I also had a meeting with our education and outreach team. We discussed ways to promote iGEM in high schools and universities. | ||
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| - | + | Competent Cell Testing: Results! | |
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| - | + | The competents cells appeared to be very competent (or at least satisfactory to lab standards!). | |
| + | |||
| + | [[Image:Calgary_XL_Gold_test.png|400px]] | ||
| + | [[Image:Calgary_TOP10_test.png|400px]] | ||
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| + | I also contacted Worley Parsons and Spectra Energy for sponsorship opportunity (to no avail). | ||
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Plasmid was isolated from an overnight culture of cl lamba using Sigma GenElute Mini Prep Kit. The concentration and purity of the isolated plasmid was then measured with the NanoDrop spectrophotometer. Restriction digest was performed with XbaI and PstI in React 2 (Invitrogen) buffer for 2 hours at 37ºC. Cut plasmid and uncut plasmid were then run on a 1.0% agarose gel, no bands were seen for either column. The isolated plasmid was then specked again, to verify the initial concentration, which turned out to be accurate. An overnight Restriction digest was then set up as described above; however, 600ng of DNA were used instead of 200ng. | Plasmid was isolated from an overnight culture of cl lamba using Sigma GenElute Mini Prep Kit. The concentration and purity of the isolated plasmid was then measured with the NanoDrop spectrophotometer. Restriction digest was performed with XbaI and PstI in React 2 (Invitrogen) buffer for 2 hours at 37ºC. Cut plasmid and uncut plasmid were then run on a 1.0% agarose gel, no bands were seen for either column. The isolated plasmid was then specked again, to verify the initial concentration, which turned out to be accurate. An overnight Restriction digest was then set up as described above; however, 600ng of DNA were used instead of 200ng. | ||
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| - | A colony PCR was set up with colonies of the construction LuxPQ-B0015-R0040-LuxOU-B0015 in psB1AC3 as the template. Seven colonies were chosen. This PCR was performed with BBK CP F/R primers (these anneal on the BBK vector just outside the cloning site) and LuxPQ F / LuxOU R primers (these are gene specific primers). Due to the long extension time for this PCR (6min and 20 seconds) it is run overnight and then held at 4ºC upon completion. For PCR conditions, refer to PCR performed on July | + | <br> |
| + | A colony PCR was set up with colonies of the construction LuxPQ-B0015-R0040-LuxOU-B0015 in psB1AC3 as the template. Seven colonies were chosen. This PCR was performed with BBK CP F/R primers (these anneal on the BBK vector just outside the cloning site) and LuxPQ F / LuxOU R primers (these are gene specific primers). Due to the long extension time for this PCR (6min and 20 seconds) it is run overnight and then held at 4ºC upon completion. For PCR conditions, refer to PCR performed on July 27 2009. Restreaks of the seven colonies were made and overnight liquid cultures were made so that plasmid can be isolated tomorrow. | ||
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| - | + | A Second Look through Lab Activities | |
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| - | + | Today was spent going through the lab activities and modifying the scripts along the way. I revised the whole PCR script so that all of it behaves like the general PCR activity and there is not a problem with locating items and the script will no longer proceed if anything is missing, which I believe happened because of a function modification since it definitely did not do that before. Mandy has been having difficulty with the items we have to add although I have not even when we try the same activity, which leads us to believe that the names of her items and the items we need do not match up so we will be comparing names of objects tomorrow. | |
| + | |||
| + | I also changed the scripts within: | ||
| + | * Phosphatase treatment | ||
| + | * Restriction digest | ||
| + | * Gel electrophoresis (Mandy made some changes here too) | ||
| + | |||
| + | They now search for items the same way as the new PCR machine so the order items are added does not make any difference. I then had to change the PCR machine’s cleaning methods so that more objects were accounted for as necessary and were not deleted before they could be used. I believe this could have also been a problem for some of the items in the PCR machine since they were possibly being deleted before they could be checked since the machine did not recognize the materials as something that needed to be kept. | ||
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| + | Unfortunately, I was unable to make significant headway on the DNA replication animation due to all of my time spent in the lab. I was able to build a primase that rez the RNA primers, but I still need to set the positions the enzymes will have to get to along the single-stranded DNA. | ||
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| + | I plan to insert instructions into the bacterial transformation activity tomorrow, which now allows for bacteria to be grown on plates if more than six questions are answered correctly, each plate having their own specific textures. When the plates with growth are touched avatars are given a short note card out of the three I made on Tuesday and each still require a little modification, but now avatars can complete this activity. I still need to add deletion to the activity so that inventory can be cleaned out though, but the rest is operational and Mandy was able to complete it successfully. Providing significant instruction for avatars will be very important within the lab so I will get the other group members to try and follow the instructions I have made tomorrow. | ||
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| - | + | 1. Plasmid Isolation | |
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| - | + | Plasmid isolation of K082003 (GFP + LVA) and Q04510 (inverter) from TOP10 cells | |
| - | + | The purpose of isolating these parts is to construct the reporter circuit with K082003 and response circuit with Q04510. Because the plasmids were relatively pure and high in concentration, I can continue with the construction of the Reporter circuit. | |
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| + | 2. Construction of the reporter circuit | ||
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| + | <b>a. Restriction digest</b> | ||
| + | There were two ways of constructing this circuit: one being cutting out Pqrr4 + B0034 and inserting it into K082003, and another one is K082003 being the insert and Pqrr4 + B0034 being the vector. However, I forgot that K082003 is in a plasmid that has kanamycin resistance, and just went ahead with putting K082003 into the Pqrr4 + B0034 plasmid. This was done by cutting the sequenced Pqrr4 + B0034 with speI and PstI, and cutting K082003 with XbaI and PstI. Apparently though, the BBK CP primers that were made by Thane are only compatible with pSB1A__ vectors, meaning if I had put Pqrr4 + B0034 into K082003 vector, I would not be able to verify it using the current BBK CP primers. | ||
| + | <br> | ||
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| + | <b>b. Construction</b> | ||
| + | The insert, being the K082003, was put away into the -20 freezer, and the vector, being the Pqrr4+B0034, was phosphotased. The two were than ligated together, then transformed into TOP10 cells. | ||
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| + | 3. Verification of K082003 | ||
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| + | Because I have to make sure the cells that iGEM provided/sent us contain the right parts, I have ran the restriction digested K082003 on a 1% gel. This part is about 756bp long, and the following is the picture of the gel: | ||
| + | <center> | ||
| + | [[Image:Calgary_2009.07.29.GFP+LVA.png|200px]] | ||
| + | </center> | ||
| + | As you can see, there is a band slightly above 700bp region in the cut K082003 lane, meaning the piece of interest is likely to be present in the plasmid. In the Uncut lane, multiple bands are visible, some of them being nicked and supercoiled plasmids, resulting in running at different speeds on the gel. | ||
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| - | + | Second Life: Trouble shooting notecards and scripting | |
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| - | + | Today, I tried running all of the lab missions and 'general' procedures for the equipment, I noted when things weren't quite working with one another, or if instructions weren't clear enough. I was able to fix a few typos and scripting errors, but we still need to streamline a lot of the activities. That is what I'll be working on tomorrow with Katie. We also introduced Emily and Carol to Second Life, hopefully Emily and Fahd will get a better idea of how to run conferences and hosted events in Second Life after a bit of exploration. | |
| + | <br> | ||
| + | Tomorrow, Katie and I will also be giving our notecards some more editting and readthroughs and then sending them to over (every time I read mine, I think of something else I have to add, which is kind of bad...) | ||
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| + | Wiki Updates and Clean-up | ||
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| + | Did the daily blog/news update for the wiki. I also converted a few elements on all the pages to templates (eg. the notebook sidebar) to make it easier to edit multiple pages at once, and make the code cleaner so people can read it more easily. We figured out that the easiest way to make tables is to type them in excel, save them as a html file and then copy the source. | ||
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| - | + | A new day, a new bunch of code to write | |
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| - | + | Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day. | |
| + | |||
| + | Toyed with the idea of making enzymes which produce some small molecules or other, or degrade the same (AiiA for example). Couldn't figure out a good way to control such objects, once again, clicking is already reserved for movement, so it would be necessary to create yet another screen in the HUD to manage these enzymes. | ||
| + | |||
| + | Today was a day of choices though, most of the rough edges on the previously implemented features had been sanded away, now I got to choose what to try to implement next. The two big contenders were cofactors for dna binding proteins, and the biobrick designer aka biobricker, with the interaction checker a distant third. | ||
| + | |||
| + | I settled on the biobricker, because the ability to easily build devices of any configuration is such a huge part of what the simulator is meant to be, and it will actually be an extremely useful tool for building and testing all of the remaining parts as well. Cofactors would increase the range of the simulation, allowing lac, tet and lux operons to be more accurately described. But that just means more extremely painful assembly by hand of even more biobricks for each level! Interaction checker would have the same problem, to really put the checker through its paces and test lots of different systems in different configurations means having lots of biobrick devices available, which means a working biobricker. | ||
| + | |||
| + | So, I got right to work, and produced this basic outline for the biobrick display by the end of the day: | ||
| + | |||
| + | display += "Biobricker "; | ||
| + | display += "X Name "; | ||
| + | display += "X Name "; | ||
| + | display += "X Name "; | ||
| + | display += "X Name "; | ||
| + | display += "< P T A R S C >"; | ||
| + | display += " "; | ||
| + | display += "< M H R B D h >"; | ||
| + | |||
| + | The last and third to last rows are all command buttons (more than on any other screen!), whose functions have changed very little to this day. | ||
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| - | + | Today: | |
| + | I called the following companies: | ||
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| + | Let’s start with the bad news: Called Company 1– she spoke to Dr. Moloney and he said they spent all the money that was allocated for charity/sponsorship so they can’t sponsor us. When I asked to meet him anyways to discuss next year’s sponsorship…she said they allocate money in January so call back to meet up sometime in November | ||
| + | |||
| + | Company 2 : Called VP but he he wasn’t there so left a msg follow up tomorrow | ||
| + | |||
| + | Company 3: Called left a msg, I need an email address to send a msg follow up tomorrow | ||
| + | |||
| + | Company 4: looking over pkg …..left a msg follow up july 31 or tomorrow | ||
| + | |||
| + | Company 5: Called President and left a msg follow up july 31 | ||
| + | |||
| + | Company 6: sent sponsorship pkg before to the Marketing leader …called today and left a voicemail…follow up tomorrow | ||
| + | Cayman chemicals needs more time to think it over…follow up july 31 or august 3 | ||
| + | |||
| + | BioAlberta’s president wants to meet me next week and he said he’ll call me when he comes back to town. | ||
| + | |||
| + | Then I sent out the July newsletter to about 20 companies (I didn’t list them all so plz refer to Gmail excel sheet b/c I updated it so you’ll know which companies I’ve sent the newsletter to) | ||
| + | |||
| + | After our outreach meeting, I started jotting down ideas for outreach activities which I’ll share with Emily and fahd tomorrow since they didn’t have time today. | ||
| + | |||
| + | Also I confirmed the bake sale for next Wednesday with the rest of the team. If you guys are ok with this then I’ll go book tables tomorrow. Oh and I also have to call Linden Labs tomorrow to see if they’ll sponsor us. | ||
| + | Yesterday I wrote the general acknowledgement blurb and today I wrote up the acknowledgement for each individual on the wiki. | ||
| + | |||
| + | I also wrote up an email to Ms Ellechuk (my high school bio teacher) which I need to finish and edit tomorrow. | ||
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| - | + | Order in the Kingdom | |
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| - | + | I managed to set up the exhibits somewhat and develop a semblance of | |
| + | order. I moved everything to eye level since the person is walking | ||
| + | along the path rather than flying. First is the <i>E.coli</i> | ||
| + | that you "kill" using the chat by saying "colicin". The smelly | ||
| + | bacteria follows, although I forgot to mention this in previous | ||
| + | updates. Not that impressive...I just put a script in so the bacteria | ||
| + | emits a colored smoke periodically (smells like pizza). Next is the | ||
| + | GFP jellyfish/bacteria combo. I realized that the jellyfish from which | ||
| + | the protein is isolated from looks nothing like the abomination I have | ||
| + | created so I built an almost exact replica of <i>Aequorea victoria</i> | ||
| + | and segregated them to an area. Then the Bioremediation | ||
| + | Station (pretty good title, eh?) is next, and there's some unfinished | ||
| + | stairs leading up to it. | ||
| + | |||
| + | why did I do it like this? well we don't want to confuse our visitors | ||
| + | right off the bat. I figured "antibiotics kill bacteria" would be an | ||
| + | easy enough to concept to grasp. Also once they know that different | ||
| + | genes can do different things when expressed in bacteria (smelly), and | ||
| + | they can be extracted from animals (jellyfish) then the bioremediation | ||
| + | (an actual application) becomes easier to understand. | ||
| + | |||
| + | There was also a brief ethics meeting today. We all decided that it's | ||
| + | best if we planned out the Second Life conference as soon as possible. | ||
| + | Fahd and Emily are going to look up some ethics places in SL and | ||
| + | listen to some talks to see how they are organized. We are also | ||
| + | brainstorming about what people should be involved in our conference | ||
| + | as speakers. We are hoping to get people like Andrew from DIY Biology | ||
| + | and other important figures. Audience will also be important and other | ||
| + | than SL residents we are hoping to invite some educators that we met | ||
| + | along the way. Also, now that we're helping TU Delft, I bet at least | ||
| + | one person on their team would be willing to help us. | ||
| + | |||
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| - | + | Notebook writing | |
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| - | + | * The earlier entries took a long time because I was a lot less detailed than I should have been. The latter entries are easier to deal with because I actually took the time to write down what I was supposed to. Also, I had a better sense of what I was actually doing and where it fit into the project, so the better understanding is reflected in the entries. | |
| + | * I have finished typing up my notebook up to June 29. I will finish the rest over the weekend and upload it to the wiki shortly after that. | ||
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Latest revision as of 03:35, 14 September 2009
UNIVERSITY OF CALGARY
