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Team:Calgary/6 July 2009
From 2009.igem.org
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| - | + | Transformation of <i>luxCDABE</i> into both pSB1AK3 and pSB1AC3 | |
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| - | + | * Ligated <i>luxCDABE</i> into both pSB1AK3 and pSB1AC3 | |
| + | * Transformed ligated products into Top 10 cells and plated on AK plates for overnight incubation. | ||
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| - | + | Search for parameter values | |
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| - | </html> | + | </html>Looked through many paper for estimates of initial values for each species in our model and estimates for the k values present in the model. |
| - | + | <br>cell volume : 0.58 -0.69 um^3 | |
| + | <br>Kubitschek HE. Cell volume increase in Escherichia coli after shifts to richer media. J Bacteriol. 1990 Jan172(1):94-101 | ||
| + | <br>RNA polymerase transcription rate :70nt/sec | ||
| + | <br>Mutation per base pair per replication : Drake JW, A constant rate of spontaneous mutation in DNA-based microbes.Proc Natl Acad Sci U S A. 1991 Aug 1588(16):7160-4 Table 1 | ||
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| - | + | Construction Begins! | |
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| + | Now that we have our gene of interest, LuxOD47E, Biobricked, we can start construction to add the J13002 promoter and the B0015 terminator. We'll do two constructions in parallel and see if one of them works. | ||
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| + | * Did a restriction digest cutting the insert with EcoRI and SpeI and cutting the recipient with EcoRI and XbaI restriction enzymes. | ||
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| + | * Performed Antarctic Phosphatase Treatment (NEB) on the recipients, followed by ligation of the inserts and recipients. | ||
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| + | *Transformed J13002-LuxOD47E into TOP10 cells and plated on AC and C plates in 25 uL and 50 uL, left plates for overnight growth. | ||
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| + | *Did restreaks and made overnight cultures of colony2X LuxOD47E BBK. | ||
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| - | + | Marketing and Ethics for July 6th 2009 | |
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| - | + | Today, I e-mailed out our sponsorship package to Shell Canada, Western Canada Oil Spill Cleanups, ConocoPhillips and Syncrude Inc. I will make follow-up calls to them next week. I also continued to work on the July newsletter. | |
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| - | + | I also read some ethics papers regarding open sources VS closed sources. | |
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| - | + | Sequencing Results Analysis! | |
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| - | + | Sequencing results indicated successful construction of the luxOU construct. Glycerol stocks made. | |
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| - | Isolated | + | Isolated Fluorescent proteins were constructed behind of R0040 or J13002, depending on whether or not the fluorescent protein contained RBS, and transformed into TOP10 cells. |
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| - | + | Building Frenzy ala Second Life | |
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| - | + | Some more building design for the labs, platforms, etc. was done today. | |
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| - | + | Half Way Point: Taking Stock | |
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| - | + | Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day. | |
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| + | The major systems I had built to date included: | ||
| + | *DNA finding each other in polymer, communicating with RNAP | ||
| + | *binding/unbinding of TetR with each other, and DNA, and control by the HUD | ||
| + | *The HUD display system | ||
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| + | The messages passed between these parts were beginning to crowd the chat channels I was using, I came up with a few rules of thumb to keep things sane. Each script state gets its own channel to listen on instead of using a single channel for everything, and wherever possible messages from one part to another use the same format: 'owner's ID','destination object's id','message label' as the first three parts of the message. Some messages don't need any more information than that, and some are broadcast messages that don't need a destination object id either. Others would have several additional pieces of information, each separated by a comma. This system has been very reliable, and it takes practically no effort to set up reliable new messages types now. | ||
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| + | I believe I actually made a big diagram of all the messages to make sure that none were conflicting with each other, before realizing that if I just followed these rules there couldn't be any problems! | ||
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| + | Also made a list of what I hadn't implemented yet: | ||
| + | *Count mode, aka the Interaction Checker | ||
| + | *Polymer maker, aka the Biobricker (later fully implemented! wohoo!) | ||
| + | *DNA binding interactions, I had RNAP and repression working, but not translation/transcription or activation, and the system for termination was a little unstable too. (later fully rectified and implemented as well. yay!) | ||
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| + | I also wrote a [http://igemcalgary.blogspot.com/2009/07/welcome-my-name-is-patrick-king-i-am.html blog post] today. | ||
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| - | + | Marketing | |
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| - | + | Today I spoke to the President from BioAlberta and he generously offered to donate money to iGEM Calgary. BioAlberta was added to our Bronze list for sponsorship. | |
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| + | I also wrote up some emails to companies after researching the companies for the past few days. The rest of the marketing team edited the emails before I sent them off. I also call 8 other companies and 5 companies were interested in looking at our sponsorship package. I sent off the package and updated the iGEM marketing excel spreadsheet. I left a voicemail to the 3 companies and I'll follow up with them tomorrow or later this week. I got the contact information of the Marketing Lead for one of those 3 companies. My plan is to call the other 2 companies tomorrow to first: get a hold of someone in-charge of sponsorship and second: speak to them about our project, potential applications and how it may benefit their company. | ||
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| - | + | Scripting progress | |
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| - | + | I have gotten some books from the library on scripting. I'm learning by using bits ans pieces from other people's free scripts and modifying them to do different things. I have managed to make a script that is adjustable to make an object move around randomly. | |
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| - | + | Sequencing results from the J13002 + LuxO D47A + B0015 on pSB1AK3 that I prepared on Thursday | |
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| - | + | The sequencing results were returned from the samples of J13002 + LuxO D47A + B0015 on pSB1AK3 that I sent down last day. These were confirmed positive. I have included the sequencing results for one of the samples and the alignments immediately thereafter. These results are the same for both of the samples that I tested, indicating that the colonies that I tested have the proper sequence for J13002 + LuxO D47A + B0015 on pSB1AK3. | |
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| + | [[Image:J13002LuxOD47AB0015sequence.jpg|700px]] | ||
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| + | Alignment results | ||
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| + | [[Image:J13002LuxOD47AB0015comparison.jpg|700px]] | ||
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Latest revision as of 21:21, 20 October 2009
UNIVERSITY OF CALGARY
