Team:Calgary/20 August 2009

From 2009.igem.org

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Human Practices for August 20th 2009
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WIKI CODING HERE
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Today, I continued to work on the ethics paper. I started reading literature on the environmental, legal and societal issues surrounding Quorum Sensing and I found a lot of interesting information that I can add on to the ethics paper. I will continue to work on the ethics paper over the next couple of days and try to improve it further.
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I also contacted some companies that were on my marketing list.  Most of these companies were Oil and gas development companies. I will do a follow-up with these companies next week.
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Using a 96 well microplate, AI-2 isolated from <i>Salmonella typhimurium</i> 14028 supernatant will be tested.  <i>V. harveyi</i> reporter strain MM32 (luxS- and luxN-) will luminesce in the presence of AI-2.  Controls used in the experiment include <i>S. typhimurium</i> SS007, <i>V. harveyi</i> BB120 and <i> E. coli</i> DH5a.  Overnight OD600 and luminescence (CPS) readings will be taken by a VICTOR (thank you Surette lab, especially Margot!). <br /><br />
Using a 96 well microplate, AI-2 isolated from <i>Salmonella typhimurium</i> 14028 supernatant will be tested.  <i>V. harveyi</i> reporter strain MM32 (luxS- and luxN-) will luminesce in the presence of AI-2.  Controls used in the experiment include <i>S. typhimurium</i> SS007, <i>V. harveyi</i> BB120 and <i> E. coli</i> DH5a.  Overnight OD600 and luminescence (CPS) readings will be taken by a VICTOR (thank you Surette lab, especially Margot!). <br /><br />
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Preliminary testing was done on the bacterial transformation and genomic DNA isolation activities in the virtual lab.  Additionally, I now have transparent wings which glow.  Cool beans.
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Preliminary testing was done on the bacterial transformation and genomic DNA isolation activities in the virtual lab.  Additionally, I now have transparent wings which glow.
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Fundamental Displays
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WIKI CODING HERE
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I recently remembered that I will have to start making cleaning scripts for a lot of the equipment in the lab to prevent clutter for easy maintenance. I started to make these and added them to bacterial transformation, the sequencing station and DNA extraction. It is not imperative that these be completed immediately since this type of abuse is not expected for testing.
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Added rho termination, but I am not satisfied with it yet and will continue to change where objects travel too although the pushing part looks satisfactory I need a better place for the mRNA strand. I have no idea how I am going to do the hairpin termination at this time but it will probably mean I have to fiddle with rotation again. There is now a complementary strand of DNA that will change its bases when an avatar makes changes to the template strand (the bottom one strand in this animation) so that the two strands will remain complementary to one another.
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Also did some more wiki updates.
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1. Measuring fluorescence of GFP
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WIKI CODING HERE
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Yesterday, varieties of bacterial cultures were grown in order to test the expression of GFP's. The fluorescence of GFP are measured by the plate reader, and following are the results that Emily and I were able to get today:
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*Fluorescent levels of different cultures (Stored in the plate reader for 5 hours after dilution of R0040+GFP)
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[[Image:Calgary_GFP_reading_5hrs_Plate_reader.png|700px]]
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*Fluorescent levels of different cultures (Stored in the shaker at 37˚C for 5 hours after dilution of R0040+GFP)
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[[Image:Calgary_GFP_reading_5hrs_shaker.png|700px]]
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We were forced to wait for 5 hours after the dilution of R0040+GFP culture as we were asked to leave the lab that had the plate reader by another organization. We were able to measure once quickly right after the dilution; however, with no accurate settings, meaning we cannot make any comparisons.
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As for the above results, it seems convincing enough for me to say those cells that have the reporter and mutant (LuxOD47E) circuits in them are doing something special that make their readings higher. Our KT1144 cells have a cosmid that contains <i>Qrr4</i> promoter driving an expression of GFP; thus, as shown by the data, express GFP in larger quantities when it has the LuxOD47E mutant than when it doesn't. Our TOP10 cells also have a plasmid containing <i>Pqrr4</i> and GFP, which behaves the same way as our KT1144 cells.
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Both data show that the fluorescence reading is about 2 times higher in cells that have both mutants and reporters than the cells that doesn't have mutant circuits. As we can see in well B7, the cells that have R0040, a constitutive promoter, driving GFP have a much higher reading than the rest, which is what was expected. Also as expected, diluting the R0040+GFP cells to 1/10, 1/50, and 1/100 ratios resulted in much lower readings of fluorescence. Two interesting things to note about the above collected data are:
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1. Both with and without mutant readings of TOP10 cells with reporter are than the readings of KT1144 cells. I do not know exactly why it is as such, but my guess is that different type of cells absorb different amount of wavelengths.
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2. The fluorescent levels of the cells that were grown for an extra 5 hours in the shaker were lower than the cells that grew in the plate reader, except for the cells that were diluted with fresh LB media. As above, I do not know the exact reason. Perhaps it is because the cells have already reached their 16~20 hours of growing overnight, growing for an extra 5 more hours at 37˚C killed the bacteria, decreasing the production of GFP, while the growth of the cells that were stored in the plate reader for an extra 5 hours slowed down due to a relatively colder temperature, thus being able to produce GFP more stably.
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As for the diluted cells with R0040+GFP, because they were transferred to a fresh media before the additional growth of 5 hours, storing them in the shaker at 37˚C led to faster replication, which sped up the production of GFP, while storing them in the colder plate reader slowed down the replication, which slowed the production of GFP.
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2. Sequencing results
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The sequencing results of <i>Pqrr4</i>+B0034+K082003 came back today. Unfortunately, B0034 was still not there despite the change of <i>Pqrr4</i>+B0034 colonies from C9 to C1. Surprisingly, the sequence for the previous construction that used <i>Pqrr4</i>+B0034 C9 actually match the sequence that came back today, which used <i>Pqrr4</i>+B0034 C1. (99% match) The following is the alignment of the two sequences:
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>lcl|28877
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Length=801
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Score = 1472 bits (797),  Expect = 0.0
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Identities = 800/801 (99%), Gaps = 1/801 (0%)
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Strand=Plus/Plus
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Query  9    GGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTATTAAGCTACTAA  68
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            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sbjct  1    GGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTATTAAGCTACTAA  60
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Query  69  AGCGTAGTTTTCGTCGTTTGCAGCAGGCCTTTTGTATAGTTCATCCATGCCATGTGTAAT  128
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            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sbjct  61  AGCGTAGTTTTCGTCGTTTGCAGCAGGCCTTTTGTATAGTTCATCCATGCCATGTGTAAT  120
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Query  129  CCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTT  188
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            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sbjct  121  CCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTT  180
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Query  189  CGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCC  248
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            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sbjct  181  CGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCC  240
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Query  249  AATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTG  308
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            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sbjct  241  AATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTG  300
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Query  309  TCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATT  368
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            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sbjct  301  TCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATT  360
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Query  369  GTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATC  428
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Sbjct  361  GTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATC  420
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Query  429  AATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACG  488
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            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sbjct  421  AATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACG  480
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Query  489  TGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGG  548
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            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sbjct  481  TGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGG  540
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Query  549  CATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTG  608
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            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sbjct  541  CATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTG  600
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Query  609  AACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGT  668
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            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sbjct  601  AACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGT  660
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Query  669  GCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGAC  728
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Sbjct  661  GCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGAC  720
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Query  729  AGAAAA-TTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAG  787
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Sbjct  721  AGAAAAATTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAG  780
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Query  788  TGAAAAGTTCTTCTCCTTTAC  808
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Sbjct  781  TGAAAAGTTCTTCTCCTTTAC  801
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And the following is the previous sequencing result that I have gotten for <i>Pqrr4</i>+B0034 that was used for construction:
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<b>Sequence of Pqrr4+B0034 (RBS) C1</b>
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Legend
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<font color="red">Vector contamination</font>
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<font color="green">BBK Restriction sites</font>
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<font color="yellow"><i>Pqrr4</i> Sequence</font>
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<font color="cyan">B0034 sequence</font>
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GGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTG</font>
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GAATTCGCGGCCGCTTCTAGA</font><font color="yellow">GTATCAGCAAAAACACTACGGTGGATAATCAGTAAAACCATGAAACTAGAGCCCCGCA
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CACTTGCGGGGCTTTTTAATTTTGAATTTCTTTCTTATTAAAACGCCATTTTTCTGATAAATGTATTAGTAGCAA
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TGCGCATGGTGGCATATTTGCATCATTTTGCATTTTGCAAATGCGATTTGCAAAATGCGTGCTCAATAAAGCACC
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AATATGCATCAGGATCGAAGAAAAAAGGCGTTTTTAAAAGTTGGCACGCATCGTGCTTTATACAGAT</font><font color="purple">ACTAGAG</font><font color="cyan">AAAGAGGAGAAA</font>
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<font color="green">TACTAGTAGCGGCCGCTGCAG</font>
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<font color="red">TCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTT
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CGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGG
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CGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCG
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TAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG
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TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC
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CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC
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GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT
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CGTCTTGAGTCCAACCCGGTAGACACGACTTATCGCCACTG</font>
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When I carefully analyzed today's <i>Pqrr4</i>+B0034(C1)+K082003, I could find part of the B0034 embeded between <i>Pqrr4</i> and K082003. The following explains this further:
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<b>Legend</b>
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<Font color="yellow">end of <i>Pqrr4</i> sequence</font>
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<Font color="green">Biobrick restriction sites</font>
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<Font color="cyan">Part of B0034 (Complete sequence is AAAGAGGAGAAA</font>
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<Font color="white">Part of scar? (Complete sequence is ACTAGAG)</font>
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<Font color="pink">Beginning of K082003</font>
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<Font color="yellow">...AGAT</font><font color="green">ACTAGAG</font><font color="cyan">AAAGAGG</font><font color="white">CTAG</font><font color="pink">ATGCGTA...</font>
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A mistake of sending two wrong colonies of <i>Pqrr4</i>+B0034 for sequencing is highly unlikely, and also a mistake of using a wrong colony for construction two times is highly unlikely, especially because I was not willing to mess up the construction the second time. I will have to investigate this further.
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The Next Big Thing: Levels
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WIKI CODING HERE
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As the iGEM season starts winding up, it is becoming very urgent to make the second life project publicly presentable! I spent today getting organized and nailing down the design for the last big feature that will live in the Biobrick Simulator heads up display object: levels.
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The simulator is not a game (though it will be fun), but the 'playing through levels' concept is widely known amongst young people these days, and it's much more appealing than something like 'concepts', or 'chapters', even though those terms are better descriptions of what a 'level' will be. Each level is a self contained set of parts, with the goal of introducing one or two new features of the simulator and concepts in molecular biology to the user. The preliminary list of levels is a good illustration of the progression I'm looking for:
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*First Transcription
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*Activators (a simple activatable device, and an activator)
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*Repressors (a simple repressable device)
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*External Activator (the activator/repressor is produced by a device, instead of given)
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*External Repressor
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*Negative Autoregulation (Control)
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*Positive Autoregulation (Memory)
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*Or Gates
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*Inverters (Not Gate)
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*And Gates (two inverters w. dual repressed promoter)
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*Bistable Toggle Switch
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*Tristable Toggle Switch (See [http://openwetware.org/wiki/IGEM:Brown/2007/Tri-Stable_Switch Brown's 2007 project.])
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*Repressilator
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Unfortunately, there are also a good number of systems I had wanted to make into levels, but whose features aren't yet supported by the simulator's engine! Including, but not limited to:
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*Lac Operon
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*Tet Operon
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*And Gates (additional varieties)
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*Lux Operon / Quorum Sensing
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*Feedforward systems (various types)
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*XOR Gates
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*Ara Operon?
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In addition, a number of the levels I will be building would benefit from features these other systems require, especially inducers for LacI and TetR. Inducers make a huge amount of sense for systems like the toggle switches, for getting from one state to another; and also for positive autoregulation, for turning on/off cellular memory. That's to say nothing of incorporating environmental signals like temperature, osmolarity, and light, each of which have sensor parts within the registry, leading to more systems and more levels... but I digress! In a perfect world I'd have a chance to implement all of these things before putting together a selection of levels. But since I would like a version of the simulator with most of its *features* in place before the end of summer, choices have to be made about content. And who knows, there might still be time.
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Fortunately, none of the code that remains to be written for the levels is very complicated. The Level Selection screen makes the levels and their parts available, the Home screen gives some basic guidance for each level, and the Parts screen makes supplementary objects, and replacement objects in case you happen to delete the ones you're working with (or wish to combine multiple levels' parts together. That will work just fine!) All three screens are necessary for the levels to work, and none have new or complicated features, it's mostly a question of adapting code I've already written for other purposes.
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And all of that isn't even the last major portion of the island that needs to be developed! To support all of this biobrickery, users will first visit a zone describing the basics of molecular biology and biochemistry (in Second Life's interactive style). Katie is already working on a display of DNA replication for this zone. Also fortunately, each of the exhibits I have in mind are more or less stand-alone displays, as compared to the biobrick simulator where every part interacts with half a dozen others, so I don't anticipate it taking as long.
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But, with development as with biology, a good safe bet is to multiply all your time estimates by three...
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I ran the gene-specifc PCR with aiiA-F/R primers and ran it with a 1% gel at 100V.
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I had contaminated my negative control (which was just the mastermix) with my aiiA gene. Since all three bands are around 756 bp, we can say that the gene exists.
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I followed up with couple pharmaceutical companies. Some of our potential sponsors are currently deciding on sponsoring iGEM Calgary. Again, I couldn't get hold of other companies by phone but I left a message with a call back number. I also emailed those companies with a follow- up email. The good news is that Beckman Coulter has generously donated $200.00 to our iGEM Team this year. Unfortuantely, Alda Phamaceuticals Corporation is unable to sponsor our team this year.  
I followed up with couple pharmaceutical companies. Some of our potential sponsors are currently deciding on sponsoring iGEM Calgary. Again, I couldn't get hold of other companies by phone but I left a message with a call back number. I also emailed those companies with a follow- up email. The good news is that Beckman Coulter has generously donated $200.00 to our iGEM Team this year. Unfortuantely, Alda Phamaceuticals Corporation is unable to sponsor our team this year.  
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Testing...Testing
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WIKI CODING HERE
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Today, a generic avatar was created (aptly named Generic Lennie) that was in a separate group that had access to our island. He was used to test each area on our sim. We don't want visitors to be able to move, objects, change script and make new permanent objects. For the synthetic Kingdom everything was in order:
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*All objects were not modifiable
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*Generic Lennie was able to move the vitamins, Bad Guy/Champion cells, quorum sensing bacteria and oil clean-up bacteria, which is ok because they have a self-destruct script inside
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*GFP station drag and drop worked!
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This means that the Synthetic Kingdom is unofficially done! 
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...And some more work on the modelling front
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WIKI CODING HERE
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* Chinee and I met with Afshin and Iman to align our models with each other. The SimBiology and Membrane Computing reactions were subsequently updated, so that we are now modelling the same thing where applicable. The only major differences lie in that the membrane computing model accounts for LuxS (which the SimBiology model omits) and the membrane computing model accounts for AI-2 molecules leaving the cell and going to another one (while ours just includes a reverse reaction for AI-2 binding to LuxPQ, as we are only modelling performance in one cell).
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* I helped Chinee download Latex and will provide a lesson on how to use that platform
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* I looked up degradation rates for GFP:LVA mRNA
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* Our sensitivity analysis crashes with the addition of more reactions to our model. I worked on fixing that, to little avail. I will try again tomorrow.
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Latest revision as of 21:40, 10 September 2009

University of Calgary

UNIVERSITY OF CALGARY



THIS MONTH

August
MTWTFSS
          [http://2009.igem.org/Team:Calgary/1_August_2009 1] [http://2009.igem.org/Team:Calgary/2_August_2009 2]
[http://2009.igem.org/Team:Calgary/3_August_2009 3] [http://2009.igem.org/Team:Calgary/4_August_2009 4] [http://2009.igem.org/Team:Calgary/5_August_2009 5] [http://2009.igem.org/Team:Calgary/6_August_2009 6] [http://2009.igem.org/Team:Calgary/7_August_2009 7] [http://2009.igem.org/Team:Calgary/8_August_2009 8] [http://2009.igem.org/Team:Calgary/9_August_2009 9]
[http://2009.igem.org/Team:Calgary/10_August_2009 10] [http://2009.igem.org/Team:Calgary/11_August_2009 11] [http://2009.igem.org/Team:Calgary/12_August_2009 12] [http://2009.igem.org/Team:Calgary/13_August_2009 13] [http://2009.igem.org/Team:Calgary/14_August_2009 14] [http://2009.igem.org/Team:Calgary/15_August_2009 15] [http://2009.igem.org/Team:Calgary/16_August_2009 16]
[http://2009.igem.org/Team:Calgary/17_August_2009 17] [http://2009.igem.org/Team:Calgary/18_August_2009 18] [http://2009.igem.org/Team:Calgary/19_August_2009 19] [http://2009.igem.org/Team:Calgary/20_August_2009 20] [http://2009.igem.org/Team:Calgary/21_August_2009 21] [http://2009.igem.org/Team:Calgary/22_August_2009 22] [http://2009.igem.org/Team:Calgary/23_August_2009 23]
[http://2009.igem.org/Team:Calgary/24_August_2009 24] [http://2009.igem.org/Team:Calgary/25_August_2009 25] [http://2009.igem.org/Team:Calgary/26_August_2009 26] [http://2009.igem.org/Team:Calgary/27_August_2009 27] [http://2009.igem.org/Team:Calgary/28_August_2009 28] [http://2009.igem.org/Team:Calgary/29_August_2009 29] [http://2009.igem.org/Team:Calgary/30_August_2009 30]
[http://2009.igem.org/Team:Calgary/31_August_2009 31]


NOTEBOOK PAGE INDEX
Carol
Chinmoyee
Emily
Fahd
Iman
Jamie
Jeremy
Katie
Kevin
Mandy
Patrick
Prima
Stefan
Vicki



CALENDAR

May
MTWTFSS
        [http://2009.igem.org/Team:Calgary/1_May_2009 1] [http://2009.igem.org/Team:Calgary/2_May_2009 2] [http://2009.igem.org/Team:Calgary/3_May_2009 3]
[http://2009.igem.org/Team:Calgary/4_May_2009 4] [http://2009.igem.org/Team:Calgary/5_May_2009 5] [http://2009.igem.org/Team:Calgary/6_May_2009 6] [http://2009.igem.org/Team:Calgary/7_May_2009 7] [http://2009.igem.org/Team:Calgary/8_May_2009 8] [http://2009.igem.org/Team:Calgary/9_May_2009 9] [http://2009.igem.org/Team:Calgary/10_May_2009 10]
[http://2009.igem.org/Team:Calgary/11_May_2009 11] [http://2009.igem.org/Team:Calgary/12_May_2009 12] [http://2009.igem.org/Team:Calgary/13_May_2009 13] [http://2009.igem.org/Team:Calgary/14_May_2009 14] [http://2009.igem.org/Team:Calgary/15_May_2009 15] [http://2009.igem.org/Team:Calgary/16_May_2009 16] [http://2009.igem.org/Team:Calgary/17_May_2009 17]
[http://2009.igem.org/Team:Calgary/18_May_2009 18] [http://2009.igem.org/Team:Calgary/19_May_2009 19] [http://2009.igem.org/Team:Calgary/20_May_2009 20] [http://2009.igem.org/Team:Calgary/21_May_2009 21] [http://2009.igem.org/Team:Calgary/22_May_2009 22] [http://2009.igem.org/Team:Calgary/23_May_2009 23] [http://2009.igem.org/Team:Calgary/24_May_2009 24]
[http://2009.igem.org/Team:Calgary/25_May_2009 25] [http://2009.igem.org/Team:Calgary/26_May_2009 26] [http://2009.igem.org/Team:Calgary/27_May_2009 27] [http://2009.igem.org/Team:Calgary/28_May_2009 28] [http://2009.igem.org/Team:Calgary/29_May_2009 29] [http://2009.igem.org/Team:Calgary/30_May_2009 30] [http://2009.igem.org/Team:Calgary/31_May_2009 31]


June
MTWTFSS
[http://2009.igem.org/Team:Calgary/1_June_2009 1] [http://2009.igem.org/Team:Calgary/2_June_2009 2] [http://2009.igem.org/Team:Calgary/3_June_2009 3] [http://2009.igem.org/Team:Calgary/4_June_2009 4] [http://2009.igem.org/Team:Calgary/5_June_2009 5] [http://2009.igem.org/Team:Calgary/6_June_2009 6] [http://2009.igem.org/Team:Calgary/7_June_2009 7]
[http://2009.igem.org/Team:Calgary/8_June_2009 8] [http://2009.igem.org/Team:Calgary/9_June_2009 9] [http://2009.igem.org/Team:Calgary/10_June_2009 10] [http://2009.igem.org/Team:Calgary/11_June_2009 11] [http://2009.igem.org/Team:Calgary/12_June_2009 12] [http://2009.igem.org/Team:Calgary/13_June_2009 13] [http://2009.igem.org/Team:Calgary/14_June_2009 14]
[http://2009.igem.org/Team:Calgary/15_June_2009 15] [http://2009.igem.org/Team:Calgary/16_June_2009 16] [http://2009.igem.org/Team:Calgary/17_June_2009 17] [http://2009.igem.org/Team:Calgary/18_June_2009 18] [http://2009.igem.org/Team:Calgary/19_June_2009 19] [http://2009.igem.org/Team:Calgary/20_June_2009 20] [http://2009.igem.org/Team:Calgary/21_June_2009 21]
[http://2009.igem.org/Team:Calgary/22_June_2009 22] [http://2009.igem.org/Team:Calgary/23_June_2009 23] [http://2009.igem.org/Team:Calgary/24_June_2009 24] [http://2009.igem.org/Team:Calgary/25_June_2009 25] [http://2009.igem.org/Team:Calgary/26_June_2009 26] [http://2009.igem.org/Team:Calgary/27_June_2009 27] [http://2009.igem.org/Team:Calgary/28_June_2009 28]
[http://2009.igem.org/Team:Calgary/29_June_2009 29] [http://2009.igem.org/Team:Calgary/30_June_2009 30]


July
MTWTFSS
    [http://2009.igem.org/Team:Calgary/1_July_2009 1] [http://2009.igem.org/Team:Calgary/2_July_2009 2] [http://2009.igem.org/Team:Calgary/3_July_2009 3] [http://2009.igem.org/Team:Calgary/4_July_2009 4] [http://2009.igem.org/Team:Calgary/5_July_2009 5]
[http://2009.igem.org/Team:Calgary/6_July_2009 6] [http://2009.igem.org/Team:Calgary/7_July_2009 7] [http://2009.igem.org/Team:Calgary/8_July_2009 8] [http://2009.igem.org/Team:Calgary/9_July_2009 9] [http://2009.igem.org/Team:Calgary/10_July_2009 10] [http://2009.igem.org/Team:Calgary/11_July_2009 11] [http://2009.igem.org/Team:Calgary/12_July_2009 12]
[http://2009.igem.org/Team:Calgary/13_July_2009 13] [http://2009.igem.org/Team:Calgary/14_July_2009 14] [http://2009.igem.org/Team:Calgary/15_July_2009 15] [http://2009.igem.org/Team:Calgary/16_July_2009 16] [http://2009.igem.org/Team:Calgary/17_July_2009 17] [http://2009.igem.org/Team:Calgary/18_July_2009 18] [http://2009.igem.org/Team:Calgary/19_July_2009 19]
[http://2009.igem.org/Team:Calgary/20_July_2009 20] [http://2009.igem.org/Team:Calgary/21_July_2009 21] [http://2009.igem.org/Team:Calgary/22_July_2009 22] [http://2009.igem.org/Team:Calgary/23_July_2009 23] [http://2009.igem.org/Team:Calgary/24_July_2009 24] [http://2009.igem.org/Team:Calgary/25_July_2009 25] [http://2009.igem.org/Team:Calgary/26_July_2009 26]
[http://2009.igem.org/Team:Calgary/27_July_2009 27] [http://2009.igem.org/Team:Calgary/28_July_2009 28] [http://2009.igem.org/Team:Calgary/29_July_2009 29] [http://2009.igem.org/Team:Calgary/30_July_2009 30] [http://2009.igem.org/Team:Calgary/31_July_2009 31]


August
MTWTFSS
          [http://2009.igem.org/Team:Calgary/1_August_2009 1] [http://2009.igem.org/Team:Calgary/2_August_2009 2]
[http://2009.igem.org/Team:Calgary/3_August_2009 3] [http://2009.igem.org/Team:Calgary/4_August_2009 4] [http://2009.igem.org/Team:Calgary/5_August_2009 5] [http://2009.igem.org/Team:Calgary/6_August_2009 6] [http://2009.igem.org/Team:Calgary/7_August_2009 7] [http://2009.igem.org/Team:Calgary/8_August_2009 8] [http://2009.igem.org/Team:Calgary/9_August_2009 9]
[http://2009.igem.org/Team:Calgary/10_August_2009 10] [http://2009.igem.org/Team:Calgary/11_August_2009 11] [http://2009.igem.org/Team:Calgary/12_August_2009 12] [http://2009.igem.org/Team:Calgary/13_August_2009 13] [http://2009.igem.org/Team:Calgary/14_August_2009 14] [http://2009.igem.org/Team:Calgary/15_August_2009 15] [http://2009.igem.org/Team:Calgary/16_August_2009 16]
[http://2009.igem.org/Team:Calgary/17_August_2009 17] [http://2009.igem.org/Team:Calgary/18_August_2009 18] [http://2009.igem.org/Team:Calgary/19_August_2009 19] [http://2009.igem.org/Team:Calgary/20_August_2009 20] [http://2009.igem.org/Team:Calgary/21_August_2009 21] [http://2009.igem.org/Team:Calgary/22_August_2009 22] [http://2009.igem.org/Team:Calgary/23_August_2009 23]
[http://2009.igem.org/Team:Calgary/24_August_2009 24] [http://2009.igem.org/Team:Calgary/25_August_2009 25] [http://2009.igem.org/Team:Calgary/26_August_2009 26] [http://2009.igem.org/Team:Calgary/27_August_2009 27] [http://2009.igem.org/Team:Calgary/28_August_2009 28] [http://2009.igem.org/Team:Calgary/29_August_2009 29] [http://2009.igem.org/Team:Calgary/30_August_2009 30]
[http://2009.igem.org/Team:Calgary/31_August_2009 31]


September
MTWTFSS
  [http://2009.igem.org/Team:Calgary/1_September_2009 1] [http://2009.igem.org/Team:Calgary/2_September_2009 2] [http://2009.igem.org/Team:Calgary/3_September_2009 3] [http://2009.igem.org/Team:Calgary/4_September_2009 4] [http://2009.igem.org/Team:Calgary/5_September_2009 5] [http://2009.igem.org/Team:Calgary/6_September_2009 6]
[http://2009.igem.org/Team:Calgary/7_September_2009 7] [http://2009.igem.org/Team:Calgary/8_September_2009 8] [http://2009.igem.org/Team:Calgary/9_September_2009 9] [http://2009.igem.org/Team:Calgary/10_September_2009 10] [http://2009.igem.org/Team:Calgary/11_September_2009 11] [http://2009.igem.org/Team:Calgary/12_September_2009 12] [http://2009.igem.org/Team:Calgary/13_September_2009 13]
[http://2009.igem.org/Team:Calgary/14_September_2009 14] [http://2009.igem.org/Team:Calgary/15_September_2009 15] [http://2009.igem.org/Team:Calgary/16_September_2009 16] [http://2009.igem.org/Team:Calgary/17_September_2009 17] [http://2009.igem.org/Team:Calgary/18_September_2009 18] [http://2009.igem.org/Team:Calgary/19_September_2009 19] [http://2009.igem.org/Team:Calgary/20_September_2009 20]
[http://2009.igem.org/Team:Calgary/21_September_2009 21] [http://2009.igem.org/Team:Calgary/22_September_2009 22] [http://2009.igem.org/Team:Calgary/23_September_2009 23] [http://2009.igem.org/Team:Calgary/24_September_2009 24] [http://2009.igem.org/Team:Calgary/25_September_2009 25] [http://2009.igem.org/Team:Calgary/26_September_2009 26] [http://2009.igem.org/Team:Calgary/27_September_2009 27]
[http://2009.igem.org/Team:Calgary/28_September_2009 28] [http://2009.igem.org/Team:Calgary/29_September_2009 29] [http://2009.igem.org/Team:Calgary/30_September_2009 30]


October
MTWTFSS
      [http://2009.igem.org/Team:Calgary/1_October_2009 1] [http://2009.igem.org/Team:Calgary/2_October_2009 2] [http://2009.igem.org/Team:Calgary/3_October_2009 3] [http://2009.igem.org/Team:Calgary/4_October_2009 4]
[http://2009.igem.org/Team:Calgary/5_October_2009 5] [http://2009.igem.org/Team:Calgary/6_October_2009 6] [http://2009.igem.org/Team:Calgary/7_October_2009 7] [http://2009.igem.org/Team:Calgary/8_October_2009 8] [http://2009.igem.org/Team:Calgary/9_October_2009 9] [http://2009.igem.org/Team:Calgary/10_October_2009 10] [http://2009.igem.org/Team:Calgary/11_October_2009 11]
[http://2009.igem.org/Team:Calgary/12_October_2009 12] [http://2009.igem.org/Team:Calgary/13_October_2009 13] [http://2009.igem.org/Team:Calgary/14_October_2009 14] [http://2009.igem.org/Team:Calgary/15_October_2009 15] [http://2009.igem.org/Team:Calgary/16_October_2009 16] [http://2009.igem.org/Team:Calgary/17_October_2009 17] [http://2009.igem.org/Team:Calgary/18_October_2009 18]
[http://2009.igem.org/wiki/index.php?title=Team:Calgary/19_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/20_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/21_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/22_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/23_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/24_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/25_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 25]
[http://2009.igem.org/wiki/index.php?title=Team:Calgary/26_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/27_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/28_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/29_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/30_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/31_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 31]



AUGUST 20, 2009


CAROL

Descriptive Title of What You’re Doing

WIKI CODING HERE

CHINMOYEE

Wiki Modelling page planning and Modelling meeting

MatLab paper formatting
Abstract
Introduction – talk about our system , have a system diagram
Methodology —algorithms , lab procedures etc
Results and discussions —graphs , pics of glow bac
Conclusions

(Do in LaTeX)

Wiki Formatting
 Main frame (put pics beside overview )

 Left frame Matlab modeling --- The tab contains definitions and overview of methodology like
Robustness
transfer function
response time
Parameter sensitivity
Optimization
Chemical kinetic equation / Law of mass action
more...

Membrane computing--- this tab contain pics and relavant information for overview of concepts

Paper 1 Matlab Modelling---

Paper 2 Membrane computing--- pdf file , made with LaTeX

The Meeting
Meeting with Afshin and Iman in main campus . Compared reactions present in both of our systems . Need to inquire into mRNA degradation .


EMILY

Descriptive Title of What You're Doing

WIKI CODING HERE


FAHD

Human Practices for August 20th 2009

Today, I continued to work on the ethics paper. I started reading literature on the environmental, legal and societal issues surrounding Quorum Sensing and I found a lot of interesting information that I can add on to the ethics paper. I will continue to work on the ethics paper over the next couple of days and try to improve it further.

I also contacted some companies that were on my marketing list. Most of these companies were Oil and gas development companies. I will do a follow-up with these companies next week.



IMAN

Descriptive Title of What You're Doing

WIKI CODING HERE


JAMIE

Setting up the V. harveyi MM32 AI-2 Assay and Testing Experiments in Second Life's Virtual Lab

Using a 96 well microplate, AI-2 isolated from Salmonella typhimurium 14028 supernatant will be tested. V. harveyi reporter strain MM32 (luxS- and luxN-) will luminesce in the presence of AI-2. Controls used in the experiment include S. typhimurium SS007, V. harveyi BB120 and E. coli DH5a. Overnight OD600 and luminescence (CPS) readings will be taken by a VICTOR (thank you Surette lab, especially Margot!).

Preliminary testing was done on the bacterial transformation and genomic DNA isolation activities in the virtual lab. Additionally, I now have transparent wings which glow.

JEREMY

Descriptive Title of What You're Doing

WIKI CODING HERE


KATIE

Fundamental Displays

I recently remembered that I will have to start making cleaning scripts for a lot of the equipment in the lab to prevent clutter for easy maintenance. I started to make these and added them to bacterial transformation, the sequencing station and DNA extraction. It is not imperative that these be completed immediately since this type of abuse is not expected for testing.

Added rho termination, but I am not satisfied with it yet and will continue to change where objects travel too although the pushing part looks satisfactory I need a better place for the mRNA strand. I have no idea how I am going to do the hairpin termination at this time but it will probably mean I have to fiddle with rotation again. There is now a complementary strand of DNA that will change its bases when an avatar makes changes to the template strand (the bottom one strand in this animation) so that the two strands will remain complementary to one another.

Also did some more wiki updates.


KEVIN

1. Measuring fluorescence of GFP

Yesterday, varieties of bacterial cultures were grown in order to test the expression of GFP's. The fluorescence of GFP are measured by the plate reader, and following are the results that Emily and I were able to get today:

  • Fluorescent levels of different cultures (Stored in the plate reader for 5 hours after dilution of R0040+GFP)


Calgary GFP reading 5hrs Plate reader.png

  • Fluorescent levels of different cultures (Stored in the shaker at 37˚C for 5 hours after dilution of R0040+GFP)


Calgary GFP reading 5hrs shaker.png
We were forced to wait for 5 hours after the dilution of R0040+GFP culture as we were asked to leave the lab that had the plate reader by another organization. We were able to measure once quickly right after the dilution; however, with no accurate settings, meaning we cannot make any comparisons. As for the above results, it seems convincing enough for me to say those cells that have the reporter and mutant (LuxOD47E) circuits in them are doing something special that make their readings higher. Our KT1144 cells have a cosmid that contains Qrr4 promoter driving an expression of GFP; thus, as shown by the data, express GFP in larger quantities when it has the LuxOD47E mutant than when it doesn't. Our TOP10 cells also have a plasmid containing Pqrr4 and GFP, which behaves the same way as our KT1144 cells. Both data show that the fluorescence reading is about 2 times higher in cells that have both mutants and reporters than the cells that doesn't have mutant circuits. As we can see in well B7, the cells that have R0040, a constitutive promoter, driving GFP have a much higher reading than the rest, which is what was expected. Also as expected, diluting the R0040+GFP cells to 1/10, 1/50, and 1/100 ratios resulted in much lower readings of fluorescence. Two interesting things to note about the above collected data are:
1. Both with and without mutant readings of TOP10 cells with reporter are than the readings of KT1144 cells. I do not know exactly why it is as such, but my guess is that different type of cells absorb different amount of wavelengths.
2. The fluorescent levels of the cells that were grown for an extra 5 hours in the shaker were lower than the cells that grew in the plate reader, except for the cells that were diluted with fresh LB media. As above, I do not know the exact reason. Perhaps it is because the cells have already reached their 16~20 hours of growing overnight, growing for an extra 5 more hours at 37˚C killed the bacteria, decreasing the production of GFP, while the growth of the cells that were stored in the plate reader for an extra 5 hours slowed down due to a relatively colder temperature, thus being able to produce GFP more stably. As for the diluted cells with R0040+GFP, because they were transferred to a fresh media before the additional growth of 5 hours, storing them in the shaker at 37˚C led to faster replication, which sped up the production of GFP, while storing them in the colder plate reader slowed down the replication, which slowed the production of GFP.


2. Sequencing results

The sequencing results of Pqrr4+B0034+K082003 came back today. Unfortunately, B0034 was still not there despite the change of Pqrr4+B0034 colonies from C9 to C1. Surprisingly, the sequence for the previous construction that used Pqrr4+B0034 C9 actually match the sequence that came back today, which used Pqrr4+B0034 C1. (99% match) The following is the alignment of the two sequences: 

>lcl|28877 
Length=801

Score = 1472 bits (797),  Expect = 0.0
Identities = 800/801 (99%), Gaps = 1/801 (0%)
Strand=Plus/Plus

Query  9    GGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTATTAAGCTACTAA  68
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  1    GGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTATTAAGCTACTAA  60

Query  69   AGCGTAGTTTTCGTCGTTTGCAGCAGGCCTTTTGTATAGTTCATCCATGCCATGTGTAAT  128
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  61   AGCGTAGTTTTCGTCGTTTGCAGCAGGCCTTTTGTATAGTTCATCCATGCCATGTGTAAT  120

Query  129  CCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTT  188
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  121  CCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTT  180

Query  189  CGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCC  248
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  181  CGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCC  240

Query  249  AATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTG  308
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  241  AATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTG  300

Query  309  TCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATT  368
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  301  TCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATT  360

Query  369  GTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATC  428
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  361  GTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATC  420

Query  429  AATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACG  488
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  421  AATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACG  480

Query  489  TGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGG  548
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  481  TGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGG  540

Query  549  CATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTG  608
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  541  CATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTG  600

Query  609  AACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGT  668
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  601  AACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGT  660

Query  669  GCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGAC  728
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  661  GCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGAC  720

Query  729  AGAAAA-TTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAG  787
            |||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  721  AGAAAAATTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAG  780

Query  788  TGAAAAGTTCTTCTCCTTTAC  808
            |||||||||||||||||||||
Sbjct  781  TGAAAAGTTCTTCTCCTTTAC  801

And the following is the previous sequencing result that I have gotten for Pqrr4+B0034 that was used for construction:

Sequence of Pqrr4+B0034 (RBS) C1
Legend
Vector contamination
BBK Restriction sites
Pqrr4 Sequence
B0034 sequence

GGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTG GAATTCGCGGCCGCTTCTAGAGTATCAGCAAAAACACTACGGTGGATAATCAGTAAAACCATGAAACTAGAGCCCCGCA CACTTGCGGGGCTTTTTAATTTTGAATTTCTTTCTTATTAAAACGCCATTTTTCTGATAAATGTATTAGTAGCAA TGCGCATGGTGGCATATTTGCATCATTTTGCATTTTGCAAATGCGATTTGCAAAATGCGTGCTCAATAAAGCACC AATATGCATCAGGATCGAAGAAAAAAGGCGTTTTTAAAAGTTGGCACGCATCGTGCTTTATACAGATACTAGAGAAAGAGGAGAAA TACTAGTAGCGGCCGCTGCAG

TCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTT CGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGG CGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCG TAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT CGTCTTGAGTCCAACCCGGTAGACACGACTTATCGCCACTG


When I carefully analyzed today's Pqrr4+B0034(C1)+K082003, I could find part of the B0034 embeded between Pqrr4 and K082003. The following explains this further:

Legend
end of Pqrr4 sequence
Biobrick restriction sites
Part of B0034 (Complete sequence is AAAGAGGAGAAA
Part of scar? (Complete sequence is ACTAGAG)
Beginning of K082003

...AGATACTAGAGAAAGAGGCTAGATGCGTA...




A mistake of sending two wrong colonies of Pqrr4+B0034 for sequencing is highly unlikely, and also a mistake of using a wrong colony for construction two times is highly unlikely, especially because I was not willing to mess up the construction the second time. I will have to investigate this further.


MANDY

Descriptive Title of What You're Doing

WIKI CODING HERE


PATRICK

The Next Big Thing: Levels

As the iGEM season starts winding up, it is becoming very urgent to make the second life project publicly presentable! I spent today getting organized and nailing down the design for the last big feature that will live in the Biobrick Simulator heads up display object: levels.

The simulator is not a game (though it will be fun), but the 'playing through levels' concept is widely known amongst young people these days, and it's much more appealing than something like 'concepts', or 'chapters', even though those terms are better descriptions of what a 'level' will be. Each level is a self contained set of parts, with the goal of introducing one or two new features of the simulator and concepts in molecular biology to the user. The preliminary list of levels is a good illustration of the progression I'm looking for:

  • First Transcription
  • Activators (a simple activatable device, and an activator)
  • Repressors (a simple repressable device)
  • External Activator (the activator/repressor is produced by a device, instead of given)
  • External Repressor
  • Negative Autoregulation (Control)
  • Positive Autoregulation (Memory)
  • Or Gates
  • Inverters (Not Gate)
  • And Gates (two inverters w. dual repressed promoter)
  • Bistable Toggle Switch
  • Tristable Toggle Switch (See [http://openwetware.org/wiki/IGEM:Brown/2007/Tri-Stable_Switch Brown's 2007 project.])
  • Repressilator

Unfortunately, there are also a good number of systems I had wanted to make into levels, but whose features aren't yet supported by the simulator's engine! Including, but not limited to:

  • Lac Operon
  • Tet Operon
  • And Gates (additional varieties)
  • Lux Operon / Quorum Sensing
  • Feedforward systems (various types)
  • XOR Gates
  • Ara Operon?

In addition, a number of the levels I will be building would benefit from features these other systems require, especially inducers for LacI and TetR. Inducers make a huge amount of sense for systems like the toggle switches, for getting from one state to another; and also for positive autoregulation, for turning on/off cellular memory. That's to say nothing of incorporating environmental signals like temperature, osmolarity, and light, each of which have sensor parts within the registry, leading to more systems and more levels... but I digress! In a perfect world I'd have a chance to implement all of these things before putting together a selection of levels. But since I would like a version of the simulator with most of its *features* in place before the end of summer, choices have to be made about content. And who knows, there might still be time.

Fortunately, none of the code that remains to be written for the levels is very complicated. The Level Selection screen makes the levels and their parts available, the Home screen gives some basic guidance for each level, and the Parts screen makes supplementary objects, and replacement objects in case you happen to delete the ones you're working with (or wish to combine multiple levels' parts together. That will work just fine!) All three screens are necessary for the levels to work, and none have new or complicated features, it's mostly a question of adapting code I've already written for other purposes.

And all of that isn't even the last major portion of the island that needs to be developed! To support all of this biobrickery, users will first visit a zone describing the basics of molecular biology and biochemistry (in Second Life's interactive style). Katie is already working on a display of DNA replication for this zone. Also fortunately, each of the exhibits I have in mind are more or less stand-alone displays, as compared to the biobrick simulator where every part interacts with half a dozen others, so I don't anticipate it taking as long.

But, with development as with biology, a good safe bet is to multiply all your time estimates by three...


PRIMA

Marketing and tieing up loose ends

I ran the gene-specifc PCR with aiiA-F/R primers and ran it with a 1% gel at 100V.

Calgary 2009.08.19.gene.specific.aiiapcr.png


I had contaminated my negative control (which was just the mastermix) with my aiiA gene. Since all three bands are around 756 bp, we can say that the gene exists.

I followed up with couple pharmaceutical companies. Some of our potential sponsors are currently deciding on sponsoring iGEM Calgary. Again, I couldn't get hold of other companies by phone but I left a message with a call back number. I also emailed those companies with a follow- up email. The good news is that Beckman Coulter has generously donated $200.00 to our iGEM Team this year. Unfortuantely, Alda Phamaceuticals Corporation is unable to sponsor our team this year.

I updated some of the days on the wiki notebook. I also worked on my lab notebook to make sure all the gels were labeled and all pages are numbered.

Finally, I began working on the iGEM articles for media purposes and for our meeting with Leanne next week. I should have that completed by this weekend. Tomorrow, I'll discuss my gel with Thane and Sonja and decide where to go from here in the lab. For marketing, I'll continue to contact companies (mostly via email) for sponsorship for this year and next year.


STEFAN

Testing...Testing

Today, a generic avatar was created (aptly named Generic Lennie) that was in a separate group that had access to our island. He was used to test each area on our sim. We don't want visitors to be able to move, objects, change script and make new permanent objects. For the synthetic Kingdom everything was in order:

  • All objects were not modifiable
  • Generic Lennie was able to move the vitamins, Bad Guy/Champion cells, quorum sensing bacteria and oil clean-up bacteria, which is ok because they have a self-destruct script inside
  • GFP station drag and drop worked!


This means that the Synthetic Kingdom is unofficially done!


VICKI

...And some more work on the modelling front

  • Chinee and I met with Afshin and Iman to align our models with each other. The SimBiology and Membrane Computing reactions were subsequently updated, so that we are now modelling the same thing where applicable. The only major differences lie in that the membrane computing model accounts for LuxS (which the SimBiology model omits) and the membrane computing model accounts for AI-2 molecules leaving the cell and going to another one (while ours just includes a reverse reaction for AI-2 binding to LuxPQ, as we are only modelling performance in one cell).
  • I helped Chinee download Latex and will provide a lesson on how to use that platform
  • I looked up degradation rates for GFP:LVA mRNA
  • Our sensitivity analysis crashes with the addition of more reactions to our model. I worked on fixing that, to little avail. I will try again tomorrow.

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