Team:Calgary/2 July 2009

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Transformation of <i>luxCDABE</i> into pSB1AK3
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WIKI CODING HERE
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* Attempted to transformed <i>luxCDABE</i> into pSB1AK3 and plated on AK plates at 37 degrees Celsius overnight.
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CHINMOYEE
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Digest Verification
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*Performed a verification digest with NotI of BBK LuxOD47E in psB1AC3- colonies 3/4 cut with EcoRI/PstI and colonies 2/3 cut with XbaI/PstI.  This is one last verification that our gene of interest (LuxOD47E) is in the psB1AC3 vector.  If successful, cut lanes should contain 2 bands at ~1.4 for the gene and ~3 kb for the vector, Uncut lanes should only contain one band at ~4 kb, representing the vector with the gene of interest inside.
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*Protocol: Used GenElute Plasmid Miniprep Kit (Sigma), lot # 048k6062, used as according to the manufacturers instructions, eluting in 50 μL ddH2O.
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*Ran products on a 1% agarose gel with 1.0kb+ GeneRuler DNA Ladder.
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*Ran each cut colony with a uncut colony as a control.
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*See gel photo below.  Lanes 1,3,5 and 7 are cut DNA- EcoRI/PstI colonies 2 and 4 and XbaI/PstI colonies 2 and 3 respectively, lanes 2,4,6 and 8 are all uncut colonies.
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*Results
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*Analysis: Colonies cut with EcoRI/PstI have bands much higher than expected (~1.4 kb and ~3.0 kb in the cut lanes).  XbaI/PstI-cut colony 3 does not show two bands in the cut lane and the bands are not the expected size.  XbaI/PstI-cut colony 2 does show two bands in the cut lane at around the expected sizes and the uncut lane has only 1 band at around 4 kb, as expected.  We will proceed with this colony, sending it down for sequencing.
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*Sent down 10uL XbaI-cut Colony 2 with BBK_CP_F and BBK_CP_R primers, results should be in by Monday.
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{| border="1"
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| '''Plasmid''' || '''260/280'''    || '''260/230''' || '''Concentration [ng/μL]'''
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| ''psB1AC3 1  || 1.96 || 3.48 || 76.9
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| ''psB1AC3 2  || 1.93 || 2.29 || 98.1
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| ''psB1AC3 3  || 1.87|| 1.88|| 48.5
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| ''psB1AC3 4|| 1.90|| 2.11|| 118.9
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| ''psB1AK3 1  || 2.05 || 2.42 || 134.4
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| ''psB1AK3 2 || 1.95 || 2.23 || 55.6
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| ''psB1AK3 3  || 1.91 || 2.22 || 97.9
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| ''psB1AK3 4 || 2.03 || 1.92 || 44.5
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Verification digest to verify the presence of restriction sites in the psB1AC3, psB1AK3 vectors as well as in our TOPO TA vector with LuxOD47E.
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*Protocol:
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5 reactions for psB1AC3 vector, 5 reactions for psB1AK3 vector, 1 reaction for gene of interest (LuxOD47E in pCR2.1-TOPO vector).
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BBK Vector Reaction List
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Reaction 1- Not1 + React 3 Buffer
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Reaction 2- EcoRI + SpeI + React Buffer 4
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Reaction 3- XbaI + PstI + React 2 Buffer
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Reaction 4- EcoRI + PstI + React 2 Buffer
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Reaction 5- XbaI + SpeI + React 4 Buffer
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BBK vector tube preparation
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8 uL Plasmid (psB1AC3 or psB1AK3)
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2 uL appropriate React Buffer
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1 uL of each appropriate restrcition enzyme
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ddH20 up to 20 uL
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TOPO vector tube preparation
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10 uL DNA (LuxOD47E in pCR2.1- TOPO vector)
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7 uL ddH20
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2 uL React 3 Buffer
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1 uL NotI restriction enzymes
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Left to digest overnight in waterbath at 37°C.
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Phosphatase, Ligation and Transformation of LuxPQ into BBK vector
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Purpose: to construct and transform the LuxPQ insert and psB1AC3 insert in order to standardize the genetic part for future construction. The products from the overnight digest (July 1) underwent phosphatase treatment and subsequent ligation and transformation into TOP10 competent cells. The cells were then plated on LB+chlor plates.
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For example, when cryopreserving a tissue, you want to have a uniform level of antifreeze activity across the tissue; thus if you integrate the bacteria with AI2 signalling system to produce these antifreeze proteins all at once, then you would have a better chance of spreading the antifreeze protein across the tissue.  
For example, when cryopreserving a tissue, you want to have a uniform level of antifreeze activity across the tissue; thus if you integrate the bacteria with AI2 signalling system to produce these antifreeze proteins all at once, then you would have a better chance of spreading the antifreeze protein across the tissue.  
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Research in Antifreeze Activity
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We want to look at AFP, which has a very different structure in different organisms. One that we would be able to work with best (or more efficiently with) would be from gram negative bacteria. So I looked at <i>Pseudomonas Putida</i>, <i>Micrococcus cryophilus</i>, <i>Rhodococcus erythropolis</i>, and <i>Marinomonas protea primoryensis</i>. Some are psychrophilic and halophilic, so those might not be ideal for us.
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PRIMA
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The 1% agarose gel is shown below. Lanes 1 and 15 are a GeneElute 1kb+ DNA ladder; lanes 2-5 are 4 different colonies of the recently-PCR'd samples (2uL of PCR product); lanes 9-12 are those same colonies but with only 1uL of PCR product; lanes 6 and 8 are 2 different colonies of LuxOD47A + B0015 as a size control; lane 7 is LuxOD47A BBk as a further size control; and lanes 13 and 14 are both negative controls.
The 1% agarose gel is shown below. Lanes 1 and 15 are a GeneElute 1kb+ DNA ladder; lanes 2-5 are 4 different colonies of the recently-PCR'd samples (2uL of PCR product); lanes 9-12 are those same colonies but with only 1uL of PCR product; lanes 6 and 8 are 2 different colonies of LuxOD47A + B0015 as a size control; lane 7 is LuxOD47A BBk as a further size control; and lanes 13 and 14 are both negative controls.
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These are attached below. Colonies 1 and 5 are encouraging, and were sent down for sequencing later today.
These are attached below. Colonies 1 and 5 are encouraging, and were sent down for sequencing later today.
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[[Image:J13002LuxOD47AB0015RD.jpg|700px]]
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Latest revision as of 18:46, 20 October 2009

University of Calgary

UNIVERSITY OF CALGARY



THIS MONTH

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]


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]



JULY 2, 2009


CAROL

Transformation of luxCDABE into pSB1AK3

  • Attempted to transformed luxCDABE into pSB1AK3 and plated on AK plates at 37 degrees Celsius overnight.


EMILY

Digest Verification

  • Performed a verification digest with NotI of BBK LuxOD47E in psB1AC3- colonies 3/4 cut with EcoRI/PstI and colonies 2/3 cut with XbaI/PstI. This is one last verification that our gene of interest (LuxOD47E) is in the psB1AC3 vector. If successful, cut lanes should contain 2 bands at ~1.4 for the gene and ~3 kb for the vector, Uncut lanes should only contain one band at ~4 kb, representing the vector with the gene of interest inside.
  • Ran products on a 1% agarose gel with 1.0kb+ GeneRuler DNA Ladder.
  • Ran each cut colony with a uncut colony as a control.
  • See gel photo below. Lanes 1,3,5 and 7 are cut DNA- EcoRI/PstI colonies 2 and 4 and XbaI/PstI colonies 2 and 3 respectively, lanes 2,4,6 and 8 are all uncut colonies.
  • Analysis: Colonies cut with EcoRI/PstI have bands much higher than expected (~1.4 kb and ~3.0 kb in the cut lanes). XbaI/PstI-cut colony 3 does not show two bands in the cut lane and the bands are not the expected size. XbaI/PstI-cut colony 2 does show two bands in the cut lane at around the expected sizes and the uncut lane has only 1 band at around 4 kb, as expected. We will proceed with this colony, sending it down for sequencing.
  • Sent down 10uL XbaI-cut Colony 2 with BBK_CP_F and BBK_CP_R primers, results should be in by Monday.



JEREMY

Phosphatase, Ligation and Transformation of LuxPQ into BBK vector

Purpose: to construct and transform the LuxPQ insert and psB1AC3 insert in order to standardize the genetic part for future construction. The products from the overnight digest (July 1) underwent phosphatase treatment and subsequent ligation and transformation into TOP10 competent cells. The cells were then plated on LB+chlor plates.


KEVIN

Continuation of the research in Antifreeze proteins

Usefulness of the signalling system in antifreeze activity
For example, when cryopreserving a tissue, you want to have a uniform level of antifreeze activity across the tissue; thus if you integrate the bacteria with AI2 signalling system to produce these antifreeze proteins all at once, then you would have a better chance of spreading the antifreeze protein across the tissue.


MANDY

Research in Antifreeze Activity

We want to look at AFP, which has a very different structure in different organisms. One that we would be able to work with best (or more efficiently with) would be from gram negative bacteria. So I looked at Pseudomonas Putida, Micrococcus cryophilus, Rhodococcus erythropolis, and Marinomonas protea primoryensis. Some are psychrophilic and halophilic, so those might not be ideal for us.


VICKI

Repeat of colony PCR of J13002 + LuxOD47A + B0015

The purpose and materials/methods are the same as they were yesterday. This time, only 4 colonies were tested, along with the same size controls as yesterday.

Results:

The 1% agarose gel is shown below. Lanes 1 and 15 are a GeneElute 1kb+ DNA ladder; lanes 2-5 are 4 different colonies of the recently-PCR'd samples (2uL of PCR product); lanes 9-12 are those same colonies but with only 1uL of PCR product; lanes 6 and 8 are 2 different colonies of LuxOD47A + B0015 as a size control; lane 7 is LuxOD47A BBk as a further size control; and lanes 13 and 14 are both negative controls.



J13002LuxOD47AB0015gel.jpg


Plasmid isolation and NotI restriction digest

Purpose: To further verify if the J13002 + LuxOD47A + B0015 was successfully biobricked into the colonies.

Materials and methods:

These were done in accordance with the procedure outlined on the protocol page.

Results:

These are attached below. Colonies 1 and 5 are encouraging, and were sent down for sequencing later today.

J13002LuxOD47AB0015RD.jpg

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