Team:Calgary/30 June 2009

From 2009.igem.org

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June 30, 2009
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JUNE 30, 2009
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PCR product Purification and Restriction Digest
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WIKI CODING HERE
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* PCR product was purified using sigma PCR purification kit and PCR product 1-6 were pooled together as well as PCR product 7-12 was pooled together.
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* Digested the plasmid pSB1AK3 and the PCR product using both XbaI+PstI and EcoRI+PstI enzymes for 2 hours.
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* Phosphatase the plasmid and then ligated product with PCR product using Quick Ligase. The transformation was done as well into Top 10 cells, and plates were incubated at 37 degrees Celsius overnight. 
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Isolating Plasmid and Modified Colony PCR of LuxOD47E BBK
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WIKI CODING HERE
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Today I isolated plasmid from my overnight cultures.  The best concentrations that looked the most pure was BBK LuxOD47E colony 2 cut with XbaI/PstI and BBK LuxOD47E colony 4 as cut with EcoRI/PstI.
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*I then set up a modified version of my colony PCR from yesterday, using only these 2 colonies and LuxOD47E C4-8 in the pCR2.1-TOPO vector and the psB1AC3 vector as positive controls.  Cycling conditions were as follows: 94ºC for m minutes, 36x (94ºC for 45s, 55ºC for 45s and 72ºC for 2 minutes), 72ºC for 10 minutes.  Held at 4ºC.
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Today, I helped organize our second bake sale (Cultural Bake Sale: The Sequel) of our marketing campaign. The bake sale managed to raise more than 370 dollars. We had cool baked items such as samosas, spring rolls, muffins and lots lots and lots of other stuff. I want to thank my entire iGEM calgary team for their tremndous support and countless effort the put in for the event.
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Today, I helped organize our second bake sale (Cultural Bake Sale: The Sequel) of our marketing campaign. The bake sale managed to raise more than 370 dollars. We had cool baked items such as samosas, spring rolls, muffins and lots lots and lots of other stuff. I want to thank my entire University of Calgary iGEM team for their tremendous support and countless effort the put in for the event.
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The Beginning of a Short Lab Hiatus to Write!
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WIKI CODING HERE
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Time was spent writing a paper detailing the trials and tribulations of luxOU construction to help internalize and review microbiology concepts and techniques.  Next pressing update: July 6, 2009.
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Purpose: to move linear LuxPQ with BBK restriction sites into the standard psB1AC3 vector. The LuxPQ PCR product was purified by pooling 4 X 40uL of product and following the Qiaquick PCR purification protocol. This productwas then digested with XbaI and PstI, as was psB1AC3 for 2 hours at 37ºC. After phosphotase treatment and ligation, the product was transformed into TOP10 competent cells and plated on LB+chlor plates overnight.  
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Purpose: to move linear LuxPQ with BBK restriction sites into the standard psB1AC3 vector. The LuxPQ PCR product was purified by pooling 4 X 40uL of product and following the Qiaquick PCR purification protocol. This product was then digested with XbaI and PstI, as was psB1AC3 for 2 hours at 37ºC. After phosphotase treatment and ligation, the product was transformed into TOP10 competent cells and plated on LB+chlor plates overnight.  
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*1099bp long
*1099bp long
*They have a relatively weak TH activity of ~0.1-0.5°C, which is less than that of fish AFPs and significantly less than that of insect AFPs.
*They have a relatively weak TH activity of ~0.1-0.5°C, which is less than that of fish AFPs and significantly less than that of insect AFPs.
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*The TH activity of recombinant proteins from an E.coli expression system is weak
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*The TH activity of recombinant proteins from an <i>E. coli</i> expression system is weak.
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Today, I followed up with almost all the companies which I was in charge of contacting and sent them the June Newsletter. I thanked all those companies who have sponsored our project and encourageed everyone to visit our wiki for details on our progress. I also attached the CTV link to every email.  
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Today, I followed up with almost all the companies which I was in charge of contacting and sent them the June Newsletter. I thanked all those companies who have sponsored our project and encouraged everyone to visit our wiki for details on our progress. I also attached the CTV link to every email.  
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[[Image:LuxOD47AB0015sequence.jpg|700px]]
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Latest revision as of 06:59, 20 October 2009

University of Calgary

UNIVERSITY OF CALGARY



THIS MONTH

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]


NOTEBOOK PAGE INDEX



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]



JUNE 30, 2009


CAROL

PCR product Purification and Restriction Digest

  • PCR product was purified using sigma PCR purification kit and PCR product 1-6 were pooled together as well as PCR product 7-12 was pooled together.
  • Digested the plasmid pSB1AK3 and the PCR product using both XbaI+PstI and EcoRI+PstI enzymes for 2 hours.
  • Phosphatase the plasmid and then ligated product with PCR product using Quick Ligase. The transformation was done as well into Top 10 cells, and plates were incubated at 37 degrees Celsius overnight.


CHINMOYEE

Bioprocess Engineering

Looked into my bioprocess engineering book for useful information and different aspects to consider for the model.


EMILY

Isolating Plasmid and Modified Colony PCR of LuxOD47E BBK

Today I isolated plasmid from my overnight cultures. The best concentrations that looked the most pure was BBK LuxOD47E colony 2 cut with XbaI/PstI and BBK LuxOD47E colony 4 as cut with EcoRI/PstI.

  • I then set up a modified version of my colony PCR from yesterday, using only these 2 colonies and LuxOD47E C4-8 in the pCR2.1-TOPO vector and the psB1AC3 vector as positive controls. Cycling conditions were as follows: 94ºC for m minutes, 36x (94ºC for 45s, 55ºC for 45s and 72ºC for 2 minutes), 72ºC for 10 minutes. Held at 4ºC.


FAHD

Bake Sale Sequel, June 30th 2009

Today, I helped organize our second bake sale (Cultural Bake Sale: The Sequel) of our marketing campaign. The bake sale managed to raise more than 370 dollars. We had cool baked items such as samosas, spring rolls, muffins and lots lots and lots of other stuff. I want to thank my entire University of Calgary iGEM team for their tremendous support and countless effort the put in for the event.


JAMIE

The Beginning of a Short Lab Hiatus to Write!

Time was spent writing a paper detailing the trials and tribulations of luxOU construction to help internalize and review microbiology concepts and techniques. Next pressing update: July 6, 2009.


JEREMY

Moving linear LuxPQ into BBK vector

Purpose: to move linear LuxPQ with BBK restriction sites into the standard psB1AC3 vector. The LuxPQ PCR product was purified by pooling 4 X 40uL of product and following the Qiaquick PCR purification protocol. This product was then digested with XbaI and PstI, as was psB1AC3 for 2 hours at 37ºC. After phosphotase treatment and ligation, the product was transformed into TOP10 competent cells and plated on LB+chlor plates overnight.


KEVIN

Search for other antifreeze proteins

Carrot's antifreeze proteins were also looked into.

  • 1099bp long
  • They have a relatively weak TH activity of ~0.1-0.5°C, which is less than that of fish AFPs and significantly less than that of insect AFPs.
  • The TH activity of recombinant proteins from an E. coli expression system is weak.


MANDY

Meeting with Dr. Jacob for Second Life

Today, we went over to main campus to meet up with Dr. Jacob and discuss our current progress. We also continued discussion about when and how we would beta test our project, and how to make it more intuitive and educational. Katie and I looked at a Biobrick circuit assignment given to us by Thane from the beginning of the year. We began modelling possible biobricker levels based on the concepts shown in these circuits.



PATRICK

HUD Details

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

Spent today working out some details of how the tile control system would work. Should I erase the entire screen before drawing the next one, or just let the next screen's text and buttons replace all of the current display? Decided to blank first instead of relying on the next screen to replace it all. Sending a single message to all display tiles to pre-blank had a low overhead cost, the update process looked cleaner on the HUD, and I could then skip space characters when processing the next screen's text. (No need to tell tile x to display a space character, if it has been pre-blanked).

Set up a system for parsing lists of characters into lines on the UI, so that I could easily visualize what the display would look like in the program's code. e.g., an early version of the main menu:

       display += "   Biobrick    ";
       display += "   Simulator   ";
       display += "               ";
       display += " X Introduction";
       display += " X Levels	   ";
       display += " X Sandbox     ";
       display += " X Help        ";
       display += "               ";

The Xes here stand in for buttons.


PRIMA

Marketing

Today, I followed up with almost all the companies which I was in charge of contacting and sent them the June Newsletter. I thanked all those companies who have sponsored our project and encouraged everyone to visit our wiki for details on our progress. I also attached the CTV link to every email.


VICKI

Sequencing of the LuxOD47A BBk + B0015 colonies

Based on yesterday's NotI restriction digest results, the LuxOD47A + B0015 colonies look promising. These were prepared with BBk construction primers and sent for sequencing, with positive results eventually confirmed. These are attached below.



LuxOD47AB0015sequence.jpg


Construction protocol: adding J13002 to LuxOD47A + B0015

Cutting with (EcoRI+SpeI for the J13002 part) and (EcoRI+XbaI for the LuxOD47A + B0015 part) restriction enzymes, Antarctic phosphatase treatment, ligation, transformation into competent TOP10 cells and plating on AK plates were all done in accordance with the procedures outlined on the protocol page. If this is done properly, the net J13002 + LuxOD47A + B0015 will be on an pSB1AK3 plasmid.