Team:Alberta/Notebook/TransformingCells

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(Rubidium chloride competent cells and lyophilization)
 
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==TSS Transformation Protocol==
==TSS Transformation Protocol==
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The TSS transformation was tested. The procedure is listed here.
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The TSS transformation protocol was tested. The procedure is listed <html><a href="http://www.pnas.org/content/86/7/2172.abstract">here</a></html>.
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One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same
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solution
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http://www.pnas.org/content/86/7/2172.abstract
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*Acquisition of reagents- June 3, 2010  
*Acquisition of reagents- June 3, 2010  
*Preparation of buffer - June 7, 2010
*Preparation of buffer - June 7, 2010
*Cell preparation - June 7, 2010
*Cell preparation - June 7, 2010
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*Competency - June 10, 2010
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*Protocol trial - June 10, 2010
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*Assessment of efficiency - June 11, 2010
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Results:
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Transformation with pSB1C3 gave a transformation efficiency of 4.8*10^7 colonies/μL. All colonies on the plate were strongly pink, as expected. Preparation of the cells took only 120 minutes, with a single transformation taking 90 minutes. On the other hand, a typical stock Inoue transformation cells takes a full day of work, with a single transformation taking about 120 minutes.
==Rubidium chloride competent cells and lyophilization==
==Rubidium chloride competent cells and lyophilization==
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This method was adapted from a method used by Justin Fedor.
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This method was adapted from a competent cell protocol found <html><a href="http://www.unc.edu/depts/marzluff/protocols/rubidium_competent_cells.pdf" >here</a></html>, which was suggested to us by former iGEM team member Justin Fedor. A small modification to include 200mM of sucrose in each of the transformation buffers. The cells were then lyophilized overnight at 4°C on a FTS Systems Dura-Dry Lyophilizer.
*Preparation of transformation buffer - July 6, 2010  
*Preparation of transformation buffer - July 6, 2010  
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*Lyophilization - August 2, 2010  
*Lyophilization - August 2, 2010  
*Transformation - August 5, 2010  
*Transformation - August 5, 2010  
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Results:
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Lyophilization proved to greater impair transformation efficiency. The determined transformation efficiency was found to be a 2*10^-1 colonies/μg.
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{{Team:Alberta/endMainContent}}

Latest revision as of 03:02, 28 October 2010

TEAM ALBERTA

Transforming Cells

Project Timeline: Click on an image to see more information


Transformation typically requires expensive equipment and storing competent cells is difficult for many high schools who do not have -80oC freezers. We tried a few methods of transforming cells and making them competent to attempt to solve this problem in our kit.

TSS Transformation Protocol

The TSS transformation protocol was tested. The procedure is listed here.

  • Acquisition of reagents- June 3, 2010
  • Preparation of buffer - June 7, 2010
  • Cell preparation - June 7, 2010
  • Protocol trial - June 10, 2010
  • Assessment of efficiency - June 11, 2010

Results: Transformation with pSB1C3 gave a transformation efficiency of 4.8*10^7 colonies/μL. All colonies on the plate were strongly pink, as expected. Preparation of the cells took only 120 minutes, with a single transformation taking 90 minutes. On the other hand, a typical stock Inoue transformation cells takes a full day of work, with a single transformation taking about 120 minutes.

Rubidium chloride competent cells and lyophilization

This method was adapted from a competent cell protocol found here, which was suggested to us by former iGEM team member Justin Fedor. A small modification to include 200mM of sucrose in each of the transformation buffers. The cells were then lyophilized overnight at 4°C on a FTS Systems Dura-Dry Lyophilizer.

  • Preparation of transformation buffer - July 6, 2010
  • Preparation of cells - July 10, 2010
  • Preparing competent cells - July 11, 2010
  • Lyophilization - July 13, 2010
  • Transformation of FD cells - July 14, 2010
  • Preparation of cells 2 - July 30, 2010
  • Lyophilization - August 2, 2010
  • Transformation - August 5, 2010

Results:

Lyophilization proved to greater impair transformation efficiency. The determined transformation efficiency was found to be a 2*10^-1 colonies/μg.