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 -80^oC 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.

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-Transformation typically requires expensive equipment and storing competent cells is difficult for many high schools who do not have -80C 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==
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+{{Team:Alberta/navbar|notebook=selected}}
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 {{Team:Alberta/beginMainContent}}
+=Transforming Cells=
+===Project Timeline: Click on an image to see more information===
+<html>
+<center>
+<img src="https://static.igem.org/mediawiki/2010/8/8b/TransformingCellsMap.jpg" border="0" usemap="#Notebook_Top">
+<map name="Notebook_Top">
+<area shape="rect" coords="0, 0, 171, 167" href="https://2010.igem.org/wiki/index.php?title=Team:Alberta/Notebook/TransformingCells#TSS_Transformation_Protocol">
+<area shape="rect" coords="277, 0, 466, 167" href="https://2010.igem.org/wiki/index.php?title=Team:Alberta/Notebook/TransformingCells#Rubidium_chloride_competent_cells_and_lyophilization">
+</map>
+</center>
+</html>
+----------
+Transformation typically requires expensive equipment and storing competent cells is difficult for many high schools who do not have -80<sup>o</sup>C freezers.
+We tried a few methods of transforming cells and making them competent to attempt to solve this problem in our kit.
-The TSS transformation was tested. The procedure is listed here.
+==TSS Transformation Protocol==
-One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same
+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>.
-solution
-http://www.pnas.org/content/86/7/2172.abstract
+*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
-Acquistion of reagents- June 3, 2010
+Results:
-Preparation of buffer - June 7, 2010
+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.
-Cell preparation - June 7, 2010
-Competency - June 10, 2010
 ==Rubidium chloride competent cells and lyophilization==
-This method was adapted from a method used by Justin Fedor.
+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 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.
-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
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