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.

@@ Line 28: / Line 28: @@
 ==TSS Transformation Protocol==
-The TSS transformation was tested. The procedure is listed here.
+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>.
-<html><a href="http://www.pnas.org/content/86/7/2172.abstract">One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution</a></html>
 *Acquisition of reagents- June 3, 2010
@@ Line 39: / Line 37: @@
 Results:
-Transformation with pSB1C3 gave a transformation efficiency of 4.8*10^7 colonies/&muL.
+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 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
@@ Line 53: / Line 51: @@
 *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.
 {{Team:Alberta/endMainContent}}

Team:Alberta/Notebook/TransformingCells

From 2010.igem.org