Transformation!

Home Editor: Erica Cunningham

Transformation occurs when a cell incorporates a new, separate gene into its DNA. For the transformation to work, the cell must be "competent" and able to accept a DNA insert. Then, the cell must express the gene. Specifically, in our experiment, we would like the L4440 plasmid in the DH5α //E. coli// strain to accept the rol-6 gene. //E. coli,// Escherichia coli//,// is a bacteria often found in stomachs of warm-blooded organisms, and the DH5α is a commonly used effective and convenient strain of //E. coli//. The rol-6 gene is a gene that causes the worm, //C. Elegens//, to have a right roller phenotype, or to be twisted into a right handed helix.

Before transformation can occur, the digested L4440 and the digested rol-6 (isolated genomic DNA) must be ligated together. Only then can the L4440 plasmid and rol-6 insert undergo transformation. Then, the recombinant vector must be added to competent DH5α cells on an ampicillin plate. Ampicillin is an antibiotic that will weed out the transformant cells from the non-transformant cells. The cells with the rol-6 insert will be resistant to ampicillin, while the cells without the rol-6 insert will not be resistant to ampicillin. Subsequently, the transformed cells that survive and grow on the plate will be plucked and used for a polymerase chain reaction (PCR). After PCR has been completed and a verification gel has been run, we can ascertain specifically which colonies have been transformed with the recombinant plasmid. Then, a miniprep must be performed on the transformed cells to isolate the plasmid; now, the HT115 strain of //E. coli// must be transformed with the recombinant plasmid. Learn more about the //E. coli// HT115 strain by clicking here. To verify that the HT115 cells have been transformed, minipreps must be performed on selected colonies and run on a gel. The DNA from these colonies would then become the worm "feeding strain" and the rol-6 gene can be expressed in the worms.

Prior to actual transformation, competent cells must be created. We use a the //Z-Competent E. Coli// Transformation Kit from G-Biosciences. This method facilitates high transformation efficiency (# of transformants per μg of plasmid DNA).

PROCEDURE:

__Kit Components__
 * Wash Buffer (2x) ||= 600 μl ||
 * Competent Buffer (2x) ||= 600 μl ||
 * Dilution Buffer ||= 1200 μl ||
 * SOB Culture Medium ||= 2 x 25 ml ||

__Protocol for creating competent cells:__ -Inoculate 500μl of fresh overnight //E. Coli// culture grown in LB to 25ml SOB in a shaking water bath at 20-25°C for 10-36 hours -Dilute all 2X buffers to 1X by adding an equal volume of dilution buffer (Keep buffers at 0-25°C) -Place culture on ice for 10 minutes -Spin cells in the centrifuge for 6 minutes -Remove supernatant and re-suspend cells in 500μl 1X Wash Buffer and re-pellet as in the two prior steps -Remove supernatant amd re-suspend cells in 500μl 1X Competent Buffer -Aliquot 100 μl of the competent cells into sterile centrifuge tubes on ice

The newly prepared competent cells must be stored on ice and are ready for transformation.

__Transformation Protocol:__ -Add 5 μl DNA and gently mix -Incubate on ice for 15-60 minutes -Spread 50-100μl on AMP/LB plates pre-warmed to 37°C

The same competent cell and transformation protocol is used for the DH5α cells and the DT115 cells.

Flow Chart:

RESULTS:

Picture of Gel Electrophoresis from Transformation of HT115 cells:

Unfortunately, we did not successfully transform the HT115 cells. The bands indicate that the L4440 plasmid is in each cell; however, the inserted vector did not create a recombinant vector. This can be determined by noting that no bands are smaller than the L4440 band. Although PCR worked successfully, the transformation of the cells did not. No cells have a recombinant plasmid.