Transformation

I. Introduction

The purpose of the lab is to calculate the transformation efficiency of bacteria exposed to plasmid conferring antibiotic Ampicillin resistance and bioluminescence.

Transformation is the uptake of foreign DNA by competent cells to express a foreign gene. In order to be competent, the cell must be in logarithmic growth. It also must have a weak cell wall and plasma membrane. The cell then should undergo rapid binary fission. The foreign DNA is transferred to the competent cell via a vector. The primary vector used is the plasmid. Plasmids are extra chromosomal and contain the gene of interest, in this case bioluminescence. The R plasmid, which causes the resistance to Ampicillin, confirms antibiotic resistance. It makes it able to identify which cells under went transformation. Restriction endonucleases can be used to cut and insert pieces of foreign DNA into the plasmid. Once the bacteria or competent cell takes up the plasmid, the restriction enzymes in the host cell immediately begin cutting up the foreign DNA. The DNA can then fit in the host DNA due to sticky ends. Once the host cell accepts the plasmid, it has been transformed. The transformation efficiency can then be calculated by the following calculation:

Number of transformants * final volume at recovery (ml) = number of mg of DNA volume plated (ml) transformants per mg

Those cells that accept the plasmid will be identified by growing on an Ampicillin positive plate and glow in the dark.

If the exposure of bacteria to plasmid pAMP/pBlu is increased, then the transformation efficiency will increase.

The dependent variable is the rate of transformation efficiency as measured by number of colonies glowing.

The independent variable is the amount of exposure to pAMP/pBlu plasmid as defined by 10 ml of plasmid.

The control is the rate of transformation efficiency of bacteria without exposure to plasmids pAMP/pBlu.

II. Procedure

Materials

· 2- 15 ml tubes
· 2- micropipettes
· 500 ml- CaCl2
· 2- cotton swabs
· 10 ml- pAMP solution (.005mg/ml)
· 1- 500 ml beaker
· 250 ml- ice cold water
· 10- ice cubes
· 1- clock
· 1- LB agar plate
· 1- LB agar plate with Ampicillin
· 1- permanent marker
· 1- hear shock bath with water at 42 Celsius
· 1- thermometer
· 500 ml- Luria
· 1- cell spreader
· 1- Bunsen burner
· 1- 250 ml beaker
· 50 ml- alcohol

Experimental Design

For experiment procedure, refer to pages 66-67 of the lab packet with the following modifications:

Instead of using a sample of e-coli from a starter plate, take a sample from one's self using a cotton throat swab.

1. Open sterile cotton swab and place down throat.
2. Rub the cotton swab for optimal transfer.
3. Swirl the cotton swab in the solution to dislodge the cells.

Instead of using three control agar plates, only use one (LB agar).

1. While the tubes are on ice, obtain one LB agar plate and one LB/Amp agar plate. Label each plate on the bottom as follows: one LB agar plate as "LB+" and label the Lb/Amp plate "LB/AMP+"

Instead of placing 100ml of "+" cells on to the agar plate labeled "LB/Amp +" and placing 100ml of "-" cells on the "LB" plate, place all of the cells in the test tube.

1. Place all of the "+" cells on the "LB/Amp +" plate. Place all of the "-" cells on the "LB-" plate.

Instead of allowing the plates to sit overnight, allow to sit for two nights.

1. Tape your plates together and incubate inverted for 2 days at 37° C.

Constants

The following must remain constant during the experiment: the type of e-coli, the type of plasmid, and the type of antibiotic.

III. Results

Data Table 1.1: Amount of Colonies Found in Agar Plates
Refer to page 67 in lab: Molecular Biology
Data Table 1.2: Amount of Colonies Transformed Calculated into Transformation Efficiency
Refer to page 67 in lab: Molecular Biology
Chart 1.1: The Flow of Transformation and Process of Making Cells Competent
Refer to page 72 in lab: Molecular Biology

IV. Conclusion

The hypothesis was supported by the data. After increased exposure to plasmid pAMP/pBlu, the transformation efficiency increased. There were .4*10^2 colonies per mg of plasmid. One hundred and eighty colonies were found in the LB- agar plate with no ampicillin or plasmid. Two colonies were found on the LB/AMP+ plate with ampicillin and plasmid.
The results of this experiment may be inconclusive due to the following errors: The competent cells could have been damaged in the process of making them competent. The heat bath was to be set exactly at 42 degrees Celsius. Any higher could have damaged the cells making them incompetent and unable to pick up foreign DNA from the plasmid. The cells may have been left in the ice bath too long damaging the cell wall causing them also to not be able to pick up foreign DNA from the plasmid. If the cells were not able to pick up DNA, then they could not be transformed, sharply decreasing the transformation efficiency. A final error possibly could have been that there were not enough e-coli to transform. Since the group decided to use e-coli from their own throat, the number of cells transferred may have been minimal. This would also cause a decrease in the transformation efficiency.
I learned that transformation is necessary in the process of manufacturing human insulin from bacteria cells. In diabetic humans, the body cannot produce the needed amount of natural insulin. In order to obtain the needed amount, patients must look to an outside source. Scientists can actually make insulin. A plasmid, with the gene of interest as insulin, can be transformed into a bacteria cell. The cell will then accept the foreign DNA and begin producing insulin. The manufactured human DNA will save the patients life.http://www.oppapers.com/essays/Transformation/127999

Transformation 8.2 of 10 on the basis of 3096 Review.