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Rapid Colony Transformation of E -Coli with Plasmid DNA - Lab Report Example

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This report provides E-Coli transformed by plasmid DNA using a rapid method. This is to demonstrate that e-coli in combination with a foreign gene-in this case-plasmid DNA- shows the ability to multiply into an antibiotic resistant phenotype. The method can be categorized into four stages.
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Rapid Colony Transformation of E -Coli with Plasmid DNA
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E-Coli transformed by plasmid DNA using a rapid method Purpose: This is to demonstrate that e-coli in combination with a foreign gene-in this case-plasmid DNA- shows the ability to multiply into an antibiotic resistant phenotype.
The transformation method can be categorized into four:
1.Pre-incubation stage: In this the cells are immersed in a cat ions solution and incubated at 0CThis immersion is supposed to help the negatively charged phosphates of lipids in the E-Coli membrane. The low temperature gels the cell membrane, thereby stabilizing the distribution of charged phosphates and allowing them a more effective shield from the cat ions.
2. Incubation: The DNA is added and the cell suspension is kept at 0C.the cat ions are thought to neutralize negatively charged phosphates in the DNA and the cell membrane.
3. Heat Shock: The cell +DNA suspension is briefly incubated at 42C and then returned to 0C. The rapid temperature change creates a heat imbalance on either side of the E-Coli membrane and is supposed to create a wave that sweeps plasmids into the cell.
4. Recovery: LB broth is added to the DNA/cell suspension and incubated at 37C before being put on plates with different selected antibiotic resistant markers. Transformed cells recover from the treatment, amplify the transformed plasmid and begin to express the antibiotic resistant strain.


Method: Samples of E-coli cells are taken from a nutrient agar plate (LB agar) and suspended in two tubes containing a solution of calcium chloride. Plasmid pAMP is added to one cell suspensionBothe the tubes are then incubated at 0C for 15 minutes. After this a brief heat shock is administered at 42 .the samples are cooled and LB broth is added. Samples of both the cell suspensions, one with just the E-coli and the other with E-Coli with added plasmids-are put on two plates with two types of growth agent, LB agar and LB agar ampicillin (LB /amp)


The plates are then incubated for 12 to 24hours at 37and then checked for bacterial growth. Only cells that have been transformed by absorbing the plasmid DNA with the ampicillin resistant gene will grow on theLB/amp plate.


RRR
Results:
Subsequent division of an antibiotic resistant cell will produce a colony of resistant clones. Thus, each colony on an ampicillin plate represents a single transformation event.
Ampicillin is the most practical antibiotic -resistance marker because of it capability to rapidly transform.Ampicillin interferes with the construction of the layer of cell wall and restricts itself to killing only replicating cells that are creating new cell envelopes. On-replicating E-coli are untouched. Thus, cells can be put on plates with the ampicillin medium directly after heat shock, omitting the recovery step.

Conclusion: By itself-Coli cannot form ampicillin resistance, but the addition of plasmid DNA that contains an ampicillin resistant gene (pAMP) produce a colony of resistant clones. Plates containing E-coli are used for the laboratory source. These cells are best used within 12-24 hours of incubation.However, it can be noted that good results are obtained when cells are also stored at room temperature for 1-2 additional days. This causes the E-coli colonies to proliferate and makes it easy to take samples. Read More
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