Question

Pre-la question 1. Is it possible to transform an organism? Which Organism is most appropriate 2. How can you confirm that organism has been genetically trudomed? 3. Predict the effect of ampicillin on the transformed and non-transformed E. coli cells? 4. What is the function of CaCl on Ecoli cells? 5. Define competent cells 6. What is the function of heat shock during transformation 7. On which of the plates would you expect to find bacteria most like the original non-tradormed collcome you intially bened? Explain your predictions

Answer 1

1. Its possible to transform an organism, Bacteria is the most appropriate one

2. By the process of selection marker

3. E Coli cells contains that a normal E colli cells do not grow in the presence of ampicillin. and Transformed cells are resistance to Ampicillin

4. It increases the ability of a prokaryotic cell to incorporate plasmid DNA allowing them to be genetically transformed.

5. Competent cells are Ecoli cells that have been specially treated to transform efficiently. There are two types of competent cells: chemically competent and electrocompetent. If plasmid is simply added to E. coli, nothing happens! The cells must be competent

CHEMICALLY COMPETENT CELL

Chemically competent cells are treated with a buffer that contains CaCl2 and other salts that disrupt the cell membrane creating “holes” that allow the plasmids to pass into the cell. Most researchers use chemically competent cells because they are less expensive, and also can be made in the lab and do not require special equipment.

ELECTRO COMPETENT CELL

• Electrocompetent transformations require a high density of cells and a non-ionic buffer. The cells are placed in an electroporation device that delivers a pulse of electricity to disrupt the membranes of the cells allowing the plasmids to enter the cells. Electrocompetent formats provide the highest transformation efficiencies, but do require an electroporation device.

6, During the preparation of competent cells, chemical treatments are expected to alter the cell surface charge, in order to improve DNA interaction with the cells under cold conditions. The heat shock step is expected to destabilise the membrane and increase its permeability, allowing entry of DNA. The cells are then replaced on ice to allow the membrane to restore, followed by recovery in nutritional medium.

7.  Based on the colonies observed a predicted plate that untransformed E. coli bacteria can be foundon the LB/ (-) pGLO plate. No plasmid was added to this plate.

8. LB/ amp and LB/amp/aa plates is where transformed cells are found. Ampicillin gene resistance is express when genetically transforms cells are taken by the pGLO plasmid. These cells can survive on these plates which contain ampicillin.