Lecture 4--Transformation--14.08.19.pptx

transformation

1. Bacterial Transformation

2. Griffith’s experiment Unlike conjugation and transduction where physical contact is necessary for DNA transfer, Transformation is a process, where a recipient bacteria can directly take up a piece of DNA from its surroundings. Bacteria →uptake → DNA fragment (from its surroundings)

3. Unlike conjugation and transduction where physical contact is necessary for DNA transfer, Transformation is a process, where a recipient bacteria can directly take up a piece of DNA from its surroundings. Bacteria →uptake → DNA fragment (from its surroundings)

4. From where DNA comes? Bacteria lyse → release → DNA fragments ↓ ‘Competent’ cell ↓ taken up ↓ integrate ↓ Chromosome

5. What is competent cell Normal bacterial cell is not permeable to DNA fragments directly. It cannot pass through the membrane of bacteria. But during the growth of bacterial cycle, i.e. During exponential phase some bacteria shows permeability means they can pass DNA through their membrane. Such phase are called competent phase

6. Mechanism of transformation Transformation: DNA in the environment of a cell is taken into the recipient cell forming a merozygote ; then recombination occurs occurs naturally in some bacteria ( e.g., Pneumococcus ) occurs rarely in others, but can be promoted by treating cells to destabilize their membranes ( e.g., in recombinant DNA work) can map genes by co-transformation (frequency with which two genes are simultaneously transferred

7. Mechanism of Transformation

8. Cotransfromation Genes located close together are often transferred as a unit to recipient cell = cotransformation Genes that are far apart are less likely to be transferred together Cotransformation is used to map gene order

9. How do we get the plasmid inside of the bacteria? Obtain E. Coli bacteria cells + Add to CaCl 2 (helps plasmid attach to bacteria) Add plasmid to same microtube 1. E. Coli 2. pBlu plasmid

10. How do we get the plasmid inside the bacteria? 3. Heat shock: This temporarily opens pores to allow the plasmid to enter the bacteria… timing is critical and very important

11. Growing the bacteria After they have received the plasmid Placed on a growth media and allowed to grow.