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Transformation MISS : SALSABEEL H. AL- JOUJOU.

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Presentation on theme: "Transformation MISS : SALSABEEL H. AL- JOUJOU."— Presentation transcript:

1 Transformation MISS : SALSABEEL H. AL- JOUJOU

2 Transformation :- is a process in which cells take up foreign DNA from their environment. bacteria take up exogenous (foreign) DNA and produce the genetic products (proteins) encoded in the foreign DNA. Under proper conditions, a cell that is incubated with plasmid DNA can absorb the plasmid into its cytoplasm. E.coli HB 101 strain is an excellent host for cloning experiment. because they do not have any natural antibiotic resistance .

3 Transformation enables inexpensive and reliable production of important medical products such as insulin, human growth hormone, and other replacement hormone and gene therapies.

4 For transformation to occur, bacterial cells must be in a particular physiological state, referred to as competency. Much current research in molecular biology involves the transformation of E. coli, an organism that does not naturally enter a state of competency. E. coli can artificially be made competent when treated with chloride salts of the metal cations calcium, magnesium . DNA and bacteria are kept on ice for half an hour to enable the DNA to increase interactions between calcium ions and the negative components of the cell.

5 Characteristics of plasmid to be suitable for the transformation process:-
In genetic research, plasmids must be large enough to manipulate, small enough for the cell to take up, and include a marker gene after the targeted gene. The marker gene allows the researcher to readily identify which cells successfully acquired the new genetic material. For example, if a phosphorescence gene follows the gene for insulin production, then glowing cells successfully took up the plasmid and can produce insulin

6 Note:- The process of Genetic engineering is made effective by the action of two enzymes namely, Restriction enzymes and ligases. Restriction enzymes, called molecular scissors are precisely cut the desired DNA and its carrying vector, at specific sites , whereas the ligase enzyme, called Molecular glue are pasted these DNA fragments into the carrying vector.

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8 Steps of Bacterial Transformation
Competency Transformation Recovery period Growth & Isolation

9 After make cell competent we make heat shock
Then the tube in which the cells are kept is put at 42°C for 90 seconds (this time can be adjusted depending on the size of the tube) . This action is called the 'Heat shock. Immediate chilling on ice ensures closure of pores.

10 3- Recovery period Allow the cell to regain strength and start to multiply Necessary to allow expression of the antibiotic resistance gene on the plasmid and insert. Add sterile luria broth. Move tubes to water bath at 37oc for 30min.

11 4- Growth & Isolation` Mark four Luria Broth agar plates

12 This concept can be demonstrated by plating the same volume of recovered cells on selective and nonselective agar medium. The nonselective medium will have many more growing cells since all the untransformed cells survive, in addition to the transformed cells.

13 The nonselective bacterial agar plates will be covered heavily with untransformed cells, forming a “lawn”, in contrast to individual colonies obtained on the selective agar plate.

14 Transformation Efficiency:-
The number of cells transformed per 1 microgram (μg) of DNA is known as the transformation efficiency. In practice, small amounts of DNA are used (5 to 100 nanograms, ng) since excessive DNA (>100 ng) inhibits the transformation process Too little DNA can result in low transformation efficiencies

15 Calculate the transformation efficiency:-
Transformation Efficiency = no. of colonies observed/ µg Plasmid

16 Note that each colony originally grew from one transformed cell

17 In this lab we will perform transformation of E Coli
pUC8 = Ampicillin Resistant plasmid will be inserted into E coli

18 4 plates Luria Broth - No antibiotic, no plasmid
Luria Broth + No antibiotic, With plasmid AMP Broth - antibiotic, No plasmid AMP Broth + antibiotic, With plasmid

19 What do you expect Luria Broth - No antibiotic, no plasmid
Normal growth = lawn of bacteria Luria Broth + No antibiotic, With plasmid Normal growth = lawn of bacteria AMP Broth - antibiotic, No plasmid NO growth, all killed AMP Broth + antibiotic, With plasmid Individual Colony growth of transformed resistant bacteria

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