BCH 462 [practical]

1. 1st +2nd and 3rd labs Palsmid isolation and purification.+competent cells formation and transformation of competent cells with DNA.++extraction and purification of bacterial proteins. BCH 462 [practical]

2. DNAcloning: is a technique used to create copies of DNA fragments. 1-PCR[polymerase chain reaction]. 2-cell based [using a vector carrying the DNA of interest ,which eventually inserted to A host cell “usually bacteria” and self replicate]. Isolated and amplified of DNA fragment “e.g.: gene A”

3. DNA cloning involve: “cell based” 1.Insertion of DNA fragments in to a cloning vector ”e.g. plasmid”. 2. Introducing a vector in to bacterial cells” the host”. 3. Amplifying the vector DNA using bacterial DNA replication machinery. 1 DNA fragment cloning vector Recombinant DNA 2 Recombinant DNA the host Transformed bacteria 3

4. Plasmid: Are small, double stranded, closed circular DNA molecules that can be isolated from bacterial cells. Features: 1. Found in a wild variety of bacterial species. 2.Extra-chromosomal elements, which replicate independently of the bacterial chromosome. 3. Are not essential for the bacterium but may confer a selective advantage. 4.Using the enzymes and proteins encoded by their host for their replication and transcription. 5. Are inherited. 6. Used in many applications e.g. Drugs production.

6. At least one R.E recognition sites Origin of replication EcoR1 Bam H1 Anti-bacterial resistance gene. e.g: ampicillin resistance gene. Hind III Generally plasmid vectors should contain three important parts.

7. Some of plasmid applications : Gene therapy. Molecular cloning.

8. Using same R.E used For cutting gene A Using R.E Animal DNA “gene A” “gene A” DNA cloning using plasmid 1 Ligation [using ligase enzymes] 2 Bacterial cell Introducing 3 cloning Chromosomal DNA Culture plate [media containing appropriate antibiotic ] Amplified Recombinant plasmid Transformed bacteria

9. Restriction endonucleases Enzymes[R.E]: Are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases. Restriction Enzyme, e.g: EcoR1

10. DNA ligase: is a specific type of enzyme, that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond.

11. What is cloning vector? A DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial cell and produces many copies of itself and the foreign DNA. They must be: capable of independent replication within the bacterial host cells they most contain at least one specific nucleotide sequence recognized by a restriction endonuclease. Tow major types of cloning vector can be found in bacterial cells they are: -plasmid. -bacteriophages.

12. Bacteria can acquire new genetic information by: 1.During conjugation  direct contact. Conjugation: DNA is transferred directly from one organism to another and it requires direct cell-cell contact.

13. 2.Transduction  bacteriophages Transduction: is the process by which DNA is transferred from one bacterium to another by a virus[bacteriophges].

14. Introducing Bacterial cell Chromosomal DNA Transformed bacteria 3.Transformation DNA, plasmid DNA Transformation: acquisition of extracellular DNA from the environment.

15. -Competenceis the ability of a cell to take up extracellular ("naked") DNA from its environment. -It is the ability to undergo transformation which means the ability of a cell to take the DNA from the environment. -There are natural competence: a genetically specified ability of bacteria that is occur under natural condition. -Artificial competence: when cells in laboratory cultures are treated to be permeable to DNA.

16. Methods of transformation: 1. Electroporation, or Electropermeabilization

17. 2. Chemical transformation. Insertion 1.CaCl2 treatment To permeabilize the bacterial cell membrane 2. Brief heat shock to facilitate The DNA up take. Transformed bacteria

18. Practical part

19. Lab#1 Plasmid isolation and purification Principle of the experiment “Alkaline lysis method “: using SDS in the alkaline solution: -The SDS: will lyse the bacterial cell membrane and denature the proteins too. -The alkaline pH :denature the genomic DNA and denature the proteins too. -The degraded cell wall, denatured chromosomal DNA and bacterial proteins form large aggregated complex which will precipitated during the plasmid isolation and removed by centrifugation. -Native plasmid DNA can be collected from the supernatant.

20. Lab#2 Competent Cells formation and transformation of competent Cells with DNA Principle of the experiment: “ chemical transformation method “ Cells are incubated in CaCl2 solution that help the cells to take up the DNA plasmid by increasing the bacterial cell s membranes permeability [renders them competent to take up DNA], then applying brief heat shock will facilitate the DNA up take. Note: the transformed cells are then grown in LB agar plate containing appropriate antibiotic to be able to count the transformed colonies only , “which they are colonies containing transformed cells -containing the DNA plasmid-” , each colony on an antibiotic plate presents a single transformation event. -Then calculations of the transformation efficiency will be done.

21. Principle of the experiment: Insertion 1.CaCl2 treatment To permeabilize the bacterial cell membrane 2. Brief heat shock to facilitate The DNA up take. Transformed bacteria

22. 3.Positive selection on LB plates with the appropriate antibiotic. Transformation efficiency= total number of colonies on LB/Amp plate CFU/µg amount of DNA plated [µg/ml] CFU: colony –forming units. Animation: http://www.dnai.org/b/index.html

23. Lab#3 Extraction and determination of bacterial protein Isolation of bacterial proteins involves several steps : growth and induction of bacterial cultures. Lysis of cells in a suitable buffer containing a detergent. DNase and RNase treatment for the removal of the nucleic acids. Passage of the extract through an affinity resin and finally elution of proteins. -Biuret method is used to determine the protein concentration, using standard curve. - Biuret method is based on copper ions binding to peptide bonds of protein under alkaline condition to give a violet to purple color. -The intensity of the color resulting from the Cu-protein complex is linearly proportional to the mass of protein present in the solution.

24. Depending upon nutritional status, bacteria exhibit different growth patterns which include: Lag phase: in this phase bacteria adapt themselves to growth conditions and synthesis its own DNA,RNA and proteins. Log phase: it is exponential phase, bacterial cells divide and the production of new cells is proportion to increased time. Stationary phase: the growth rate slows as nutrients become limited, waste products accumulate and the rate of cell division equals the rate of death. Death phase: due to continuous accumulation of toxic metabolites and the lack of nutrients, death occurs of the bacteria.