competent cells formation and transformation of competent cells with DNA.

BCH 462 [practical]

2nd lab

DNA cloning involve: “cell based” DNA cloning is a method of rapid isolation and amplification of DNA fragmaents

Cloning involves constriction of hybrid DNA molecules that are able to self-replicate in a host cell , this is accomplished by :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

2

Recombinant DNA the host

3

Recombinant DNA

Transformed bacteria

Cloning Vector •Molecule of DNA to which the fragment of DNA to be cloned is

joined. •Three features of all cloning vectors

* They must be capable of independent replication within the bacterial host cells

* They must contain at least one specific nucleotide sequencerecognized by restriction endonuclease

* They must contain at least selectable markers for drug Resistance

•Types of Cloning Vectors Plasmid

Phage - derivatives of bacteriophage lambda; linear DNA molecules ,

Anti-bacterial resistance gene.e.g: ampicillin resistance gene.

Origin of replication

Generally plasmid vectors should contain three important parts.

Generally plasmid vectors should contain three important parts.

At least one R.E recognition sites

EcoR1

Bam H1

Hind III

e.g: ampicillin resistance gene

Introducing

Transformed bacteria

cloning

“gene A”

“gene A”

Bacterial cell

Chromosomal DNA

Culture plate[media containing appropriate

antibiotic ]

Animal DNA

Amplified Recombinant plasmid

1

23

Ligation[using ligase enzymes]

Using R.EUsing same R.E used For cutting gene A

DNA cloning using plasmid

Restriction endonucleases Enzymes[R.E]:

Are DNA-cutting enzymes is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites.

Restriction Enzyme, e.g: EcoR1

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

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.

2.Transduction bacteriophages

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

Introducing

Transformed bacteria

Bacterial cell

Chromosomal DNA

3.Transformation DNA, plasmid DNA

Transformation: acquisition of extracellular DNA from the environment.

-Competence-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

Chilling cells in the presence of divalent cations such as CaCl2 or MgCl2 prepares the cell walls to become permeable to plasmid DNA .

Cells that are undergoing very rapid growth are made competent more easily than cells in other stages of growth .Thus cells are brought into log phase before the procedure is begun .The log phase cells are all living, healthy, and actively metabolizing .

Because this procedure can be very harsh on cells, the log-phase cells are more able to withstand this treatment .

OrElectroporation .-

Why we use CaCl2 ?

1 .Electroporation, or Electropermeabilization

Methods of transformation:

1.CaCl2 treatment To permeabilize the bacterial cell membrane

Insertion

Transformed bacteria

2.Chemical transformation.Less efficient than electroporation.

2.Brief heat shock to facilitate The DNA up take.

Practical part

Competent Cells formation Theory :

Since DNA is a very hydrophilic molecule, it won't normally pass through a bacterial cell's membrane .

In order to make bacteria take in the plasmid, this is done by creating small holes in the bacterial cells by suspending them in a solution with a high concentration of calcium.

There are two main methods for transformation of competent bacterial cells, the calcium chloride and the electroporation method. We will be using the

calcium chloride method .

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.

1.CaCl2 treatment To permeabilize the bacterial cell membrane

Insertion

Transformed bacteria

[Chemical transformation]

2.Brief heat shock to facilitate The DNA up take.

Principle of the experiment:

CompetentBacterial cell

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.