Molecular Biology and Genetic ENGINEERING

Experiment 5

Aim of the Experiment

To learn the process of cloning a foreign gene into a vector.

Introduction:

The cloning experiments of Herbert Boyer, Stanley Cohen, Paul Berg, and their colleagues in the early 1970s ushered in the era of recombinant DNA technology. Recombinant DNA is a DNA that has been created artificially. DNA from two or more sources is incorporated into a single recombinant molecule. The basic procedure of molecular cloning involves a series of steps. First, the DNA fragments to be cloned are generated by using restriction endonucleases. Second, the fragments produced by digestion with restriction enzymes are ligated to other DNA molecules that serve as vectors. Vectors can replicate autonomously (independent of host genome replication) in host cells and facilitate the manipulation of the newly created recombinant DNA molecule. Third, the recombinant DNA molecule is transferred to a host cell. Within this cell, the recombinant DNA molecule replicates, producing dozens of identical copies known as clones. As the host cells replicate, the recombinant DNA is passed on to all progeny cells, creating a population of identical cells, all carrying the cloned sequence. Finally, the cloned DNA segments can be recovered from the host cell, purified, and analyzed in various ways.

Principle:

Cloning refers to the exact copy of an organ, whole organism, single cell or a piece of DNA. Gene cloning is a process through which an exact copy of a particular gene is made. The process through which a foreign piece of DNA is transferred into another DNA led to the development of recombinant DNA technologies. The entire cloning procedure involves the following steps:

1. Isolation of pure vector and insert DNA

2. Restriction digestion of the DNAs

3. Ligation of the two linear DNA fragments

4. Transformation of the ligated product

5. Screening for the right clone

1. Isolation of pure vector and insert DNA:

During cloning the foreign DNA is isolated after following the specific DNA isolation procedure. The insert DNA which contains the gene of interest is obtained after restriction enzyme digestion. Several naturally occurring plasmids have been engineered to make different types of cloning vectors. Cloning vectors are circular DNA molecules in which DNA fragments/insert are maintained and amplified. Vectors should have the following features that make them compatible for a variety of uses in recombinant DNA procedures:

Size: Vectors are relatively small molecules; most are only 2.5 - 3 kb in size.

Ori: A vector should contain an origin of replication (ori) in order to maintain its autonomous state.

Marker: To facilitate selection of cells that contain the vector, a vector should carry at least one genetic marker for which an easy assay exists (e.g., ampicillin resistance, Ampr).

Cloning sites: A vector should contain several different restriction endonuclease sites that allow the insertion of a gene of interest during cloning. Many cloning vectors like pUC19 contain a synthetic DNA sequence called a polylinker, which consists of 21 closely-packed RE recognition sites in a small region of DNA.

2. Restriction digestion of the DNAs: During cloning procedure an insert is added to a vector and for this reason both of them have to be cleaved by restriction endonuceases (REs). Restriction endonucleases recognize a specific, rather short, nucleotide sequence on a double-stranded DNA molecule, called a restriction site, and cleave the DNA at this recognition site or elsewhere, depending on the type of enzyme. The target site or sequence which the restriction enzyme recognizes is generally from 4 to 6 base pairs and arranged in a palindromic sequence. Generally both the vector and the foreign DNA are digested with the same RE. The restriction digested vector DNA has to be dephosphorylated to prevent self-ligation.

3. Ligation of two linear DNA fragments: Two linear DNA molecule ends (vector and insert) can be joined together through a process called ligation. This process involves the formation of a covalent bond between two DNA fragments (having blunt or overhanging, complementary, 'sticky' ends) with the help of an enzyme named as ligase. DNA ligase forms a phosphodiester bond between the 3’ hydroxyl of one nucleotide and the 5’ phosphate of another. Ligation can be directional or non-directional based upon the restriction enzyme used. When both the vector and the insert are digested with a single RE then the ligation can occur in either direction and when they are digested with two REs then ligation takes place only in one direction.

4. Transformation of the ligated product: The ligation reaction mixture is introduced into bacterial cells in a process called as transformation. In this method cells are incubated in a concentrated calcium salt solution to make their membranes leaky. The permeable “competent” cells are then mixed with DNA (ligation mix) to allow entry of the DNA into the bacterial cell. Alternatively, a process called as electroporation can be used that drives DNA into cells by a strong electric current. Successfully transformed bacteria will carry either recombinant or non- recombinant plasmid DNA. Multiplication of the plasmid DNA occurs within each transformed cell. A single bacterial cell placed on a solid surface (agar plate) containing nutrients can multiply to form a visible colony made of millions of identical cells. As the host cell divides, the plasmid vectors are passed on to progeny, where they continue to replicate.

5. Screening of the right clone: The last step during cloning is screening for the right clone. Screening means to detect the right clone among a population of colonies and the screening procedure depends upon the vector used for cloning. The E. coli plasmid pUC19 carries sites for different restriction enzymes (Multiple Cloning Site, MCS) within the N-terminal coding sequence for β-galactosidase of the lac operon. When pUC19 is transformed into a competent host cell (which has a deletion at the amino terminal end of the LacZ gene, which codes for β-galactosidase), the truncated products from both complement each other and as a result enzymatically active β-galactosidase is produced. This is called α-complementation. The tranformants turn blue on X-gal and IPTG containing plates due to the production of β-galactosidase. X-gal is a chromogenic substrate of β-galactosidase and IPTG acts as an inducer for the expression of this enzyme. If an insert is cloned within the MCS of pUC19 then β-galactosidase is not produced and as a result the transformants turn white on X-gal and IPTG containing plates. As a result one can directly screen for the insert containing vector (recombinant).

Materials required

Glasswares: Conical flask, Measuring cylinder, Beaker

Other requirements: Micropipettes, Tips, 50 ml Centrifuge Tubes, Water bath (42oC), 37oC Incubator, 37oC Shaker, Centrifuge, UV Trans illuminator, Crushed ice, Sterile double distilled water, Sterile loop and spreader.

Procedure

Day 1:

1 Open the vial containing culture and resuspend the cells with 0.25 ml of LB broth.

2 Pick up a loopful of culture and streak onto LB agar plate

3 Incubate overnight at 370C.

Day 2:

1. Inoculate a single colony from the revived plate in 1 ml LB broth.

2. Incubate at 37oC overnight.

3. Thaw 10X Ligase Buffer, T4 DNA Ligase, Vector DNA and insert DNA on ice and then set up the ligation reaction as follows:

Molecular biology grade water : 2 μl

Vector DNA : 3 μl

Insert DNA : 3 μl

10X Ligase buffer : 1 μl

T4 DNA ligase : 1 μl

Total reaction volume : 10ul

Mix the contents by tapping gently and incubate at 16°C water bath, overnight.

Day 3:

1. Take 50 ml of LB broth in a sterile flask. Transfer 1 ml overnight grown culture into this flask.

2. Incubate at 37oC shaker at 250 rpm for 2-3 hours till the O.D reaches ~ 0.6.

A) Preparation of Competent Cells:

Note: Prepare competent cells within 3 days of reviving the strain.

1. Transfer the above culture into a prechilled 50 ml polypropylene tube (not provided).

2. Allow the culture to cool down to 4oC by storing on ice for 10 minutes.

3. Centrifuge at 5000 rpm for 10 minutes at 4oC.

4. Decant the medium completely. No traces of medium should be left.

5. Resuspend the cell pellet in 30 ml prechilled sterile 0.1 M Calcium chloride solution.

6. Incubate on ice for 30 minutes.

7. Centrifuge at 5000 rpm for 10 minutes at 4oC.

8. Decant the calcium chloride solution completely. No traces of solution should be left.

9. Resuspend the pellet in 2 ml prechilled sterile 0.1M Calcium chloride solution.

10. This cell suspension contains competent cells and can be used for transformation.

B) Transformation of cells:

1. Take 200 μl of the above cell suspension in four 2.0 ml collection tubes and label them as control, positive control plasmid, negative control plasmid, and ligation mix.

2. Add 2 μl of positive control plasmid DNA to the tube labeled as positive control plasmid and

2 μl of negative control plasmid DNA to the tube labeled as negative control plasmid mix well.

3. Add 10 μl of the ligation mix to the tube labeled as ligation mix and mix well.

4. Incubate all the 4 tubes on ice for 30 minutes.

5. Transfer them to a preheated water bath set at a temperature of 42oC for 2 minutes (heat shock).

6. Rapidly transfer the tubes on ice. Allow the cells to chill for 5 minutes.

7. Add 800 μl of LB Broth to all the tubes. Incubate the tubes for 1 hour at 37oC to allow the

bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.

8. Plate 200 μl of cell culture from each tube on LB agar plate containing ampicillin, Xgal, IPTG using a sterile spreader.

9. Store at room temperature till the plates are dry.

10. Incubate the plates overnight at 37oC.

Observation & Result:

After incubation observe the bacterial growth on plates and look for the blue colonies on the plate with positive control plasmid and white colonies on plate with negative control plasmid.

Interpretation:

On transformation of cells with plasmids, antibiotic resistance is conferred on the host as these plasmids carry gene for ampicillin resistance. As a result, those cells that grow in presence of ampicillin are transformed cells. In contrast, no growth is observed on plating competent cells untransformed with plasmid as the host E.coli cell is sensitive to ampicillin. The E. coli plasmid pUC19 (here positive control) encodes the N-terminal coding sequence for β-galactosidase gene of the lac operon. The E. coli host strain has a deletion at the amino terminal end of the LacZ gene, which codes for β-galactosidase. When pUC19 is transformed into the competent host cells, the truncated products from both complement each other and as a result enzymatically active β-galactosidase is produced. This is called α- complementation. The transformants turn blue on X-gal and IPTG containing plates due to the production of β- galactosidase. X-gal is the chromogenic substrate of β-galactosidase and IPTG acts as the inducer for the expression of this enzyme. The negative control plasmid has one insert cloned in the N-terminal coding sequence for β- galactosidase gene. As a result enzymatically active β-galactosidase is not produced and the transformants are white in colour. When the insert DNA of the kit is cloned in the pUC19 vector then we get white colonies and if the vector DNA is self- ligated then we get blue colonies on X-Gal and IPTG containing plates.

Reference Material

T.A Brown Cloning.

Techniques in Biochemistry and Molecular Biology

Molecular cloning by sambrook Russell

Questions

1. Why does transformants from ligation does not mix. Explain giving possible solution?

2. How can one avoid bacterial growth on plates?

3. What are the possible reasons if there is contamination observed on plates?