Answer

Introduction

            Gene cloning is the process of isolating a piece of a given DNA from an organism and introducing the DNA into a host referred to as clone that is able to grow and divide rapidly. The study of the cloned DNA is thus possible as well as encoded protein by the gene. The cDNA can be inserted into a given vector and cloned (Krebs et al 2010). Bacteria species possess plasmid that is capable of carrying genes responsible for antibodies resistance, metabolism or conjugation of the unusual substrates. PUC19 is one of the plasmids and is generally adopted for the cloning of DNA fragments that are small in E.coli (Lodge et al 2010, p.71). The plasmid vectors have the principle of permitting the identification of the recombinant clones by the look of insertion deactivation of lacZ or antibiotic resistance genes.

            Plasmid DNA is cut by the use of restricted enzymes to analyze the size of the produced fragments. The enzyme restriction endonucleases recognize the certain DNA sequences thereby cleaving them in a well-defined pattern. The ends of two strands as exemplified by Strachan 1999, is joined by covalent bonds brought about by the action of enzyme ligase that enables the construction of the recombinant fragments of DNA.  Ligation process involves joining of the two ends of molecule DNA between the 3’ hydroxyl of one of the nucleotide and also the 5’ of the phosphate group of the other by phosphodiester bond. The ligation step is always carried out with the bacteriophage of T4 DNA ligase enzyme by using ATP that is required for the reaction and for the suitable buffer condition. The event of ligation is actually of two types; blunt or sticky based on types of the restriction enzymes that are used for vector digestion and insertion. Transformation is the process that takes place after ligation, whereby the recombinant plasmid that is the ligation product is transformed into bacteria for the process of propagation. The bacteria that are transformed are then plated on selected agar so as to select for bacteria containing plasmid of interest. The individual colonies are then picked up and tested for the process of cloning (Sales et al 2007, p.821).

Aim

To transfer the transfer the fungal gene CIH-1 from the plasmid vector into the different vector called the PUC19 using different techniques.

Method

(Green 2013)

Results

The gel electrophoresis was used to measure the fragments sizes that were generated as shown in the figure below.

igure1: Agarose gel electrophoresis of restriction digest plasmids pUC19 and  pBKCMV

pBK CMV                      pUC19                                 Marker

From the results above, the nucleic acids in the gel migrated thus describing a linear movement of the fragments of the DNA. The ethidium bromide, that is able to fluoresces under the ultraviolet light, is used to stain the DNA to enable them visualize. From the results obtained therefore, the distance moved by fragments of DNA markers from well can be measured using a ruler. 

Results determined from transformation plates

The electrophoresis of agarose of B1, W1 and W2 of the restricted plasmids were shown on the figure below. The W1 and W2 contained recombinant DNA thus forming two DNA fragments inside the gel. Conversely, PUC19 showed a single line because it is non-recombinant DNA.

Figure2: Agarose gel electrophoresis of the restricted plasmids B1, W1 and W2

W1             W2                  B                 Marker

Discussion

            In order for the DNA to be cloned, enzymes must be used to cut the fragment in a repeatable and précised way. As a result, the foreign gene that is CIH-1 should be cut out of the vector pBK-CMV with the use of the restriction endonuclease, EcoR1 and the Xbal. The same procedure applied also on PUC19 (Sorenses 2005, p.116).

            The bacteria with plasmid will thus grow in the presence of the ampicillin. A bacterium that hydrolyzes the X-gal produces galaxtose and a compound indigo in color. The compound is responsible for turning the colonies blue. Bacteria that is not able to hydrolyze the X-gal produces the white colonies. The plasmid that has the lacZ gene encodes with the enzyme β-galactosidase and the ampicillin resistance. The foreign DNA will then insert into the gene for the lactose hydrolysis that is lacZ. The bacterium that receives the plasmid vector will at the end not be able to produce β-galactosidase enzyme when the foreign DNA has been inserted (Young et al., 2009, p.1615).

            The fungal gene CIH-1 that is isolated needs to be inserted into the pCU19 vector. The cDNA was cloned in the plasmid vector pBK-CMV. The cloned DNA was cut in repeated and precise way by the use of enzymes. The foreign gene should be cut out of the pBK-CMV with the enzyme restriction endonuclease. The enzyme recognizes certain sequences of DNA that are palindromic that are about 4 to 6 bases pairs and cleaved in pattern. This therefore suggested that reading of nucleotide sequence is similar both in the directions on each strand. When cleaved, they leave a blunt end depending on the enzyme used Pingoudet al.,2005, p.699.

When the restricted enzymes are inactivated, the enzyme and the plasmid fragments are mixed with the action of T4 DNA ligase. The foreign gene that is CIH-1 from the pBK-CMV is ligated into MCS of the vector pCU19. A new recombinant molecule is formed when a vector molecule is joined to fragments of genomic DNA so as to circulate. The recombinant molecule is introduced into the bacteria E.coli. The DNA that is associated with lipopolysaccharide found on the outer surface of competent cells for DNA uptake (Chow 2005, p.11)

            Conclusively, new improvements have been advanced for isolation, analysis and also the manipulation of the nucleic acids. The cloning strategies have heralded exciting a new era in exploitation of molecules of DNA. The process of gene cloning has enabled several discoveries to be obtained thus providing precious insights into the structure of the gene, regulation and function (Strachan, 1999).