pUC19 is a circular double-stranded plasmid cloning vector widely used for routine cloning in bacterium Escherichia Coli (E. coli) (Griffiths et al. 1999). It is used for its structure that allows visual selection when a foreign DNA molecule is inserted. (Becker, Caldwell, Zachgo 1996). The plasmid has 2686 base pairs (bp) and contains fourteen unique restriction sites, an origin of replication and two selectable markers: antibiotic ampicillin resistance gene and a lacZ’ gene that codes for the enzyme ?-galactosidase (Pasternak 2005).
Restriction endonucleases are used to cleave the plasmid DNA in several places by recognizing specific short sequence elements. By cleaving, restriction endonucleases leave the ends of the resulting fragments blunt (symmetric) or asymmetric (overhanging 5′ or 3′ ends). The plasmids that are cut with the same restriction enzymes will result with complementary in base sequence ends that will enable the donor-DNA to be inserted, by a process called ligation, and cloned. (Strachan, Read 2011). Ligation is carried out by the enzyme DNA ligase, which conjoin the sticky ends resulted in digestion (Trun, Trempy 2004). The two restriction enzymes that cut the multiple cloning sites of pUC19 plasmid are ECoR1 and Xba1. They cut the DNA molecule in two fragments, one measuring 27 bp and the other measuring 4491 bp. These two restriction endonucleases also cut the pBK-CMV plasmid with the DNA insertion. The CIH-1 gene measures approximately 600 bp, while the plasmid pBK-CMV measures 4518 bp (Burke 2018).
For the cell to take up the DNA insertion, it has to be competent. The E. coli is treated with TSS buffer which contains MgCl2 and undergoes heat shocks for the DNA insertion to enter into the host cell through the temporarily altered cell membrane (Burke 2018). Extremely often, the bacteria does not take up the DNA insertion, making the transformation process very inefficient. Therefore, selectable markers are used to differentiate the cells that have been transformed from the cells that did not take the foreign DNA.
To differentiate the recombinant cells, special plates are used. Ampicillin and tetracycline are used as antibiotics to make the distinction between ampicillin resistant cells and cells that did not have the gene (E. coli used is tetracycline resistant). IPTG (isopropylthiogalactosidase) is a reagent which allows the transcription of lacZ’ gene. It is used because it cannot be hydrolysed by ?-galactosidase. X-gal (5-bromo-4-chloro-3-indolyl-B-D-galactoside) is an analog of lactose. By using IPTG and X-gal, the recombinants can be identified. X-gal turns blue when it is hydrolysed by ?-galactosidase which is active in the non-recombinant cells, resulting blue colonies on the agar plates. X-gal turns white when the fungal gene is inserted and disrupts the lacZ’ multiple cloning site (insertional inactivation), meaning the recombinant plasmids cannot produce ?-galactosidase. Therefore, the colonies on the agar plates are white (Pasternak 2005).
The aim of the experiment is to transfer the cDNA molecule CIH-1 from the plasmid vector pBK-CMV into the plasmid vector pUC19. Two restriction enzymes, EcoR1 and Xba1, will cut the plasmid in specific sites. The resulted cuts will be ligated and transformed into competent E. coli. The colonies that contain the recombinant pUC19 plasmids will be selected and will undergo restriction analysis.