Expression of Beta-Galactosidase Open Reading Frame Under Cmv Promoter in Bacterial and Mammalian Cells

Combined Lab Report:

Expression of β-galactosidase Open Reading Frame under CMV Promoter in Bacterial and Mammalian Cells

Ryan Nguyen

Section 103

4/6/16

Abstract

Recombinant plasmids containing β-galactosidase open reading frames (ORF) under mammalian CMV promoters were sub-cloned using a donor plasmid, pTETβ, and a receiver (vector) plasmid, pTUD. These recombinant plasmids were transformed into competent bacterial cells and transfected into mammalian cells to observe the expression of β-galactosidase ORF. To obtain the recombinant plasmid a restriction digest was performed on pTETβ to excise the ORF and pTUD to open the vector near the CMV promoter. The restriction fragments were then ligated together to produce recombinant plasmids, transformed into competent E. coli cells to produce viable clones, and plated on various media to observe for β-galactosidase expression. There was no expression of β-galactosidase after growth of the bacterial colonies based on the lack of blue colonies on a plate containing X-gal. The plasmid DNA of the bacterial clones were then isolated and then transfected into mammalian, Human Embryonic Kidney (HEK 293t) cells. The cells were stained with X-gal solution resulting in the growth of blue cells giving a 50% transfection efficiency. The overall experimental results demonstrate that plasmids containing mammalian cytomegalovirus (CMV) promoters are unable to express ORFs within bacterial cells, but can do so within mammalian cells.  

Introduction

        When transferring genetic information between cells its necessary for the DNA to contain a selectable marker in order to confirm a successful transfer and whether or not the cell is expressing the desired ORF under a promoter. The selectable marker chosen for this experiment was β-galactosidase ORF as well as the β-lactamase gene (bla gene) that allows for ampicillin resistance. When β-galactosidase is expressed by a cell and introduced to X-gal, X-gal is cleaved into galactose and an X-dimer resulting in blue pigmentation. In regards to this experiment, cells that have been successfully transformed with β-galactosidase ORF under an exogenous promoter will result in blue growth when exposed to X-gal.

        The recombinant plasmid that we aimed to form from cloning was pTUDβ, which contains the selectable marker (β-galactosidase ORF) under an exogenous, mammalian CMV promoter and bla gene, which allows for ampicillin resistance. The vector or receiving plasmid, pTUD, contained the CMV promoter and bla gene, and the donor plasmid, pTETβ, contained β-galactosidase ORF. To form pTUDβ a SacII restriction digest was performed to excise β-galactosidase from pTETβ and open up the vector. A “dirty” ligation was then performed with all produced restriction fragments with pTUDβ resulting as one of the recombinant plasmids.