Lab: Differential Expression Differential gene expression provides the ability for a cell or organism to respond to a constantly changing external environment. The specific constellation of proteins required for optimal function and growth varies with cellular age and environmental context. Thus, protein production is carefully regulated by multiple mechanisms that modulate both transcriptional and translational pathways. Control of transcription initiation by RNA polymerase is a predominant mechanism for regulating expression of specific proteins, presumably because it provides maximal conservation of energy for the cell. We can often observe the consequence of differential transcription due to the presence or absence of particular proteins or the growth in particular environments. Control can also occur at translation; the mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at the level of protein function; the protein is inactive, or activity is not observed due to the lack of the substrate. In this lab we will observe differential expression of two different genes encoded on plasmids. We will analyze transcriptional activity, translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers. The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the abbreviation for the University of California, where early work on the plasmid series had been conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the most widely used vector molecules as the recombinants, or the cells into which foreign DNA has been introduced, can be easily distinguished from the non-recombinants based on color differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. - pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.