Using Plasmids in Biotechnology Plasmids have special natural abilities that are commonly manipulated for two main functions: Production of protein products…insulin Gene therapy or modification…GMO plants, experimental therapies for genetic disorders (CFTR) Plasmids that are used to store and amplify genes are referred to as cloning vectors while plasmids that are used to produce viable products are referred to as expression vectors

Advantages to Housing DNA in Plasmids Plasmids are far more stable than linear DNA Plasmids can be inserted into bacteria to efficiently make many clones Plasmids can be engineered to add restriction sites to existing genes for modification Many commercially available contain multiple cloning sites (MCS) for ease of gene insertion Ensures that the sticky ends on the gene to be inserted will match a restriction site

Transcriptional Regulation of Plasmids The start codon must be inserted downstream form the promotor and in the correct reading frame (AUG) Review…a codon is a 3 letter nucleotide sequence for a specific amino acid Frameshifts (out of reading frame) lead to useless amino acid sequences or truncated proteins Constituative genes…always turned on (products needed for normal functions) Facultative genes…turned on only when needed (often for substrate metabolism) Transcriptional Regulation of Plasmids

Transcriptional Regulation of Plasmids Operons Natural occurring control units that induce (activate) or repress (turn off) groups of genes The operon (repressor) hides the promotor region making it inaccessible to RNA polymerase. Initially discovered in 1961 in E. coli. Lac operon Repressor is inactivated by the presence of lactose (inducer) Although the operon was originally discovered as part of E. coli main chromosome it has been engineered and inserted into plasmids for better control of transcription

Engineering of pGLO plasmid – pBAD18 The pGLO green fluorescent marker was incorporated into the araBAD operon promotor so that it would only be expressed when other genes of interest are transcribed Turned on in the presence of arabinose Includes the bla gene for antibiotic resistance The use of the operon allows scientists to check for proper insertion of genes and expression Engineering of pGLO plasmid – pBAD18