1. 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

2. 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

3. 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

4. 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

5. 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