Transcript Plasmids and Minipreps

Plasmids and Minipreps
Biotechnology
Plasmid- has instructions to make antibiotics
Plasmids
• Aren’t required for the bacteria to live under standard conditions.
• Plasmids produce materials during harsh living conditions.
• Plasmids replicate independently from the Bacterial DNA.
• 1 to 100 copies.
• In a plasmid the name is represented with a small p before the name.
• pBLU, pAMP, pUC19
Genetic engineering
• Take a gene from one organism and transfer it to another organism.
• https://www.youtube.com/watch?v=2YxTcBimxlw#t=1.521
The first plasmid transfer
• Somatostatin by the company Genentech 1972
• https://www.youtube.com/watch?v=TrxWCXpf8IU
• https://www.dnalc.org/view/15075-Challenge-of-isolating-a-gene-StanleyCohen.html
• http://www.smithsonianmag.com/smithsonian-institution/a-history-ofbiotechnology-in-seven-objects-180947559/?no-ist
• Video on Restriction enzymes
https://www.youtube.com/watch?v=pDHCHa1C85Y
• Remove DNA from bacteria cells
• Used a blender to shear the plasmid
Plasmid transfer
• https://www.youtube.com/watch?v=KRpik9mNRm0
Gene therapy
• Target the right cells
• Integrate the gene into the cell without being trashed.
• Activate the gene.
• Protein must function properly.
• Avoid harmful side effects. Lytic viruses are bad. Lysogenic are good.
• In vivo- in living organism. Bone marrow transplants.
• Ex vivo- in petri dish.
Gene delivery methods:
• Retrovirus- accepted only in dividing cells (RNA). 8000 bp max. Has side
effects.
• Adenovirus- double stranded DNA. Both dividing and nondividing cells.
7500 bp max. Immune system reaction. Not integrated in DNA.
• Adeno associated virus- No illness. Single DNA. Helper virus. 5000 bp max.
Integrates into DNA.
• Herpes simplex virus- dsDNA. Nervous system. 20000 bp. Not integrated.
Immune and protein reactions.
• Liposome-circular, cut dsDNA into. Less than virus max bp. Toxic effects.
Done ex-vivo (outside living body)
• DNA plasmid- all types, no maximum, not integrated. Ex vivo.
Successful cases of Gene Therapy
http://learn.genetics.utah.edu/content/genetherapy/gtsuccess/
• Severe Combined Immuno Deficiency- Rhys Evans in 2001.
• Sickle cell anemia
• Adenosine deaminase deficiency
• Hemophilia- Sebastian Misztal in 2011
• Leukemia
• Reverse vision loss in mouse, rat, and dog
• Beta Thalassemia blood disease
• Fat metabolism
• Parkinson’s disease
Bad cases of gene therapy
• Jesse Gelsinger died of a liver disorder 1999 using an adenovirus
vector.
• David Vetter- boy in the plastic bubble had SCID.
Gene Therapy simulation
• http://learn.genetics.utah.edu/content/genetherapy/
• Click what is gene therapy, learn about Cystic fibrosis by clicking the
forward buttons.
• Phenylalanine is missing from transport protein in Cystic Fibrosis
lungs, so Chlorine doesn’t get moved, too much Sodium moves into
lungs and mucus layer dries out and plugs lungs.
Enzyme finder
• https://www.neb.com/tools-and-resources/interactive-tools/enzymefinder?searchType=2&recognitionSite=&matchType=1
How to figure out what your DNA will look like
after being cut by restriction enzymes.
• Find your DNA sequence at http://www.ncbi.nlm.nih.gov/
• Select genome to find the entire sequence of all chromosomes.
• Select gene to find just one sequence. For a human there are 2999
gene sequences in the database. Click FASTA to go to the base pair
sequence.
• Copy and paste the sequence to NEBcutter and see what it will look
like in gel electrophoresis.
• http://nc2.neb.com/NEBcutter2/