Upload
erica-stace
View
215
Download
0
Embed Size (px)
Citation preview
Genetic JigsawClass instructions
Start of lesson
• Divide the classes into 6 groups:– Origin of replication– Repressor gene– Promoter– Multiple cloning site– Antibiotic resistance gene– Insert
• Each group should collect the bases they need according to following slides
Make the plasmid parts
Make the plasmid parts
• Make the following parts of the plasmid:– Origin of replication– Repressor gene– Promoter– Multiple cloning site– Antibiotic resistance gene– GFP/insulin insert
Plasmid map
Each group makes one part
• Remember to make the sense strand in black• Antisense strand in red• Make sure you get the 5’ – 3’ orientation
correct
Correct base pairing is critical!
• Green (Guanine) pairs with yellow (Cytosine)
• Blue (Adenine) pairs with orange (Thymine)
The devil is in the detail!
• The 5’ prime and 3’ prime ends of the bases must be round the right way!
Origin of replication
• Plasmid DNA replication starts here• Determines how many plasmid copies there
are in each bacterial cell:– Can be low copy number 25 – 50– High copy number can be > 500 per cell
• A-T rich region where strands are separated for DNA replication
Origin of replication
Sense strand
Anti-sense strand
Repressor gene
Sense strand
Anti-sense strand
Repressor gene
• As the repressor gene is expressed in other direction it must be inserted upside down
Sense strand
Anti-sense strand
Repressor gene
• Blocks genetic switch (promoter)• Moves when “food” present– Lactose or arabinose
• Causes conformation change• RNA polymerase can then bind to promoter
Promoter
• Genetic switch• Switched off until “food” present– Lactose or arabinose
• Repressor undergoes conformation change• RNA polymerase can then bind to promoter• Switches on genes “downstream”• Concensus sequence
Promoter
Sense strand
Anti-sense strand
Multiple Cloning Site (MCS)
• Series of unique recognitions sites• Using combinations of enzymes allows you to
directionally insert a gene• Ensures gene is correctly expressed• NheI and EcoRI sites
Multiple Cloning Site (MCS)
Sense strand
Anti-sense strand
NheI recognition site
Sense strand
Anti-sense strand
EcoRI recognition site
Sense strand
Anti-sense strand
Antibiotic resistance gene
• Most bacteria don’t take up DNA when transformed
• Identify those with plasmid with selection marker
• Ampicillin resistance gene Beta-lactamase• Note start codon ATG
Antibiotic resistance gene
Sense strand
Anti-sense strand
GFP/insulin insert
• Insert represents either:– Green Fluorescent Protein (GFP) is used a marker
gene as glows!– Human insulin used to treat diabetes
• EcoRI and NheI restriction sites at ends
GFP/insulin insert
Sense strand
Anti-sense strand
NheI site GFP/insulin sequence EcoRI site
Make the complete plasmid
Make the complete plasmid!
ori repressor promoter MCS AmpR
Genetically engineer the plasmid
Genetically engineer the plasmid
• Identify the MCS by looking for the sites:
• Align the insert with the plasmid at the MCS
NheI EcoRI
Digest the plasmid & insert
• With EcoRI
• With NheI
Line up insert and plasmid
• Put the insert in the correct way round
• And join together (ligate)
Rejoin the plasmid into a loop
• The plasmid is now ready to be transformed!
Gene regulation
How is the gene switched on?
• Locate the promoter and insert• Repressor protein blocks the promoter– Place a hand over the promoter
• Food source binds to the repressor protein– Second hand on repressor protein hand
• Conformation change occurs to repressor protein and promoter is switched on