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 the teacher’s notes

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”• Consensus 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