Genetic Jigsaw Class 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

Repressor gene group x3 x1 x2 x3x1 x3 A G C T T A C G

Multiple cloning site group x3 A G C T T A C G

Antibiotic resistance gene group x3 x2 x4 x2 x3 A G T T A C

Origin of replication group x5 A T

Promoter group x3 A T T A

Insert group x12 A T C G x9

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 siteGFP/insulin sequenceEcoRI site

Make the complete plasmid

Make the complete plasmid! orirepressorpromoterMCSAmp R

Genetically engineer the plasmid

Identify the MCS by looking for the sites: Align the insert with the plasmid at the MCS NheIEcoRI

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