1. Genetic Jigsaw Class instructions

2. 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

3. Make the plasmid parts

4. Make the following parts of the plasmid: – Origin of replication – Repressor gene – Promoter – Multiple cloning site – Antibiotic resistance gene – GFP/insulin insert

5. Plasmid map

6. 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

7. Correct base pairing is critical! Green (Guanine) pairs with yellow (Cytosine) Blue (Adenine) pairs with orange (Thymine)

8. The devil is in the detail! The 5’ prime and 3’ prime ends of the bases must be round the right way!

9. 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

10. Origin of replication Sense strand Anti-sense strand

11. Repressor gene Sense strand Anti-sense strand

12. Repressor gene As the repressor gene is expressed in other direction it must be inserted upside down Sense strand Anti-sense strand

13. Repressor gene Blocks genetic switch (promoter) Moves when “food” present – Lactose or arabinose Causes conformation change RNA polymerase can then bind to promoter

14. 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

15. Promoter Sense strand Anti-sense strand

16. 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

17. Multiple Cloning Site (MCS) Sense strand Anti-sense strand

18. NheI recognition site Sense strand Anti-sense strand

19. EcoRI recognition site Sense strand Anti-sense strand

20. 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

21. Antibiotic resistance gene Sense strand Anti-sense strand

22. 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

23. GFP/insulin insert Sense strand Anti-sense strand NheI siteGFP/insulin sequenceEcoRI site

24. Make the complete plasmid

25. Make the complete plasmid! orirepressorpromoterMCSAmp R

26. Genetically engineer the plasmid

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

28. Digest the plasmid & insert With EcoRI With NheI

29. Line up insert and plasmid Put the insert in the correct way round And join together (ligate)

30. Rejoin the plasmid into a loop The plasmid is now ready to be transformed!

31. Gene regulation

32. 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