Genetic Jigsaw

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

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

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

6. Origin of replication group A T x5 x5

7. Promoter group T A A T x3 x3 x3 x3

8. Insert group G C T A x9 x12 x12 x9

9. Make the plasmid parts

10. 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

11. Plasmid map

12. 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

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

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

15. 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

16. Origin of replication Sense strand Anti-sense strand

17. Repressor gene Sense strand Anti-sense strand

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

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

20. 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

21. Promoter Sense strand Anti-sense strand

22. 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

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

24. NheI recognition site Sense strand Anti-sense strand

25. EcoRI recognition site Sense strand Anti-sense strand

26. 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

27. Antibiotic resistance gene Sense strand Anti-sense strand

28. 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

29. GFP/insulin insert NheI site GFP/insulin sequence EcoRI site Sense strand Anti-sense strand

30. Make the complete plasmid

31. Make the complete plasmid! ori repressor promoter MCS AmpR

32. Genetically engineer the plasmid

33. Genetically engineer the plasmid • Identify the MCS by looking for the sites: • Align the insert with the plasmid at the MCS NheI EcoRI

34. Digest the plasmid & insert • With EcoRI • With NheI

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

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

37. Gene regulation

38. 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