1 / 32

Genetic Jigsaw

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 the teacher’s notes.

barid
Download Presentation

Genetic Jigsaw

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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 the teacher’s notes

  3. Make the plasmid parts

  4. 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

  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 Anti-sense strand 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” • Consensus 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 NheI site GFP/insulin sequence EcoRI site Sense strand Anti-sense strand

  24. Make the complete plasmid

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

  26. Genetically engineer the plasmid

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

  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

More Related