Changing the living world

1. Changing the living world

2. How we change the living world… Selective breeding: crossing organisms with desired traits to produce the next generation.

3. How we change the living world… Hybridization: crossing dissimilar organisms to get the best of both.

4. How we change the living world… Inbreeding: continually breeding individuals with similar characteristics.

5. GENETIC ENGINEERING

6. Genetic engineering vocab • Recombinant DNA- nucleotide sequences from two different sources to form a single DNA molecule. • Transgenic organism – contains a gene from another organism, typically a different species • Genetically modified organisms (GMOs)- organisms that have acquired one or more genes by artificial means.

7. Figure 12.1

8. Genetic Engineering • Genetic engineering: The process of manipulating genes for practical purposes. • Genetic engineering may involve building recombinant DNA DNA made from two or more different organisms.

9. Steps in a Genetic Engineering Experiment Step 1 Isolate Target DNA and plasmid and cut with restriction enzymes Step 2 Recombinant DNA is produced. Step 3 Gene cloning: the process by which many copies of the gene of interest are made each time the host cell reproduces. Step 4 Cells undergo selection and then are screened.

10. Steps in a Genetic Engineering Experiment Step 1 The DNA from the organism containing the gene of interest and the vector are cut by restriction enzymes. A vector is an agent that is used to carry the gene of interest into another cell Commonly used vectors: viruses, yeast, and plasmids. circular bacterial DNA

11. Plasmids Bacterial chromosome Remnant of bacterium Colorized TEM Figure 12.7

12. Isolate plasmids. Bacterial cell Plasmid Figure 12.8-1

13. Isolate DNA. Cell containing the gene of interest Isolate plasmids. Bacterial cell Plasmid DNA Figure 12.8-2

14. Cut both DNAs with same enzyme. DNA fragments from cell Isolate DNA. Gene of interest Other genes Cell containing the gene of interest Isolate plasmids. Bacterial cell Plasmid DNA Figure 12.8-3

15. Cut both DNAs with same enzyme. DNA fragments from cell Isolate DNA. Gene of interest Other genes Mix the DNAs and join them together. Gene of interest Cell containing the gene of interest Isolate plasmids. Bacterial cell Recombinant DNA plasmids Plasmid DNA Figure 12.8-4

16. Cut both DNAs with same enzyme. DNA fragments from cell Isolate DNA. Gene of interest Other genes Mix the DNAs and join them together. Gene of interest Cell containing the gene of interest Isolate plasmids. Bacterial cell Recombinant DNA plasmids Bacteria take up recombinant plasmids. Plasmid DNA Recombinant bacteria Figure 12.8-5

17. Cut both DNAs with same enzyme. DNA fragments from cell Isolate DNA. Gene of interest Other genes Mix the DNAs and join them together. Gene of interest Cell containing the gene of interest Isolate plasmids. Bacterial cell Recombinant DNA plasmids Bacteria take up recombinant plasmids. Plasmid DNA Recombinant bacteria Bacterial clone Clone the bacteria. Figure 12.8-6

18. Cut both DNAs with same enzyme. DNA fragments from cell Isolate DNA. Gene of interest Other genes Mix the DNAs and join them together. Gene of interest Cell containing the gene of interest Isolate plasmids. Bacterial cell Recombinant DNA plasmids Bacteria take up recombinant plasmids. Plasmid DNA Recombinant bacteria Bacterial clone Clone the bacteria. Find the clone with the gene of interest. Figure 12.8-7

19. Cut both DNAs with same enzyme. DNA fragments from cell Isolate DNA. Gene of interest Other genes Mix the DNAs and join them together. Gene of interest Cell containing the gene of interest Isolate plasmids. Bacterial cell Recombinant DNA plasmids Bacteria take up recombinant plasmids. Plasmid DNA Recombinant bacteria Bacterial clone Clone the bacteria. Find the clone with the gene of interest. Some uses of genes Some uses of proteins Gene for pest resistance Protein for dissolving clots Protein for “stone-washing” jeans Gene for toxic-cleanup bacteria The gene and protein of interest are isolated from the bacteria. Genes may be inserted into other organisms. Harvested proteins may be used directly. Figure 12.8-8

20. RESTRICTION ENZYMESmolecular scissors

21. A Closer Look: Cutting and Pasting DNA with Restriction Enzymes • Recombinant DNA is produced by combining two ingredients: • A bacterial plasmid • The gene of interest • How do we cut them? • Using restriction enzymes: bacterial enzymes which cut DNA at specific nucleotide sequences • produce pieces of DNA called restriction fragments. • Why do you think bacteria contain restriction enzymes?

22. Restriction Enzymes are palindromes: the same forward as backwards, like RACECAR. Examples: GAATTC CCCGGG AAGCTT CTTAAG GGGCCC TTCGAA G AATTC CCC GGG A AGCTT CTTAA G GGG CCC TTCGA A Sticky Ends Blunt End

23. Recognition sequence for a restriction enzyme DNA A restriction enzyme cuts the DNA into fragments. Restriction enzyme Sticky end Sticky end Figure 12.9-1

24. Recognition sequence for a restriction enzyme DNA A restriction enzyme cuts the DNA into fragments. Restriction enzyme Sticky end Sticky end A DNA fragment is added from another source. Figure 12.9-2

25. Recognition sequence for a restriction enzyme DNA A restriction enzyme cuts the DNA into fragments. Restriction enzyme Sticky end Sticky end A DNA fragment is added from another source. Fragments stick together by base pairing. Figure 12.9-3

26. DNA LIGASE • DNA ligase connects the DNA fragments into one continuous strand (DNA Glue or tape)

27. Recognition sequence for a restriction enzyme DNA A restriction enzyme cuts the DNA into fragments. Restriction enzyme Sticky end Sticky end A DNA fragment is added from another source. Fragments stick together by base pairing. DNA ligase DNA ligase joins the fragments into strands. Recombinant DNA molecule Figure 12.9-4

28. Recognition sequences DNA sequence Restriction enzyme EcoRI cuts the DNA into fragments. Sticky end

29. Your turn to try!!

30. Plasmids: • Can easily incorporate foreign DNA • Are readily taken up by bacterial cells • Can act as vectors, DNA carriers that move genes from one cell to another • Are ideal for gene cloning, the production of multiple identical copies of a gene-carrying piece of DNA

31. Bacterial cells don’t edit the RNA, so how can they make the correct protein? Genetic Engineers can eliminate the introns from mRNA and reverse the process, producing a DNA strand that is only the instructions for the protein. Use Reverse Transcriptase

32. Cell nucleus Exon Exon Exon Intron Intron DNA of eukaryotic gene Transcription Test tube Figure 12.11-1

33. Cell nucleus Exon Exon Exon Intron Intron DNA of eukaryotic gene Transcription RNA transcript Introns removed and exons spliced together mRNA Test tube Figure 12.11-2

34. Cell nucleus Exon Exon Exon Intron Intron DNA of eukaryotic gene Transcription RNA transcript Introns removed and exons spliced together mRNA Test tube Isolation of mRNA from cell and addition of reverse transcriptase Reverse transcriptase Figure 12.11-3

35. Cell nucleus Exon Exon Exon Intron Intron DNA of eukaryotic gene Transcription RNA transcript Introns removed and exons spliced together mRNA Test tube Isolation of mRNA from cell and addition of reverse transcriptase Reverse transcriptase Synthesis of cDNA strand cDNA strand being synthesized Figure 12.11-4

36. Cell nucleus Exon Exon Exon Intron Intron DNA of eukaryotic gene Transcription RNA transcript Introns removed and exons spliced together mRNA Test tube Isolation of mRNA from cell and addition of reverse transcriptase Reverse transcriptase Synthesis of cDNA strand Synthesis of second DNA strand by DNA polymerase cDNA strand being synthesized cDNA of gene without introns Figure 12.11-5