Microbiology

1. Microbiology Biotechnology Chapter 10

2. Part I Restriction Endonucleases Recombinant DNA Bacteria Transformation Transgenic Plants Gene Therapy

3. Restriction Endonucleases • Restriction Endonucleases • Enzymes which cut double stranded DNA • Cut only at specific sequences • Palindrome sequences • Naturally found in bacteria • Purpose is to cut up and destroy foreign DNA • Molecular Biologist • Have isolated these restriction endonucleases • Now they are used to manipulate DNA

4. For the sequences below write the correct corresponding sequence, box the Palindrome and mark the cut site with lines • 5’ – CAGTATCCTAGGCAT – 3’ 5’ – GTAAGCTTGCCATA – 3’

5. Restriction site Plasmid DNA From Bacteria 5 3 G A A T T C 3 5 C T T A A G 1 Add restriction endonuclease cutters G A A T T C C T T A A G Sticky ends A A T T C gene of interest from another organism G G 2 C T T A A Fragment cut by same restriction enzyme G A A T T C A A T T C G T T A A C T T A A C G G One possible combination 3 Recombinant DNA molecule

6. Test Tube Contains: DNA gene of interest cut with EcoR1 Plasmid cut with EcoR1 DNA Ligase Buffer Incubate for 1 hour 37º Every plasmid must also contain a gene for antibiotic resistance (for selection) and an origin of replication, so that this DNA can replicate during binary fission. Insulin Gene Origin or Rep Amp Amp Bacterial Plasmid Antibiotic resistance

7. Yellow: human insulin gene Green: Plasmid Bacterial Transformation +CaCl2 and heat Live Bacterial Cell Heat Shock Transformation Incubation of live bacteria cells with CaCl2 and heat. This process punches holes in the bacterial cell membrane, which allows the plasmid to enter. Another method consists of producing a brief electric shock which also punches holes in the bacterial membrane, allowing the plasmid DNA to enter. This method is known as electroporation.

8. Screening For Transformation Growth of bacterial colonies with no plasmid No AMP Transformation is not 100% efficient. Selection for the bacteria with the plasmid must be done. Remember that the plasmid contains a gene for antibiotic resistance which allows transformed bacteria to grow on the antibiotic treated plate. Only growth of bacterial colonies with plasmid AMP

9. Isolation of Purified Protein AMP Place transformed bacteria in a growth media with AMP and allow bacteria cell to transcribe and translate human insulin gene As bacteria grow and divide many insulin proteins are made. Once growth is complete lyse bacteria cells and harvest insulin AMP Insulin Protein

10. Bacterial Transformation • Many protein products today are made through recombinant DNA technology • Insulin • Human growth factor • Factor VIII – treats hemophilia • Tumor Necrosis Factor – treats cancer • Interferons – used to treat viral infections • Relaxin and Oxitocin – child birth • BST – milk production hormone for cow • Spider silk – bullet proof vests • Many Others

11. Creation of GMO’s • GMO’s • Genetically modified organisms • Uses the same recombinant technology discussed above • The plasmid or recombinant vector is put into plants or animals not bacteria • Creation of transgenic • Plants • Animals • Even humans

12. Transgentic Plants • Agrobacterium tumefaciens • bacteria found normally in the environment • Designed to infect plant cells • Contains a plasmid which causes tumors in the plant • Ti plasmid • The use of restriction cutters has allowed the removal of tumor gene and its subsequent replacement with genes for pesticide and herbicide resistance

13. Ti plasmid w/ infecting T DNA shown in red Plant Cell Wall Chloroplasts Agrobacterium Central Vacuole Stores Water Ions Plant Membrane Mitochondria Protein expression of T DNA Plant Nucleus Plant DNA T DNA codes for two compounds which benefit the bacteria and cause crown gall disease. Now, this T DNA can be removed using restriction endonucleases and a helpful gene can be inserted into the plant DNA

14. Ti plasmid - Red is the “tumor” gene Blue is a gene you want such as pesticide resistance • Steps • Remove tumor gene with restriction endonucleases • Insert your gene of interest and seal with ligase • Put genetically modified plasmid back into A. tumefaciens • Allow this modified bacteria to insert its plasmid into plant tissue • Now plant expresses the gene you want Chromosomal DNA Ti plasmid A. tumefaciens A. tumefaciens

15. Agrobacterium cell Ti plasmid w/T DNA Kanamycin resistance Gene Foreign gene Pesticide resistance Herbicide resistance Bacteria invade at Wound site Leaf segments are Transferred to agar dish Recombinant plasmid Regeneration of plants from leaf segments

16. Transgenic plant with Fluorescent gene from Jellyfish Transgenic Garden peas Pesticide resistance Transgentic Tobacco Herbicide resistant Control Plants Salt resistant and drought resistant tomato Salt resistant tomato is really interesting because the flavor of the tomato is not affected. The salt is stored in the plants leaves, which also desalinates the soil. (Good for agriculture)

17. Transgenic Animals • Advantage to bacterial systems • Transcripe and translate eukaryotic genes • Germline engineering • Foreign genes are delivered to a fertilized egg • Electroporation – foreign DNA + electric shock to egg • Viral transfection of egg • Gene Therapy • Ex vivo • Direct introduction of modified virus

18. Part II Other Biotechnology Gel electrophoresis RFLP Isolation of cDNA PCR DNA Automated Sequencing

19. Mixture of DNA Anode Gel Electrophoresis Electric Current Gel • Gel Electrophoresis • Standard laboratory technique • Used to separate DNA strands based on size • Soft Agar like jello with wells to hold DNA • An electric currant is applied • Rate of movement is determined by size • Larger fragments are trapped in gel matrix • Smaller fragments move quickly through the gel matrix • DNA is negatively charged so it travels towards the positive end Cathode Longermolecules Shortermolecules

20. Gel Electrophoresis Negative end - Larger Fragments DNA must be stained with ethidium bromide and exposed to UV light to be visualized. Smaller Fragments + Positive end

21. RFLP Analysis Incubate DNA in Test tube with restriction endonucleases “cutters” Every person’s DNA is different and only ~ 5% of DNA codes for proteins DNA contains many introns and non- coding sequences which differ in the population significantly and contain a different pattern of restriction endo- nuclease cut sites. Every person will have a different banding pattern of DNA DNA Suspect 3 DNA Suspect 2 DNA Suspect 1 The DNA is each tube is obtained via blood or tissue sample and contains all 46 human chromosomes

22. DNA Suspect 1 350bp 100bp 200bp DNA Suspect 2 400bp 100bp 50bp 25bp DNA Suspect 3 375bp 250bp Based on this information how many “bands” should each suspect have? Using the 100bp scale given, draw a sketch of this hypothetical gel

23. - 500 400 300 Size Base Pairs 200 100 75 50 + 25 Size Markers Crime Scene Sus1 Sus3 Sus2

24. Check your Knowledge Who is going to Jail? Who is the father? Child Mom A B C In reality the process is slightly more complex. The total DNA is cut and then run on a gel. However, this results in a smear of DNA down the gel. The next step is to perform a southern blot and hybridize certain sections of the DNA to a radioactively labeled probe. The differences in the banding pattern then become apparent. See text page 292-293, 520 & online movies

25. Isolation of Gene of Interest • When doing the transformation experiments above the gene of interest was isolated are ready to be inserted into the plasmid vector • How does biotechnology allow research scientists to isolate genes of interest • Creation of cDNA

26. A A G G C C C C U U A A C C G G AAAAAAAA(n) AAAAAAAA(n) T A C G G A T G C T C A G C G T PRIMER cDNA made by Reverse Transcriptase U U A A C C G G Many copies of ds cDNA made by DNA polymerase mRNA coding exons Isolate mRNA population from cell Add Reverse Transcriptase & primer mRNA coding exons Many cDNA copies of desired gene made by reverse transcriptase Now add DNA polymerase You end up with many copies of ds cDNA which contains no introns And is ready to be inserted into a plasmid vector PRIMER

27. PCR • Polymerase chain reaction • Amplifies one specific section of DNA • Copy any region of DNA using correct primers • Primers are DNA and designed by scientist • Copies exon regions in genes • Copies any section of DNA where primers box the section of interest

28. Think back to DNA replication. To copy your DNA will you need a primer? PCR is not different. You still need a primer to copy (polymerize) the gene you are interested in. Where would you design Your primers to go?? This is known as Boxing a gene Primers are DNA NOT RNA. Primers are designed by the research scientist * * * * One gene (exon) or segment of DNA that you want to copy for: research crime scene analysis This tube contains Buffers Primers Isolated Genomic DNA dNTPs Taq Polymerase * All of your DNA in a cell

29. PCR • Step 1 • Heat to 95ºC to separate DNA strands • Why do the strands separate? Gene of interest 3’ 5’ 3’ 5’ 95ºC 5’ 3’ 5’ 3’

30. PCR • Step 2 • Primer Annealing – 55ºC to 65ºC • A scientist will design a primer around the gene of interest 3’ * 5’ * 5’ 3’

31. PCR • Step 3 • A special polymerase from a thermal bacterial will quickly copy the DNA starting at the primer regions • Thermococcus litoralis • This will continue for 1 minute • Rate of coping is 1000bp/minute • These three steps will then be repeated 40X

32. PCR • Step 3 3’ 5’ * 5’ 3’ 3’ * 5’ Polymerase Copies specific region Of DNA * 5’ 3’ * 3’ 5’ 5’ 3’

33. 3’ 5’ 5’ 3’ 3’ 5’ * 5’ 3’ 5’ * 5’ 3’ 5’ 5’ 3’ 3’ 5’ * 5’ * 5’ 5’ 3’

34. Polymerase Chain Reactions PCR allows one section of DNA to be copied many times by repeating the same three basic steps: denaturing DNA w/95º heat, primer annealing, and elongation by polymerase. There are many applications of PCR. Crime scene analysis, research, evolutionary taxonomy, species identification and so on.

35. PCR Products From 30 Samples 1500 1 2 3 4 5 and so on 1200 C C 1000 Determine the size of all of these exons 900 800 700 C 600 DNA Ladders 500 21 400 300 200 100 This gel information can determine if an exon amplified by PCR has a deletion or insertion of DNA or otherwise large scale genomic change. It can not tell you if a single base pair is mutated

36. Analysis of Gel electrophoresis • Based on the data shown above • What exons are missing? • What exons are partial deletions? • However, most samples have the appropriate exon size • How could you further analyze this DNA for mutations? • Cut out the DNA bands and sequence the DNA (see next slide)

37. Liquefy gel In tubes 1 2 3 Cut bands out using An razor blade Send off for Automated DNA sequencing See page 294 – 295 of text

38. DNA Automated Sequencing Comparing this sequence to a normal sequence would reveal any mutation to the DNA