AP Genetic Modification Continued Objectives: – Determine how organisms actually become transgenic (genetically modified). – See how gel electrophoresis allows you to determine the genetic fingerprint. Bell Work: Quick Poll – Do you have netfix? Homework: Watch Food Inc on Netflix

2chttp:// 2c gel electrophoresis animation WkU&NR=1&feature=endscreenhttp:// WkU&NR=1&feature=endscreen how to clone – review wVoIhttp:// wVoI Closure – gel electophoresis uses el/ el/ virtual lab

Chimeras Cohen and Boyer’s Chimera Take bacterial plasmid Cut with EcoRI Take 9000 bp fragment Combine the ends of the fragment into a smaller plasmid = pSC101 Replication start point Resistance gene for antibiotic tetracycline Chimera = New genome that would never have existed without humans = Recombinant DNA pSC101

Chimeras Cohen and Boyer’s Chimera Take bacterial plasmid Cut with EcoRI Take 9000 bp fragment Combine the ends of the fragment into a smaller plasmid = pSC101 Replication start point Resistance gene for antibiotic tetracycline Chimera = New genome that would never have existed without humans = Recombinant DAN How does it combine into a smaller circle after being cut with EcoRI?

Manipulate pSC101 Use EcoRI to cut DNA from frog that coded for rRNA – Open pSC101 – Add frog rRNA gene – Add bacteria Now, the bacteria that takes up the plasmid that resisted tetracycline is also making frog rRNA.

Vectors Plasmids can be induced to make hundreds of copies with their foreign genes. Can even use artificial chromosomes as vectors. Or use a virus. Vector = The genome that carries the foreign DNA into a host. HOST CELL Why do viruses make good vectors?

Real Life GM Bulls with gene for human antibacterial and iron transport. Herman Some of his calves carry the gene too. Transgenic herd capability?

Real Life GM Wilt Proof Flowers – Ethylene makes flowers wilt – Make flower insensitive to ethylene = no wilting!

Real Life GM Transgenic Salmon Salmon Embryo Growth Hormone Shortens reproductive cycle Makes salmon 11x bigger!

Steps to Genetic Engineering 1.DNA cleavage 2.Production of recombinant DNA 3.Cloning 4.Screening

Steps to Genetic Engineering 1.DNA cleavage 2.Production of recombinant DNA 3. Cloning 4. Screening a)Cut with a restriction endonuclease into fragments. i.Different endonuclease = different fragments ii.Can separate using gel electrophoresis.

Steps to Genetic Engineering 1.DNA cleavage 2.Production of recombinant DNA 3. Cloning 4. Screening a)Cut with a restriction endonuclease into fragments. i.Different endonuclease = different fragments ii.Can separate using gel electrophoresis. - demonstration Gel Electrophoresis – a process of separating DNA by size.

Steps to Genetic Engineering 1.DNA cleavage 2.Production of recombinant DNA 3. Cloning 4.Screening Cut with a restriction endonuclease into fragments. a)Different endonuclease = different fragments b)Can separate using gel electrophoresis. Fragments of DNA are inserted into plasmids or viral vectors. Why is it important to use the same restriction endonuclease?

Steps to Genetic Engineering 1.DNA cleavage 2.Production of recombinant DNA 3.Cloning 4. Screening Cut with a restriction endonuclease into fragments. a)Different endonuclease = different fragments b)Can separate using gel electrophoresis. Fragments of DNA are inserted into plasmids or viral vectors. Vector is are inserted into cells (usually bacteria). a)These are maintained separately in clone libraries. b)Some may have taken the wrong vector or not have taken one at all.

Steps to Genetic Engineering 1.DNA cleavage 2.Production of recombinant DNA 3.Cloning 4. Screening Cut with a restriction endonuclease into fragments. a)Different endonuclease = different fragments b)Can separate using gel electrophoresis. Fragments of DNA are inserted into plasmids or viral vectors. Vector is are inserted into cells (usually bacteria). a)These are maintained separately in clone libraries. b)Some may have taken the wrong vector or not have taken one at all. Screen the library to find the fragment of interest. VERY challenging. Have to get rid of clones without vectors and clones that are lacking the wanted vectors.

Steps to Genetic Engineering 1.DNA cleavage 2.Production of recombinant DNA 3.Cloning 4. Screening Screen the library to find the fragment of interest. VERY challenging. Have to get rid of clones without vectors and clones that are lacking the wanted vectors. Eliminate cells without vectors by placing in an antibiotic.

Steps to Genetic Engineering 1.DNA cleavage 2.Production of recombinant DNA 3.Cloning 4. Screening Screen the library to find the fragment of interest. VERY challenging. Have to get rid of clones without vectors and clones that are lacking the wanted vectors. Eliminate cells without vectors by placing in an antibiotic.

Steps to Genetic Engineering 1.DNA cleavage 2.Production of recombinant DNA 3.Cloning 4. Screening Screen the library to find the fragment of interest. VERY challenging. Have to get rid of clones without vectors and clones that are lacking the wanted vectors. Eliminate cells without vectors by placing in an antibiotic. We can also use the Lac Z gene

Lac Z Helps cells metabolize specific sugar (x gal) When Lac Z metabolizes x gal it creates a blue product. SO… We can use a restriction enzyme that cuts Lac Z. So the bacteria will live in antibiotics and NOT produce blue product.

Bacterial cell that did not take up plasmid Functional lac z gene Lac Z gene Non Functional Fragment of DNA Lac Z gene Functional No wanted fragment of DNA uptake Antibiotic Resistance gene Place in antibiotic. Plasmids without antibiotic resistance will die. This tells us if a plasmid was picked up. It still doesn’t tell us if the plasmid has the DNA we want. Put in X-gal sugar solution. The bacteria that have the plasmid with the correct DNA will NOT produce blue since the LacZ has been cut.

Steps to Genetic Engineering 1.DNA cleavage 2.Production of recombinant DNA 3.Cloning 4. Screening Screen the library to find the fragment of interest. VERY challenging. Have to get rid of clones without vectors and clones that are lacking the wanted vectors. Eliminate cells without vectors by placing in an antibiotic. Then in Xgal to see if they turn blue or not.

Clone Libraries They are HUGE! Many fragments of DNA possible. If you want to find a particular sequence on a particular fragment you must hybridize.

Hybridization Take colonies from clone libraries.

Hybridization Take colonies from clone libraries. Make a replica with a filter.

Hybridization Take colonies from clone libraries. Make a replica with a filter.

Hybridization Take colonies from clone libraries. Make a replica with a filter. Wash to denature the DNA and include radioactive labeled probes. A T C G A T C T A T C G

Hybridization Only the colonies with that gene will retain the probe and emit radioactivity on a film placed over the filter = autoradiography

Hybridization Compare to the original plate to find the colony of interest.

How do we get lots of copies of that gene that we’ve found and we want? Old fashioned = bacterial implant = slow = unreliable. NEW AND WONDERFUL Polymerase Chain Reactions!!!