Introduction: How to Clone a gene? Manipulation of DNA sequences in organisms Is known as genetic engineering Techniques used to engineer genes Are called recombinant DNA technology

Using Plasmids in Cloning Are small, circular DNA molecules Are often found in bacteria Replicate independently of the chromosome Plasmids can serve as a vector A vehicle for transferring recombinant genes to a new host

What makes Plasmids good vectors? Contain DNA replication initiation site (ori) Contain a promoter sequence for initiating transcription of the inserted gene Contain antibiotic resistance gene which allows for identification of bacteria that carry the plasmid Contain restriction enzymes recognition sites for gene insertion http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_2.html

Applications Recombinant Plasmids are extremely useful because it allows manufacturing mass quantity of biological molecules. For example: Protein used to dissolve blood clots in heart attack therapy (tissue plasminogen activator) Insulin Hepatitis B surface antigen, to vaccinate against the Hep B virus Gene for pest resistance that is inserted into plants Gene used to alter bacteria for cleaning up toxic waste

Cloning Genes in Recombinant Plasmids In order for recombinant DNA to be useful it must be made in vast amounts. HOW? Isolate 2 different DNA = bacterial plasmid that serves as vector, and human DNA of interest Treats both DNA with same RESTRICTION ENZYME Sticky ends of the DNA joins by DNA ligase. Result : Recombinant DNA plasmid containing gene of interest. Plasmids is inserted into bacterial cells by transformation Gene Cloning – Bacteria reproduces = mass production of the recombinant DNA http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_1.html

Restriction Endonucleases 1962: “molecular scissors” discovered in bacteria. Also called restriction enzymes E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA 3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially

Origins of Restriction Enzymes Discovered in bacteria. They restrict the growth of bacteriophage by cutting the viral DNA and rendering them harmless.

Restriction Endonucleases Why don’t bacteria destroy their own DNA with their restriction enzymes? DNA Modification

Restriction Endonucleases Named for bacterial genus, species, strain, and type Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1

Restriction Endonucleases Recognition sites have symmetry (palindromic) “Hannah” Bam H1 site: 5’-GGATCC-3’ 3’-CCTAGG-5’

Restriction Endonucleases Enzymes recognize specific 4-8 bp sequences Some enzymes cut in a staggered fashion - “sticky ends” EcoRI 5’…GAATTC…3’ 3’…CTTAAG…5’ Some enzymes cut in a direct fashion – “blunt ends” PvuII 5’…CAGCTG…3’ 3’…GTCGAC…5’

Restriction Enzyme in Action This slide shows a restriction enzyme cutting DNA. The enzyme used here is EcoRI, which cuts at any site where the base sequence is GAATTC. http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter17/restriction_endonucleases.html Sticky Ends

Restriction Enzymes for Cloning Human Insulin gene cut with EcoRI Plasmid cut with EcoRI + 5’-C-G-G-T-A-C-T-A-G-OH 3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4 PO4-A-A-T-T-C-A-G-C-T-A-C-G-3’ HO-G-T-C-G-A-T-G-C-5’ 5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’ Complementary base pairing + DNA Ligase, + rATP recombinant DNA molecule 5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’

Lab 2 (Tab B): Clone That Gene Students will prepare a recombinant plasmid by using restriction enzymes

Goals of Activity Know the characteristics of plasmids Explain how plasmids are used in cloning a gene Understand the function of restriction enzymes and how they are used in creating a recombinant plasmid

Students will ….. Lab 2: use restriction enzymes to digest the 2 plasmids OR Lab 2a: digest a plasmid with 2 restriction enzymes. Good to do a visual to help students see what they are doing: paper plasmids

The New Plasmids See ordering information in meeting binder appendix http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter17/construction_of_a_plasmid_vector.html

The Recombinant Plasmids

RFP expression Bruce Wallace araC gene PBAD rfp gene Transcription mRNA Translation araC protein 19

RFP expression araC protein prevents RFP transcription by causing Bruce Wallace araC protein prevents RFP transcription by causing a loop to form in the region of the fp gene r araC gene araC protein PBAD rfp gene 20

arabinose – araC protein RFP expression Bruce Wallace RFP (red fluorescent protein) Arabinose – araC protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site (PBAD). arabinose Translation RNA polymerase arabinose – araC protein complex araC protein mRNA Transcription araC gene rfp gene PBAD 21

Bruce Wallace RFP

Running Digested DNA Through Gel Electrophoresis Lab 4: Verification of recombinant plasmid creation Running Digested DNA Through Gel Electrophoresis

How does it work? DNA is cut into smaller fragments. Loading dye is used to sink the DNA into the wells in the gel and to track the movements of DNA The negative DNA molecule is attracted to the positive (RED) electrode. DNA moves based on size and structure; with the smallest fragments move fastest

Different Structural Forms circle “multimer” Nicked Circle Linear Supercoiled “nicked-circle” Different structural forms produce different bands.

Goals of this Hands-On Lab Compare cut vs uncut plasmid DNA by running them through a gel. Look for different banding patterns and understand how to read them. Predict what kind of banding pattern a plasmid will make based on: The restriction enzyme used. The plasmid’s structural shape.

Confirmation of restriction and ligation Restriction analysis of pKAN-R and pARA Bruce Wallace Confirmation of restriction and ligation M K+ K- A+ A- L M K+ K- A+ A- L 500 1000 1500 2000 3000 4000 5000 8000 10000 4705 bp 4495 bp 807 bp 377 bp

Lonza Flash Gel System

Why? Lab 5: Transformation Plasmid vectors can be introduced into bacteria by transformation: the process of taking up DNA from the environment Why? If a recombinant plasmid can be inserted into a bacterial or yeast cell The foreign DNA will be copied and transmitted to new cells as the host cell multiplies Produces millions of identical copies of specific genes

preparing competent cells for transformation Lipid bilayer (inner) Adhesion zone Peptidoglycan layer Lipid bilayer (outer) Calcium ions

Transforming Escherichia coli with pARA-R Bruce Wallace Competent Cells pARA-R Recombinant Plasmids

transforming Escherichia coli with pARA-R Lipid bilayer (inner) Adhesion zone Peptidoglycan layer Lipid bilayer (outer) Calcium ions pARA-R

Transgenic Colony Allowed to Grow in Selective Media

growth of transformed bacteria on various plates P+ plates LB LB/amp LB/amp/ara P- plates No growth LB LB/amp