1. INTRO. TO BIOTECHNOLOGY Gene Cloning

2. Updates  Lab Group Presentation and Lab Group Project Grades will be Completed by Next Monday.  Abstracts are Due via E-mail on Monday, November 14 th.  Written in a Word Document.  Limited to 500 Words.  Next Week :  30 Point Exam  Complete Lab 10, Mix PCR Cocktail

3. The Exam  30 Point Exam.  The Exam will have Two Components. Scenario 1: Use a Given Observation (Graph or Written) to: Derive a Hypothesis. Create Methods to Test the Hypothesis. Derive a Prediction stemming from the Hypothesis and Methods. Scenario 2: Use A dataset and scenario to: Derive Appropriate Graphs. Statistically Analyze the Data. Draw the Proper Conclusions.  Study the Practice Exams on the CD ROM.  The Exam will take 1.25 – 1.5 Hours.

4. Biotechnology  Biotechnology: using technology to manipulate living organisms  Genetic engineering: Manipulating genes in organisms  DNA cloning: putting a segment of DNA in an organism and having that organism make tons of copies of the gene, gene product or DNA segment.  Transformation: Introducing DNA from one organism (usually bacteria) into another organism.

5. Steps in DNA Cloning  Identify desired piece of DNA  Remove DNA or gene from natural chromosome and splice into foreign DNA (use restriction enzymes)  Place foreign DNA into bacteria and replicate

6. Steps to Producing Recombinant DNA  Use the same Restriction Enzyme:  Digest Foreign DNA.  Digest Plasmids.  Creates Sticky Ends!  Mix the Digests Together.  Mix in DNA ligase to Seal the DNA Strands Together.  Incubate.  * NOTE * this does not always work

7. Applications: Estrogens in Freshwater Habitats Causing Hermaphroditism in Fish  Goal: use genetic engineering and DNA cloning to make E. coli that produce an enzyme to break down estrogen.  Use engineered bacteria at sewage treatment facility.

8. In this lab we will culture…  Untransformed E. coli w/ Ampicillin  Untransformed E. coli w/ No Ampicillin  E. coli transformed with Plasmid P w/ Ampicillin  Cut with PstI restriction enzyme  E. coli transformed with Plasmid A w/ Ampicillin  Cut with Apal restriction Enzyme

9. Plasmid EcoRI (0; 3,927) HindIII Pst1 ApaI SspI LacZ fragment NdeI SspI AmpicillinRes Mutant GFP Green pigment

10. Discuss in small groups  How do you know if bacteria picked up desired DNA? (e.g. transformation occurred))  How do you know you engineered your plasmid correctly?

11. Procedure # 2: DNA cloning  Competent: bacteria that are able to take up DNA.  To make bacteria more competent, they are incubated in a solution containing calcium ions and then shocked with heat to make the membranes more permeable.  Steps in DNA Cloning: 1. Cycle heating and cooling mixture of bacteria and recombinant plasmids. 2. Plate transformed bacteria. 3. Bacteria reproduce, forming colonies, DNA is cloned. 4. Leave plates to incubate 24 hrs. 5. Check, record results, finish questions.

12. Notes on Procedure #2  Work in Groups of Three.  For step 1, you will have three tubes (untransformed, plasmid A, and plasmid B) Label them and include initials  To pipette 250 μ l, pipette 125 μ l twice, CAREFUL.  Practice pipetting first with water, if you need to.  PUT YOUR NAMES ON YOUR PLATES.  The names of each group member must be on the plate to receive credit.

13. Notes on Procedure #2  Leave plates upside down in the incubator.  Make sure they are neatly stacked, and check them by Wednesday to receive credit (4 pts)  Go to Dr Basey’s Office and use the UV light to look at your plates.  Once you’ve checked it, put a check mark on it and put it in the fridge.  For Step 7, change 100 ul to 50 ul and 50 ul to 25 ul.