Amgen Laboratory Series. Tabs C and E

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1 Amgen Laboratory Series Tabs C and E

2 Chapter 2A Goals Describe the characteristics of plasmids Explain how plasmids are used in cloning a gene Describe the function of restriction enzymes Explain how to use restriction enzymes to create a recombinant plasmid

3 Plasmid Vector

4 Parts of a Plasmid Vector Vector: carry DNA sequences from one organism to another Ori ( origin of replication ): where DNA replication initiates inside the host cell Promoter: where RNA polymerase binds and initiates transcription of mrna, insert a gene next to the promoter Antibiotic Resistant Gene: gives bacteria a selective advantage by surviving the action of antibiotics


6 Bacterial Conjugation

7 Bacterial Conjugation Antibiotic resistant gene will allow us to determine which bacteria took in the plasmid

8 Restriction Enzymes Enzymes (proteins) that recognize and destroy viral DNA without damaging host cell DNA

9 Restriction Enzymes Cut DNA at sequences known as recognition sites Use restriction enzymes as molecular tools A cut is known as a digestion Cuts leave over-hanging single strands called sticky ends


11 To Cut or Not to Cut?

12 Creating a Recombinant Plasmid Cut the plasmid at a site to insert the new gene Cut the plasmid in the correct place so that the ori site, promoter, and antibiotic resistant gene are not disrupted Cut near the promoter so that the inserted gene will be expressed Cut the human DNA close to the gene of interest

13 Selection of a Restriction Enzyme

14 Activity 2: Clone That Gene Make a paper model of a recombinant plasmid that contains an insulin gene Tape the recombinant plasmid into your notebook Answer Clone That Gene Questions on p. C11

15 NOTEBOOK Clone That Gene Activity Goals Cut the plasmid and the human DNA with the appropriate restriction enzyme Insert the insulin gene in the plasmid DNA Determine which antibiotic you would use to identify bacteria that have taken in the plasmid

16 NOTEBOOK Clone That Gene Analysis Tape the recombinant plasmid into your notebook Answer Clone That Gene Questions #1-3 on p. C11 Sign and date the page

17 Lab 2A Digesting the para-r Plasmid

18 NOTEBOOK Laboratory 2A Preparing to Verify the RFP Gene: Digesting the para-r Plasmid Purpose To ensure that the recombinant plasmid, para-r, is the correct one for making the red fluorescent protein in bacteria

19 NOTEBOOK Lab 2A Materials 2.5xB restriction buffer RP para-r plasmid RE Restriction enzymes BamHI and HindIII dh2o distilled water R + label with initials R - label with initials

20 Restriction Digest Reagents NOTEBOOK

21 NOTEBOOK Laboratory 2A Analysis Questions Answer the Chapter 2A Questions, #1-5, on p. C-17 in your lab notebook Sign and date the page

22 para-r Plasmid Components rfp gene pbad promoter ampr ampicillin resistant gene arac arabinose activator (controls the promoter)


24 Lab 4A Verification of the Recombinant Plasmid Using Gel Electrophoresis

25 NOTEBOOK Laboratory 4A Verification of the Recombinant Plasmid Using Gel Electrophoresis Purpose To use gel electrophoresis to examine the products from the restriction digest of the para-r plasmid

26 Restriction Digest Review R+ : para-r plasmid and restriction enzymes R- : para-r plasmid only (control)

27 Uncut Plasmid Configurations Most common Travels quickly for its size Does not move as quickly as supercoiled plasmids Migrates very slowly Slowest

28 Gel Electrophoresis Predictions Predict what you might expect in the R- and R+ lanes on the gel Record predictions on the DNA Ladder Diagram and glue into your lab notebook

29 Gel Electrophoresis Predictions NOTEBOOK

30 NOTEBOOK Lab 4A Analysis Questions Answer the Chapter 4A Questions, #1-7, on page C-33 in your lab notebook under the DNA Ladder Diagram Sign and date the page

31 Lab 5A Transforming Bacteria with the para-r Plasmid

32 NOTEBOOK Laboratory 5A Transforming Bacteria with the para-r Plasmid Purpose Describe the role of transformation in the gene cloning process To transform E. coli bacteria with the para-r plasmid Explain how the information encoded in a gene is expressed as a trait

33 Bacterial Transformation Treat with calcium ions first Heat shock

34 NOTEBOOK Lab 5A Materials RP para-r plasmid LB Luria Broth CC E. coli cells (keep on ice at all times!) P P + 3 Petri plates with agar LB LB/amp LB/amp/ara

35 para-r Plasmid

36 Arabinose Operon (Normal)

37 Arabinose Operon (Engineered with RFP)

38 Bacterial Growth Predictions Two Groups P- Control Group No Plasmid Added (Non-transformed) P + Treatment Group Plasmid Added (Transformed)

39 P- Control Group (Non-Transformed Bacteria) NOTEBOOK

40 P+ Experimental Group (Transformed Bacteria) NOTEBOOK

41 NOTEBOOK Lab 5A Analysis Questions Take pictures of each of your plates and then view the LB/amp/ara plate using a long wave UV light Draw a picture of your results (plates) label the plates: LB, LB/amp, LB/amp/ara use colored pencils Answer the Chapter 5A Questions, #1-6, on page C-50 in your lab notebook Sign and date the page

42 Lab 6A/B Purifying the Red Fluorescent Protein

43 NOTEBOOK Laboratory 6A/B Purifying the Fluorescent Protein Purpose To use column chromatography to purify the red fluorescent protein Lab 6A: Lyse Cells Grown in the Shaker Lab 6B: Purify the Red Fluorescent Protein with Column Chromatography

44 Lab 6A: Lyse Cells To obtain the RFP protein, we must first lyse the cells (break open cell walls) Use lysis buffer

45 NOTEBOOK Materials for Lab 6A Lyse Cells EC Tube Suspension culture of E. coli from the LB/amp/ara plate Label the EC tube with your initials and class block EB Elution Buffer LyB- Lysis Buffer Liquid waste container p200 micropipette Centrifuge

46 Lab 6B: Protein Purification with Column Chromatography Each protein has a specific shape and function Shape determines function by creating binding sites for other molecules

47 Materials for Lab 6B Column Chromatography EC Tube from lab 6A Chromatography Column Binding Buffer (BB) Wash Buffer (WB) Elution Buffer (EB) Column Equilibration Buffer (CEB) 1.5 ml microcentrifuge tube (2 tubes) Liquid waste container Disposable transfer pipettes p200 micropipette NOTEBOOK

48 Protein Properties Column chromatography separates the proteins based on whether they are hydrophobic or hydrophilic proteins

49 NOTEBOOK Buffers Used to Purify the Protein All have different concentrations of salt to unfold and separate the proteins (purification) 1. Binding Buffer: Unfolds all the proteins, hydrophobic proteins stick, hydrophilic pass through (highest salt concentration) 2. Wash Buffer: Release moderately hydrophobic proteins 3. Elution Buffer: Releases the RFP protein from the resin (lowest salt concentration)

50 Protein Purification with Column Chromatography

51 NOTEBOOK Chapter 6 Questions Answer Chapter 6 Questions #1-5 on page E- 15 in your lab notebook Sign and date the page Respond to the Amgen Student Survey