Amgen Bruce Wallace Transformation Labs (2-7)

Timeline Thursday—Lecture Tuesday—Finish Lecture, Quiz, lab 2 Thursday--Lab 3, 4, 5 (Duffy does lab 6) Monday—Lab 7 Part 1 Tuesday—Lab 7 Part 2 Finals Friday—Packet Due

Assignments Pre-lab notes worksheets: do these before we do that lab by reading the information and lab procedures Flowcharts—draw on the side or bottom of the procedures page Complete conclusion questions: do at the end of the lab each day Draw your lab 4 gel results at the end of the conclusion (use a ruler, make it nice!) Entire packet will be due 1/27/2012 (day of final)—no late work because this goes in semester grade!

Prep. for Labs Week Before Make labels 10% bleach solution Aliquot chart Day before lab Lab 2—water bath set-up for 37 C Lab 3--Water baths set-up 70 C Lab 4--Pour 8 gels (35ml each) for lab 4 Pour .8% gels, add ethidium bromide (200ng/mL final or 1uL of 10mg/mL stock in gel prepared from 50mL), 6 well comb, SB buffer (2.4 grams agarose add up to 300mL TBE buffer) lab 5--ice Lab 5—water bath set-up at 42C Lab 6--Start overnight culture for lab 6 (use update instructions) Lab 7--Container of 10% at front for waste --Set-up chromatography columns

Vocab. “transformed cell” – cell has acquired new characteristics “characteristics” – due to the expression of incorporated foreign genetic material Gene expression – process by which the information encoded in a gene is converted into an observable phenotype Gene regulation – control mechanisms that turn genes on or off Inducible proteins – synthesis is regulated depending on the bacterium’s nutritional status Thank you Francois Jacob and Jacques Monod! Prokaryote operon model of gene control Repressors and activators are “trans-acting” – affect expression of their genes no matter on which DNA molecule in the cell these are located.

Overview of Labs Lab 2—Restriction Analysis of pARA and pKAN-R Cut the 2 plasmids using restriction enzymes Lab 3—Ligation of pARA/pKAN-R Restriction Fragments Producing a Recombinant Plasmid, pARA-R Insert the gene of interest into the pARA plasmid from the pKAN-R Lab 4—Confirmation of Restriction and Ligation Using Agarose-Gel Electrophoresis Run a gel to confirm the ligation in lab 3 worked (we want to make sure the gene of interest was inserted into that plasmid, if not, there is no reason to transform the plasmid into the bacteria) Lab 5—Transforming Escherichia coli with a Recombinant Plasmid Insert pARA-R (plasmid with our gene of interest) into bacteria using shock treatment, grow bacteria on plates, plasmid will produce proteins from our gene Lab 6--Preparing an Overnight Culture of Escherichia coli Take a colony that has the gene of interest from the plate, put into broth to replicate, now we have tons of bacteria (so tons of our geneprotein!) Lab 7—Purification of mFP from an Overnight Culture Lyse the bacteria cell, isolate the desired protein using chromatography

The Big Picture 2005 Pearson Education, Inc.

2005 Pearson Education, Inc

Background Concepts What are Plasmids? How can we modify plasmids? Restriction Enzymes Origins of restriction enzymes. A close look at restriction enzymes. Understanding plasmid diagrams. This slide is meant to introduce the first part of the lecture. Every question proposed here will be answered shortly. 9

What are Plasmids? In this Lecture… Circular DNA that is used by bacteria to store their genetic information. Modifying plasmids to include extra genes allows for the production of new proteins. This slide is meant to introduce the first part of the lecture. Plasmids are circular DNA used by bacteria to store their genetic information. Modifying plasmids to include extra genes allows for the production of new proteins. 10

How Can We Modify Plasmids? In this Lecture… Restriction Enzymes BamHI, HindIII, etc. Where do they come from? How do they work? Different restriction enzymes do different things. DNA Ligase This slide is meant to introduce the first part of the lecture. Every question proposed here will be answered shortly. Restriction Enzyme attached to DNA before cleavage 11

Origins of Restriction Enzymes Bacteria produce restriction enzymes to protect against invading viral DNA/RNA. More details can be given if necessary, however all the students need to know is that the bacteria have biologically designed the restriction enzymes as a defense mechanism. Many restriction enzymes exist, and they all do similar things, however their restriction sites (the site of DNA cleavage) differ in their base recognition sequences. 12

Origins of Restriction Enzymes The enzymes cut the invading DNA/RNA, rendering it harmless. 13

Restriction Enzyme in Action Sticky Ends 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. DNA strand with EcoRI restriction site highlighted. EcoRI restriction enzyme added (outline of separation about to occur). Restriction fragments separate, with “sticky ends” at each edge. 14

Adding DNA Ligase Sticky Ends DNA ligase bonds sticky ends cut with the same restriction enzyme. Sticky ends cut with different restriction enzymes will not bond together. Why? Because the base pair sequence of the two sticky ends will be different and not match up. 15

Plasmids Can Be Drawn to Show the Genes They Carry In this diagram: Blue and Orange are drawn as genes. Triangles are indicating the known restriction sites for a restriction enzyme. (shapes can vary) Plasmid Maps are more complex. Plasmid Name Bp size The purpose of this slide is to show the students how plasmids can be diagramed to indicate certain areas. In this particular image, the blue and orange regions were added to represent genes. The lighter tan colored areas indicate the rest of the plasmid. The red triangles (which can also be lines or some other shape) indicate areas of restriction by some arbitrary restriction enzyme. If a different restriction enzyme (with a different restriction site recognition) were to be used, the red triangles would appear in different locations (obviously). The plasmid’s name and base pair size (Bp size) is found in the center of the plasmid, but it can also be written elsewhere. 16

Plasmid Maps Indicate Restriction Sites and Genes This slide is meant to show the complexities in mapping DNA. Notice the many mapped restriction sites and the 3 genes labeled on the plasmid. This plasmid is 2686 base pairs in size. 17

Make Recombinant DNA Using Restriction Enzymes Application Exercise Make Recombinant DNA Using Restriction Enzymes This next series of slides is meant to use the knowledge from the previous slides and simply make recombinant DNA. 18

DNA From Two Sources (Restriction Sites Labeled) Here we are presented with the DNA to be cut. The restriction sites for a particular restriction enzyme have been labeled. Take note of the red area in the linear DNA. The goal of this series of slides is to produce a plasmid containing a single copy of this red sequence. Circular DNA Linear DNA 19

Application of Restriction Enzymes 20

Adding DNA Ligase Adding DNA ligase will cause the “sticky ends” to come together, and many combinations are possible. 21

Recombinant DNA Plasmid Many possible recombinant DNA plasmids can be produced, but this was the desired plasmid for the experiment. 22

Many Other Recombinant Possibilities …and many more! 23

Plasmid DNA Insertion DNA plasmids can be inserted into bacteria using a variety of laboratory processes. 24

Transgenic Colony Allowed to Grow 25

How Do We Get the Desired Plasmid? Recombinant plasmids Restriction fragments will ligate randomly, producing many plasmid forms. Bacterial insertion would be necessary, then colony growth, and further testing to isolate bacteria with the desired plasmid. Transformation of bacterial cells through electroporation. Bacteria are then moved to a growth plate, and grown on selective media to “weed out” cells that have not picked up the desired plasmid. 26

Running Digested DNA Through Gel Electrophoresis 27

Goals of this Hands-On Lab Take plasmid DNA that has been previously cut with restriction enzymes and compare that to a plasmid NOT cut with restriction enzymes, 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. 28

Gel Box Loading Techniques Look directly down the axis of the pipette. Loading dye makes the sample heavy, but it can still easily swish out of the well. Squirt down slowly. Take the tip out of the buffer. Then release the plunger. If you don’t do that, you will suck the sample back up. 29

Add DNA samples and ladder to the wells and “run to red!” You RUN TO RED because DNA holds a slightly negative charge, and when electrical current is added to the system, the DNA will migrate to the positive end. Add DNA samples and ladder to the wells and “run to red!” 30

Sample fragments move toward positive end. 10 kb 8 kb 6 kb 5 kb 4 kb 3 kb 2 kb MAKE SURE TO WARN THE STUDENTS OF THE HIGH VOLTAGE RUNNING THROUGH THE GEL BOXES. 1 kb .5 kb Sample fragments move toward positive end. 31

Analyzing Your Gel 32

What Makes Up the Banding Pattern in Restricted DNA? The restriction enzyme cleaves the DNA into fragments of various sizes. Each different size fragment will produce a different band in the gel. Remember that fragments separate into bands based on size. 1400 Bp 2000 Bp Lancer Plasmid 6700 Bp 3300 Bp 33

What Makes Up the Banding Pattern After Adding DNA Ligase? Several combinations of plasmids will result from the reaction. The many forms will contribute to different bands. (See following slides for chemical and structural forms) 34

Different Recombinant Forms Adding DNA Ligase does not always make the desired plasmid! Few if any could be what you wanted. Think about the large number of possible combinations. 35

Different Structural Forms circle “multimer” Nicked Circle Linear Supercoiled “nicked-circle” Different structural forms produce different bands. 36

A- A+ 10 Kb Ladder 10 Kb Ladder 10 Kb Ladder Multimer Nicked Super Coiled 5 Kb Linear Fragment This image was taken with a digital camera, zooming, low light settings, and super macro settings. This is NOT an image from the gel imager that was supplied with the kit. This is what the gel will look like when fluoresced under UV light. Linear Fragment 37

What Are Some Applications of Recombinant DNA Technology? Bacteria, Yeasts, and Plants can all be modified to produce important pharmaceuticals, enriched foods, and industrial products. Novalin-R (Insulin Regular) is a product of recombinant DNA technology, and helps diabetics to regulate blood sugar. Synagis (Palivizumab) is a product of recombinant DNA technology and is given to babies who may be at risk of respiratory syncytial virus (RSV). The flask and dish image is meant to illustrate yeasts modified with recombinant DNA to produce any number of products. The product is produced and then purified out of the growth media. The corn cob is representative of the Genetically Modified Food Industry, where food crops have been modified to resist certain pests (including insects and fungi), or certain chemicals like Roundup (glyphosphate herbicide), and to reduce costs and improve yields of agricultural crops. 38

pKAN-R/pARA Sequence Biotechnology Lab Program Marty Ikkanda Bruce Wallace pKAN-R/pARA Sequence Laboratory Protocols by: Marty Ikkanda Powerpoint by: Anthony Daulo Pierce College, Woodland Hills, CA V.1.2.4 39

pKAN-R pARA Restriction analysis of pKAN-R and pARA rfp Bruce Wallace 5408 bp BamH I rfp 702 bp pARA 4058 bp Hind III BamH I Hind III 40 bp 40

Restriction fragments after digest with Hind III and BamH I Restriction analysis of pKAN-R and pARA Bruce Wallace Restriction fragments after digest with Hind III and BamH I BamH I Hind III 4018 bp BamH I Hind III 4706 bp Hind III BamH I 702bp Hind III BamH I 40 bp

Prediction for restriction gel Restriction analysis of pKAN-R and pARA Bruce Wallace Prediction for restriction gel M K+ K- A+ A- M K+ K- A+ A- 500 1000 1500 2000 3000 4000 5000 8000 10000 42

Ligation of pKAN-R/pARA restriction fragments Bruce Wallace sticky end BamH I 3’ 5’ 3’ sticky end Hind III 5’ sticky end Hind III 5’ 3’ sticky end BamH I 5’ 5’ 3’ 3’ 3’ 5’

Recombinant plasmid of interest Ligation of pKAN-R/pARA restriction fragments Bruce Wallace Recombinant plasmid of interest pARA-R 4720 bp BamH I rfp 702bp Hind III

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 45

Preparing competent cells for transformation Bruce Wallace 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 Bruce Wallace Lipid bilayer (inner) Adhesion zone Peptidoglycan layer Lipid bilayer (outer) Calcium ions pARA-R

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

Why do we need arabinose? Why don’t we see the red protein in any LB growth media? Cells conserve energy and resources The rfp gene requires a specific substrate (arabinose) to be turned on (expressed)

Colony isolation and culture Preparing an overnight culture of E. Coli for RFP expression Bruce Wallace Colony isolation and culture LB/amp/ara broth

Many of the red colonies picked from a Lab 5 plate appear to contain cells that are interfering with rfp expression. When there is a mixed culture of red and white (nonexpressing) cells, the white cells will grow faster than those that are using their resources producing mFP. = less mFP produced for purification.

RFP expression Bruce Wallace araC gene PBAD rfp gene Transcription mRNA Translation araC protein 54

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 55

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 56

Bruce Wallace RFP

Bruce Wallace RFP

Purification of RFP from an overnight culture Bruce Wallace Overnight culture Cell pellet with RFP Lysed cells Pellet cell debris RFP with binding buffer

Lab Tips/Reminders Add initials or group # to tubes Anything that touches bacteria must go in sterilizer Sterile technique Using bacteria Contamination may affect results Carefully READ and FOLLOW the lab protocol. Be sure lab partners communicate No Food or Drinks

Agar Plate tips (Lab 5) Label the bottom of the plates at the edges Note the plate markings: I=LB, II=LB/amp, III=LB/amp/ara Samples go on the agar, not the lid Open like clam shells Agar is like finger jello, firm but not invincible, be gentle Turn the plates upside down (lids down) for incubation

Sterile technique tips Always follow the protocol carefully – know what you’re doing Work quickly – less time = less opportunities for contamination Do not leave any container (tube, plate) open any longer than needed Watch what your equipment touches – there is no “5 second rule” here. All tips, tubes and spreaders in the “contaminated waste” container at the end of the lab.

Look at labels

Clam shell technique

Lab 7 – part 1 You need to aliquot and centrifuge twice to get a sufficient number of cells = product. Be sure the centrifuge has a balanced number of tubes. Be careful not to disturb the resultant pellets. When “wicking” don’t let the towel touch the pellet Supernatant and wicking towel go in disinfectant (10% bleach solution) containing beaker Incubate in 37 C water bath (60 min.) instead of overnight at room temperature. Freeze – ice crystals also help to lyse (break open) the cells.

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