1. Amgen Bruce Wallace Transformation Labs (2-7)

2. 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

3. 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!

4. 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

5. 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.

6. 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

7. The Big Picture
2005 Pearson Education, Inc.

8. 2005 Pearson Education, Inc

9. 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

10. 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

11. 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

12. 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

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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. Application of Restriction Enzymes20

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

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

23. Many Other Recombinant Possibilities
…and many more! 23

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

25. Transgenic Colony Allowed to Grow25

26. 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

27. Running Digested DNA Through Gel Electrophoresis27

28. 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

29. 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

30. 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

31. 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

32. Analyzing Your Gel 32

33. 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

34. 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

35. 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

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

37. 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

38. 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

39. 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

40. 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

41. 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

42. 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

43. 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’

44. 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

45. 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

46. Preparing competent cells for transformation
Bruce Wallace
Lipid bilayer
(inner)
Adhesion zone
Peptidoglycan
layer
Lipid bilayer
(outer)
Calcium ions

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

48. Transforming Escherichia coli with pARA-R
Bruce Wallace
Lipid bilayer
(inner)
Adhesion zone
Peptidoglycan
layer
Lipid bilayer
(outer)
Calcium ions
pARA-R

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

50. 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)

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

52. 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.

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

55. 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

56. 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

57. Bruce Wallace
RFP

58. Bruce Wallace
RFP

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

60. 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

61. 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

62. 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.

63. Look at labels

64. Clam shell technique

65. 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.

66. Science for LIFE