1. BIOLOGY 3020 Fall 2008 Recombinant DNA Technology (DNA Cloning)
How many transcription factors (TFs) in Corn?
DNA and p53 Transcription Factor

2. Lecture Outline Traditional Cloning Method New Gateway Cloning Method
Class Project The Keys of Corn
Class Project Part 1 - Cloning a TF ORF into a Gateway entry vector
Transformation of DNA into E. coli
Sterile Technique (working with E. coli)

3. 1: Traditional Cloning
DNA Cloning (many identical copies of specific DNA molecules)
is NOT the same as Organismal Cloning
(identical genetic copies of specific individuals)

4. General Cloning Strategy for DNA
With Restriction enzymes
With Restriction enzymes
with DNA ligase enzyme
(Genetic transformation)

5. See Chapter 17 of Lecture Text (Klug and Cummings Essential s of Genetics)
Figure: 17-02
Title:
DNA Ligase
Caption:
DNA from different sources is cleaved with Eco RI and mixed to allow annealing to form recombinant molecules. The enzyme DNA ligase then chemically bonds these annealed fragments into an intact recombinant DNA molecule.

6. Traditional cloning of DNA using enzymatic restriction and ligation
Genetic Map of Lambda Phage
Head proteins
Tail
proteins
Intregration Excision
Control
Lysis kb
EcoRI Restriction Sites
Cut with EcoRI
(‘sticky ends’)
pUC18
Mix and ligate together with DNA Ligase

7. Typical Cloning Strategy to make Library
Cut with restriction enzymes, mix and ligate
Transformation of E. coli
EcoR1
EcoR1
EcoR1
Non-recombinant
Recombinant
Characterize insert (“clone”)
Blue Colonies (Discard)
White Colonies (Keep)

8. pUC18 - a common cloning vector
Figure: 16-05
Caption:
The plasmid pUC18 offers several advantages as a vector for cloning. Because of its small size, it accepts relatively large DNA fragments for cloning; it replicates to a high copy number, and has a large number of restriction sites in the polylinker, located within a lacZ gene. Bacteria carrying pUC18 produce blue colonies when grown on media containing Xgal. DNA inserted into the polylinker site disrupts the lacZ gene; this results in white colonies and allows direct identification of colonies carrying cloned DNA inserts.
Essential features
Polylinker
Selectable marker (Ampr)
Screenable Marker (LacZ)
Bacterial Origin of replication (oriR)

9. LacZ- a screenable marker
EcoR1
EcoR1
EcoR1
Lac Z gene
Interrupted
Lac gene
pUC18
pUC18
“Recombinant Molecules”
Beta-galactosidase
NO Beta-galactosidase
X-gal
(colorless)
Gal + X(Blue dye)
blue colonies
White colonies
Allows for easy visual “screening” of bacterial colonies that contain recombinant DNA molecules

10. Bacterial colonies transformed with pUC18
Figure: 16-06
Caption:
Petri plate showing growth of host cells after uptake of recombinant plasmids.
White colonies
(contain recombinant DNA molecules)
blue colonies
(contain non-recombinant DNA molecules)

11. Advantages of Traditional Cloning
1: Recombine DNA molecules from any source
“The Awesome Skill”
2: 100’s of different restriction enzymes available
Disadvantages of Traditional Cloning
1: Some restriction sites not present or present where not desired
2: Careful planning of cloning strategy required and many steps involved (including gel purification)
3: Transfer from one vector to another not straightforward (e.g. to maintain reading frame)

12. 2: A NEW way of cloning - “Gateway Cloning”
Maximize compatibility and flexibility
Minimize planning
Maintain reading frame
Eliminate the need for restriction enzyme digestion, gel purification and ligation.
Provide high-throughput compatibility– reactions are simple and robust

13. Phage l recombination in E. coli
How to avoid restriction enzymes - take advantage of Site specific recombination
Phage l recombination in E. coli
Phage l
attP 243bp
Site-specific recombination mediated by phage lambda recombination proteins.
2. The reaction is specific and directional: attB x attP ⇔attL x attR.
3. Each reaction is mediated by a different combination of enzymes.
E. coli
attB 25bp
attL 100bp
attR 168bp
Integrated prophage

14. Summary of Site Specific Recombination in E. coli
In lambda, the integration site is known as attP, in E. coli the site is attB. The attB site is short, only 25 bp, keep this in mind as it will be important later. The att sites contain the binding sites for the proteins that mediate l recombination. The integration reaction (attB x attP) is mediated by the proteins integrase (Int) and host integration factor (IHF). When integration occurs, two new sites are created, attL and attR, flanking the integrated prophage, with no loss of DNA sequence.
All the att sites are alike in that they contain a 15-bp recognition sequence for the recombinase (integrase). The reaction can also go in the opposite direction (excision). When attL x attR recombine (mediated by the proteins integrase, host integration factor and excisionase [Xis]), the lambda -DNA is excised from the E. coli genome, recreating the attB site in E. coli and the attP site in lambda

15. The GATEWAY™ Cloning System
The entry vector is recombined with a destination vector using lambda recombination enzymes
Gene
attL1
attL2
Entry
Clone
Amp
attR2
attB1 x attP1 BP
Clonase Km
ccdB
ccdB
In Gateway the specificity’s of the original lambda sites have been engineered in several ways. We now have attL1, attL2, attR1, attR2 etc. The original sites were also engineered to remove stop codons and to reduce secondary structure formation.
Entry Vector
The DNA sequence of interest (cDNA, genomic DNA, an ORF etc) is cloned into a plasmid vector, known as an Entry Vector, to generate an Entry Clone. The Entry Clone is Kanamycin resistant and transcriptionally silent. The sequence of interest is flanked by two recombination sites, L1 and L2. These two rec. sites can NOT recombine with each other.
What is the objective of our cloning reaction here?
The objective of our sub-cloning reaction is to move the DNA sequence of interest into the Destination Vector backbone. The Destination Vector is Transcriptionally active (allows protein expression), carries Ampicillin resistance and a ccdB gene (a negative selection tool) flanked by two recombination sites, R1 and R2. These two rec. sites can NOT recombine with each other.
Cloning Mechanism: Rec site L1 will only recombine with rec site R1, while rec site L2 will only combine with rec site R2. This is how orientation is maintained during the sub-cloning event. The product of the L1 x R1 and L2 x R2 reactions are B1 x P1 and B2 x P2, respectively. How do we do the cloning reaction?
Entry Vector and Destination Vector plasmids are combined in the presence of LR CLONASE - this is a proprietary mix of 3 recombination proteins (Int, IHF & Xis). The products of the reaction are the Expression Clone, which carries DNA sequence of interest now flanked by the attB1 and attB2 sites, and a by-product which carries the ccdb gene now flanked by the attP1 and attP2 sites. This reaction is effectively an In Vitro version of the phage lambda excision reaction.
The Expression clone carries the DNA sequence of interest flanked by two recombination sites (attB1 and attB2) in a new vector backbone (from the Destination Vector) which carries ampicillin resistance and is transcriptionally active.
The reaction mixture, which will contain all of the plasmid products described above, will be used to transform E.coli. By combining positive (antibiotic resistance; ampicillin) and negative selection (ccdb gene) schemes, typically 99+% of all resulting colonies will carry only the desired Expression Clone.
(Negative selection: The ccdB gene product is toxic to all standard coli strains, thereby eliminating all coli that have been transformed with non-reacted destination vector. The ccdB protein interferes with the “ligation activity” of DNA gyrase, resulting in the degradation of chromosomal DNA and subsequent cell death. Combining the positive and negative selection tools results in very high cloning efficiencies and drastically reduces the requirement to screen colonies for desired clones.
Gateway is Flexible: The yellow arrows show that the reaction can also be driven in the reverse direction by using a second recombination protein mix, known as BP Clonase. This is essentially an in vitro version of the in vivo phage lambda integration reaction. This reaction will be reviewed further later on.
Clearly the key to Gateway is generating Entry Clones. So how do you generate Entry Clones? As shown in the following slide, there are many ways of generating Entry Clones.
attR1
attR2
Co-integrate
attB1
Destination
Vector LR
Clonase
attP1
Gene
attL1 x attR1
Transform
E. coli
attL2
Amp Km
> 99% correct entry clones in Km r colonies next day

16. The intermediate cointegrate is resolved (2nd recombination event) to leave an expression clone and a by product. Select for the former on Ampicillin plates.
Gene
Amp
attB1
attB2
attR2
attB2 x attP2 BP
Clonase
Expression
Clone
ccdB
The next slide overlays our generic model and site-specific recombination of l in E. coli; this is Gateway Cloning Technology.
The Gateway reactions are in vitro versions of the integration and excision reactions. This cartoon describes the predominant pathway of the system, the Destination Reaction.
Your goal is to move your DNA sequence of interest from one vector backbone to another. This slide will introduce you to Gateway nomenclature: the sites, Entry Clones, Destination Clones, negative selection via the ccdB gene.
Key points
Combining plasmid DNAs with sequences flanked by recombination sites that do not recombine with each other, but will recombine with sites resident on another molecule.
Your sequence of interest is in a vector that is transcriptionally silent, Kmr, and is flanked by two recombination sites.
You want to move your sequence to a Destination Vector backbone; this vector contains all the sequence information required for expression (e.g., N-terminal or C-terminal fusions such as His6, GST, promoters, etc.). It also contains the negative selection marker ccdB.
Directionality and specificity conferred by att1 and att2 sites. More detail later.
The reaction is mediated by LR Clonase (this enzyme mix is comprised of Int, IHF, and Xis you are doing an in vitro excision reaction).
Resolution of the co-integrate and selection of the desired molecule: antibiotic selection and the ccdB gene.
The reaction transfers the DNA segment of interest into the Destination Vector backbone. Any unreacted Destination Vector or recombination intermediate that was taken up by the E. coli does not contribute to background because of the ccdB gene. The ccdB gene product works by interfering with the normal ligase activity associated with DNA gyrase. The consequence of its expression is degradation of the E. coli chromosome.
As you might imagine, we can also do the B x P reaction. More about that later.
Co-integrate
attB1 LR
Clonase
Amp
ccdB
attP1
Gene
attL2 x attR2
attP1
attP2
attL2
By-
Product
Transform
E. coli Km Km
>99% correct expression clones
in Ap r colonies next day

17. 3: Class Research Project Overview
Transcription Factors - The Keys of Corn

18. Corn genome is underway
Corn which was domesticated by Native Americans has become the most important cereal crop worldwide
In 2005 nearly 700 million metric tons of corn were harvested worldwide
Plant scientists aim to understand corn as much as doctors understand humans
Corn genome is underway

19. Source: National Geographic June 1993 p91-117
Avg Annual US Usage
0.2% Hybrid Seed
1.4% Food
1.8% Starch
3.7% Alcohol
5.8% Sweeteners
44.7% Animal Feed/Residual
16.8% Exports
25.6% Ending Stocks (Buffer against a bad crop)
Oil is extracted from the germ (embryo) for cooking, Starch in building materials or intravenous solutions, the shell (hull) is used in animal feed
Source: National Geographic June 1993 p91-117

20. All of the major crops worldwide are cereals (grasses)
Grain 2005 (Mt) (Mt)
Maize 694,575, ,004,683
Wheat 628,101, ,357,231
Rice 618,534, ,654,697
Barley 137,302,263 72,411,104
Sorghums 58,620, ,931,625
Millets 27,388, ,703,968
Oats 23,972, ,588,769
Rye 15,605, ,109,990
Knowledge of one grass species helps immensely in the breeding of other grass species

21. Known for some time that Transcription Factor (TF) proteins are molecular machines that turn on and of genes - like the keys of a car
Estimate that about 10% of all genes encode TFs - about 3000 in humans and maybe 6000 in corn
Scientific American (February 1995, pp )

22. With the Keys in hand, the pace of discovery will quicken
Class Project is to begin cloning all the TFs in maize as a basis for further study of global gene regulation - (Field of Regulomics)
A set of Entry clones will be made that can be used to make many diffenret constructs for molecualr biology investigation.
With the Keys in hand, the pace of discovery will quicken

23. Overview of Keys of Corn Project Strategy
Transfer clone into a variety of Gateway Destination vectors
Identify full length TF clones in Genbank
Design PCR primers to amplify ORF from flcDNA clones
Sequence and verify Entry clones
Produce blunt-end PCR products of TF ORFs
Select colonies and isolate plasmid DNA
Mix PCR product with pENTR TOPO Vector
Transform into competent E. coli cells

24. 4: Cloning full length Corn TF ORFs
1: Start with a partial sequence of an isolated corn TF cDNA (see list at end of lecture) - (cDNA should show some homology to known TFs)
2: Perform BLAST search with sequence to identify closely related overlapping sequences in Genbank database (>97% identity)
3: Organize different sequences into a contig using ContigExpress program in Vector NTI
4: Translate long contig to identify if start and stop codons are present - compare to known TFs
5: Choose the most 5’ clone and order from clone repository (e.g. Arizona Genomics Institute)

25. 6: Design PCR primers suitable to clone into pENTR/D vector
7: Amplify the Open Reading Frame (ORF) for each gene (Lab 10 Oct 30th/31st)
Lane 1 1kb DNA Ladder
Lane 2 DV bp
Lane 3 DR bp
Lane 4 EE bp
Lane 5 EE bp
Lane 6 DV bp
PCR products like these will be cloned into pENTR/D

26. (Lab 10 Nov 4th/5th) PCR of Corn TF
You will be provided with
1: a plasmid containing a TF flcDNA (this is the template)
2: PCR primers to amplify the ORF of the cDNA (designed by the course instructor and your TA)
3: Taq Polymerase, buffer, Mg solution and an optional “PCR enhancer” solution
You will set up a few PCR reactions to find the optimal Mg concentrations needed to amplify your TF gene of interest.
Next week you will clone the PCR product (if successful into a cloning vector (PENTR/D)

27. (Lab 10 Nov 4th/5th) PCR of Corn TF
PCR Optimization
Each PCR reaction must be optimized. Factors such as annealing temperature, Mg ion concentration, and Polymerase stabilizing agents all affect PCR.
Each PCR reaction is different because of the different primers that are used.
You will set up 4 reactions.
1: 2mM MgCl2,
2: 3mM MgCl2,
3: 2mM MgCl2 + Enhancer,
4: 3mM MgCl2 + Enhancer,

28. 9: Clone PCR product into Cloning Vector
(Lab 11) Nov 18th/19th)
Topoisomerase speeds up Cloning
Topoisomerase has ligase activity. Kit provides linear pENTR vector with Topo covalently bound near end - ready to ligate in insert ($20 per ligation)

29. Mix PCR product with pENTR/TOPO Incubate 5 minutes at room temperature
Place on ice
Ready for transformation into E. coli

30. PCR product ligated into pENTR/D
The GTGG overhang is displaced and the insert is directionally cloned into the entry vector (i.e. start codon is near attL1 region)

31. 5: Genetic transformation of E. coli
E. Coli is naturally unable to take up DNA efficiently
By treating rapidly growing E. coli cells with ionic solutions (CaCl2 and MgCl2) the cells are made “competent” to take up DNA.
The competent cells can be frozen at -70°C for later use (but they are very fragile and must be pipetted very slowly).We will use One Shot Competent cells.
Incubate thawed cells with DNA, then “heat-shock” at 42°C for 30 seconds (DNA is taken up by cells).
Transfer to nutrient broth (S.O.C) and allow cells to recover for 1hr.
Spread plate out on appropriate selection media

32. Heat shock transformation of E. coli
100
Heat shock transformation of E. coli
1: Transfer 2ul of TOPO cloning rxn to One Shot cells
2: Keep on ice for 30 min
3: Heat shock at 42°C 30 sec
4: Back on ice

33. Heat shock transformation of E. coli
100
Heat shock transformation of E. coli
During 1hr incubation, the kanamycin resistance gene is expressed
6: Shake transformed cells at 37°C for 1 hr
7: Plate out cells on Kanamycin selective medium
5: Add 250 ul of SOC nutrient medium

34. Performing the Spread Plate method I
100
Performing the Spread Plate method I
1: Choose appropriate nutrient agar plate with the correct antibiotic (and X-gal) if visual screening
2: Using sterile technique transfer a loopful of bacteria from a culture tube onto plate (or 100l of bacterial culture using a pipette)

35. Performing the Spread Plate method II
“Spreader” or “Hockey Stick”
Keep flame away from alcohol !!
70% EtOH
3: Dip glass “hockey stick” in 70% ethanol. Holding it DOWNWARDS flame until alcohol is burned off. DO NOT put back into alcohol
4: Remove lid of petri dish. With one hand rotate dish. With other hand move hockey stick lightly over surface to spread the inoculum evenly

36. After Incubating the plate overnight at 37°C- individual colonies of transformed bacteria should be seen
Each team will pick two individual colonies (clones) and streak on a new plate (single colony purification) for next week

37. Sterile technique
When handling E. coli and other bacteria it is essential that the live cultures do not become contaminated with other bacteria or fungi. The set of procedures used to accomplish this are known as “sterile technique”
General Points
1: Keep vials or plates containing bacteria open for a minimum amount of time.
2: Use sterilized instruments when handling the bacteria
3: Discard all bacteria in “biohazardous” waste - this will be destroyed later
4: When using an open flame never leave it unattended

38. Streak Plate method to Purify Single bacteria
Principle This is essentially a method to dilute the number of organisms, decreasing the density - individual colonies to be isolated from other colonies. Each colony is "pure," since theoretically, the colony began with an individual cell
Begin with inoculating the first, or primary, quadrant of the agar plate. Use a light touch. Don't penetrate or scrape the agar surface. Cover plate with lid.
Flame the loop, cool by touching an uninoculated portion of the surface.
3. Now rotate the plate. Open lid and streak again, remember: you are picking up growth from quadrant one, and using this as your inoculum for quadrant two.
4. Flame loop; rotate plate, and repeat procedure for quadrants three and four.

39. Performing a Plate Streak I
2-3: Using sterile technique transfer a loop of bacterial culture or single colony onto loop
4: With one hand remove lid of dish. With other hand lightly brush the loop back and forth on one quadrant of the dish
1: Flame metal inoculating loop, let cool momentarily.

40. Performing a Plate Streak II
8 :Incubate o/n at 37°C
4: Reflame metal inoculating loop, let cool momentarily.
5,6,7: Rotate petri dish 90° Use 1st streak as inoculum for 2nd streak (only pass the loop through the 1st streak once). Repeat once more rotating dish 90° and sterilizing loop again

41. Plate Streak Method
This is an example of a good streak for isolation using the "four corners" method.

42. This is not a great streak plate but it is serviceable, as there are a few isolated colonies. - would have been better if the loop had been flamed between each sector.
This is an example of how NOT to streak for isolation. Scribbling is not streaking, and most likely will not result in isolated colonies.

43.  Final Caution! Cloning corn genes may be hazardous to your health -
don’t let this happen to you! 