BIOLOGY 3020 Fall 2008 Recombinant DNA Technology (DNA Cloning)

1. BIOLOGY 3020 Fall 2008Recombinant 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)

6. Traditional cloning of DNA using enzymatic restriction and ligation Genetic Map of Lambda Phage Tail proteins Head proteins Intregration Excision Control Lysis 0 48kb 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 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 Gal + X(Blue dye) blue colonies X-gal (colorless) White colonies Allows for easy visual “screening” of bacterial colonies that contain recombinant DNA molecules

10. Bacterial colonies transformed with pUC18 blue colonies (contain non-recombinant DNA molecules) White colonies (contain 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 forrestriction enzyme digestion, gel purification and ligation. Provide high-throughput compatibility– reactions are simple and robust www.invitrogen.com/Content/Online%20Seminars/gateway/1.htm

13. 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 ccdB Km ccdB attR1 attR2 attB1 Co-integrate LR Clonase Destination Vector 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 attB1 Amp attB2 attR2 attB2 x attP2 Expression Clone BP Clonase ccdB attB1 Co-integrate LR Clonase Amp ccdB Gene attP1 attL2 x attR2 attP2 attP1 By- Product attL2 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 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. 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) 1961 (Mt) Maize694,575,552 205,004,683 Wheat628,101,035 222,357,231 Rice618,534,989 215,654,697 Barley137,302,263 72,411,104 Sorghums58,620,842 40,931,625 Millets27,388,444 25,703,968 Oats23,972,508 49,588,769 Rye15,605,370 35,109,990 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. 54-61)

22. 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 DV535460 687 bp Lane 3 DR972034 1302 bp Lane 4 EE024212 831 bp Lane 5 EE173988 1941 bp Lane 6 DV532714 1434 bp 1 2 3 4 5 6 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. 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. 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 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 DOWNWARDSflame 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! 