Exercise 5. DNA Ligation, Selection and Screening of Ligation Products

ChE 170L Laboratory

Objectives The techniques learned in Exercises 2 – 4 will be applied in this experiment in order to complete a subcloning exercise. A piece of DNA that has been cut with restriction endonucleases will be mixed with a plasmid vector that has been cut with complementary enzymes. The two pieces will be covalently linked through the action of DNA ligase, forming a new plasmid. Competent E. coli cells will be transformed with the ligation reaction. Individual colonies will then be chosen and screened to identify the ligation products.

PreLab Questions 5

Please limit your answers to a few sentences IN YOUR LAB NOTEBOOK

1. Using what you know of ligation, what factors may be important when choosing a vector for the expression of your gene of interest?

2. Why are multi-cloning sites preferred locations for the insertion of a gene?

3. What role does ATP play in the DNA ligase reaction (be specific)? How might the lack of a 5’ phosphate group affect this mechanism?

4. Is it possible for a vector that has failed to incorporate the gene during ligation to instead stick to an identical vector resulting in a cDNA twice the size expected? Why or why not? Would this be more common with sticky ends or blunt ends?

Background The advent of recombinant DNA technology is arguably the most important advancement in applied biology to be developed last century. One aspect of recombinant DNA technology allows different segments of DNA to be assembled through a “cut and paste” procedure. In this case, the molecular “scissors” are restriction endonucleases. The “glue” is the DNA ligase enzyme. When two pieces of DNA that have been cut with EcoRI, for example, are mixed together in a buffered solution, diffusion through the liquid will eventually cause the compatible “sticky” ends of the DNA to come together. The hydrogen bonds between complementary base pairs will keep the two ends in close proximity. This enables the ligase to seal each of the two strands of the DNA with a covalent phosphodiester bond, creating a new DNA molecule. In the figure below, the ligase creates the bonds indicated by the dashes between G-a and A-g.

33 ChE 170L Laboratory

 ...G a a t t c...... G-a a t t c...... C T T A A + g...... C T T A A-g...  VECTOR + insert New Fragment

This very powerful tool allows molecular biologists (and biochemical engineers!) to introduce a gene like human insulin onto a vector like a plasmid. The plasmid can then be introduced into a single-celled organism such as E. coli, and the cellular machinery of the host will produce the corresponding protein. The process by which (1) a gene is identified, (2) a DNA fragment is obtained containing the gene sequence, and (3) the gene is introduced into a new host is called cloning. Subcloning occurs when a gene which has already been cloned is transferred from one vector to another and introduced into a host organism. pUC19 is one of many plasmids which have been developed specifically for use as cloning vectors. (In this instance, cloning also refers to subcloning.) This plasmid carries a portion of the amino-terminal, or “front,” end of the E. coli gene that produces the enzyme - galactosidase. The middle of the coding region of the gene fragment has a stretch of DNA about 50 bp long which contains 11 unique recognition sequences. An enzyme which recognizes one of these sequences will only cut at this site on the plasmid. The sequences within this stretch of DNA are collectively called multi-cloning or polycloning sites because they allow several different enzymes to be used to clone into the same short region of the plasmid. The multi- cloning sites are not naturally part of the -galactosidase gene coding region, however, they are in frame. Thus, when the corresponding polypeptide is produced, some extra amino acids are incorporated but they do not interfere with the normal function of the protein. A host cell must also be chosen for the transformation step that produces the carboxy-terminal, or “back,” portion of the gene. These two protein fragments can then come together in the cell to form an active peptide. This phenomenon is called -complementation. Production of the -galactosidase enzyme is caused by the addition of a chemical called IPTG (isopropylthio--D-galactoside). The enzyme can then cleave the chemical X-gal (5- bromo-4-chloro-3-indolyl--D-galactoside), producing a blue color. Cells which contain plasmids that produce -galactosidase are therefore easily identified because they will appear blue when plated on agar which contains IPTG and X-gal. However, if a foreign DNA segment is inserted into one of the multi-cloning sites of pUC19, the coding region of the gene for - galactosidase will be interrupted and the enzyme will not be produced. In this case, the colonies will be white (colorless). pUC19 is therefore said to enable blue/white selection because cells containing plasmids which contain the insert can be selected based on their white color. In addition to pUC19, which is a cloning vector, you will be using the expression vector pBAD. This plasmid contains a bacterial promoter to drive high levels of expression in E. Coli of genes inserted in front of this promoter. Such recombinant protein production was a revolutionary advance made possible by recombinant DNA technology. Once competent cells have been transformed with new plasmids and recombinant clones have been selected, they must be screened to properly identify the plasmid. In the screening process, individual colonies are chosen and cultured in liquid medium. It is crucial that single, round colonies be obtained in this step because these represent the descendents of a single cell,

34 ChE 170L Laboratory and that one cell was presumably transformed with a single, unique plasmid. Once the colony of cells is cultured in liquid medium, the plasmid DNA is isolated and restriction endonucleases can be used once again to determine (1) whether the insert was successfully ligated with the linearized vector and (2) the orientation of the DNA insertion.

PROCEDURE

NOTES:  This procedure will require two laboratory periods to complete. You may choose to write the entire protocol prior to Day One, or you may write only the portion of the procedure which is required for a single day prior to that lab period.

 DNA ligase, like restriction enzymes, is very expensive and extremely fragile. Do not contaminate the enzyme, and do not use pipette tips used with enzymes more than once. Enzymes are generally stored at –20 °C. During this procedure, the ligase should not be held out of the ice for longer than a few seconds.

Before the lab period, the instructors digested your GFP with EcoR I and Hind III. In addition, both pBAD and pUC19 were digested with EcoRI and Hind III. In this lab, they will be electrophoresed and purified from the gel for use in the ligation reaction.

Gel Extraction and Ligation (Day One)

You will be cutting out GFP, pBAD and pUC19. Note that pBAD and GFP will be in the same lane.

1. Excise the DNA fragment from the agarose gel with a clean, sharp razor 2. Weigh the gel slice in a microcentrifuge tube. Add 100uL of buffer QG for every 100mg of gel slice (1 gel volume) 3. Incubate this mixture at 50C for 10 minutes, taking out to shake at 5 minutes. This will dissolve the agarose gel and release the DNA. 4. After gel slice is dissolved completely, add 1 gel volume of isopropyl alcohol and mix. 5. Apply sample to the spin column and centrifuge at 13k rpm for 1minute. Max volume is 800uL, if you have more than this, add 800uL, spin, then add the rest and spin again. This binds the DNA to the column 6. Add 0.5mL of buffer QG to the column and centrifuge for 1 minute. This dissolves and discards any leftover agarose 7. Add 0.75mL of buffer PE to the column and centrifuge for 1 minute. 8. Discard the flow through and centrifuge the column again for 1 minute 9. Place column in clean 1.5mL centrifuge tube. 10. Add 30uL of buffer EB to the center of the column and let sit for 1 minute. Then centrifuge for 1 minute. This 30uL contains the purified digested fragment.

11. LIGATION BEGINS HERE. Set up the following ligation reactions in 1.5 ml microfuge tubes:

Reaction 1: 6 l water 4 l digested pBAD vector 7 l digested GFP 2 l T4 DNA ligase buffer 1 l T4 DNA ligase

Reaction 2: 6 l water 4 l digested pUC19 vector 7 l digested GFP 2 l T4 DNA ligase buffer 1 l T4 DNA ligase

12. Mix the reactions by pipeting up and down gently. Incubate overnight at 14oC.

Transformation (Day Two)

13. Transform the ligation reactions and a control plasmid into competent bacteria. Use 10 l of the ligation reaction and 5 l of the plasmid control in the transformation.

Remove four tubes with 100 l competent cells from the dry ice cooler and thaw on wet ice. Label the tubes to match your ligation reactions or control plasmids.

The control plasmid is pUC19 plasmid DNA (obtained from GSI).

Spread cells onto LB-AMP-IPTG-Xgal plates.

Place plates in the 37°C incubator for overnight culture.

Observation of Cultures

14. Place your pBAD-GFP plates on the UV transilluminator. Observe the colors of the colonies present under UV light.

15. Observe the pUC19-GFP plates by room lighting, not UV.

Guidelines for Analysis & Conclusions Section (Remember, these are points you should consider and include in your analysis. This section, however, need not be limited to these specific guidelines.)

1. Why is it necessary to run the digestion mixture on a gel prior to ligation? Is it necessary to ligate your products immediately after extracting them from the gel? Why or why not?

2. Discuss why the pBAD-GFP bacterial colonies are green on LB-AMP-IPTG-Xgal plates. Discuss why the pUC19-GFP colonies are white and not green or blue.

3. pBR322 was one of the first plasmids designed for use as a cloning vector. This plasmid contains genes for resistance to two antibiotics, ampicllin and tetracycline. There are a few unique restriction sites in the coding regions of each antibiotic resistance gene. Describe a method for selection of recombinant plasmids which have been produced from pBR322.

4. The restriction enzyme AvaI has the following recognition sequence: C Py C G Pu G. “Py” indicates a pyrimidine (C or T), and “Pu” indicates a purine (A or G). How many AvaI restriction sites would you expect to observe in a 4.2 kb plasmid?

EQUIPMENT AND REAGENTS pUC19 and pQE40 plasmid vector DNA cut with Bam HI and Hind III GFP PCR insert fragment cut with Bgl II and Hind III Sterile nanopure water T4 DNA ligase, supplied with 10X reaction buffer from New England Biolabs XL-10 Gold® competent cells Sterile LB medium, with and without 100 g/ml ampicillin Six LB agar plates with 100 g/ml ampicillin, 32 g/ml IPTG, 32 g/ml X-gal Disposable cell spreaders Alcohol lamp Ice bucket w/ice 37°C and 42°C water baths 0.5 ml microcentrifuge tubes, sterile and non-sterile 1.5 ml microcentrifuge tubes, non-sterile Sterile micropipet tips Promega Wizard Miniprep DNA Isolation Kit Restriction Enzymes: Nco I, EcoRI and Hind III, from Roche Biochemicals and New England Biolabs, respectively, supplied with 10X reaction buffers Electrophoresis unit. Includes power supply, box, plate, and sample comb. Gel camera Polaroid Film for gel pictures UV light box Commercial Molecular Weight Marker, 1 kb DNA ladder from New England Biolabs Ethidium Bromide (EtBr): Stock Solution at 10 mg/ml

Agarose (Electrophoresis-grade) 10X loading buffer (see Ex 3 for composition) 50X TAE Gel Electrophoresis Buffer (see Ex 3 for composition)

REFERENCES Bailey, J.E., and Ollis, D.F. (1986) Biochemical Engineering Fundamentals, 2nd Ed., pp. 340- 344, McGraw Hill, Inc., New York. Blanch, H.W., and Clark, D.S. (1996) Biochemical Engineering, pp. 533-538, Marcel Dekker, Inc., New York. Sambrook, Fritsch, and Maniatis. (1989) Molecular Cloning - A Laboratory Manual, 2nd ed., pp. 1.85 – 1.86, Cold Spring Harbor Laboratory Press. Voet, D., Voet, J.G. (1990) Biochemistry, pp. 824-829, John Wiley and Sons, New York.