I. Overview:
In this exercise you will create a library of cloned restriction fragments from the genome of the budding yeast Saccharomyces cerevisiae. Each laboratory group will digest yeast DNA with a different restriction enzyme in order to cleave the genome into defined sets of DNA fragments with appropriate cohesive ends. The mixture of DNA fragments will then be ligated with Bluescript KS plasmid DNA that has been cut with the same restriction enzyme. The ligated DNA will then be introduced into E. coli cells that have been made competent for transformation by treatment with CaCl2. Cells containing plasmid DNA will be selected by plating the bacteria on culture media containing ampicillin. Bacterial colonies containing plasmids with recombinant DNA inserts will be identified by color selection by the presence of the chromogenic -galactosidase substrate, X-gal. Selected white colonies containing recombinant plasmids will be grown up in liquid culture and the cloned DNA fragments will be amplified by polymerase chain reaction. The nucleotide sequence of the cloned fragments will be determined by the Nevada Genomics Center. The cloned DNA fragments will be identified within the yeast genome in Exercise #6.
II. Background:
A. Construction of Recombinant DNA Libraries in Cloning Vectors:
The first step in isolating genes or mRNA sequences encoding specific proteins is the construction of a recombinant DNA library. Gene libraries are populations of cloning vectors (either plasmids or viral chromosomes) containing a large number of different recombinant DNA molecules representing the entire constellation of gene sequences of an organism. Complimentary DNA (cDNA) libraries are collections of DNA sequences complementary to the mRNA population of a tissue or cell line. Complementary DNA is produced by copying an mRNA population into cDNA sequences using the enzyme reverse transcriptase. The construction of recombinant DNA requires that the target DNA and the vector molecule must have compatible ends. This is accomplished either by digesting the target and the vector with the same restriction enzyme or by the addition of synthetic sequences to the ends of the target DNA molecules. These synthetic sequences are known as linkers or adapters. They allow the introduction of identical defined sequences at the ends of heterogeneous molecules. Any DNA molecules with compatible cohesive ends can be covalently joined together by the enzyme DNA ligase. The ligase most commonly used in gene cloning is derived from T4 bacteriophage which infects E. coli. The enzyme requires ATP and Mg++ and can joint molecules with complementary single stranded extensions. These can be either 3'- or 5'- overhangs of the type generated by restriction enzyme digestion. Molecules with blunt (non-overhanging) ends can also be joined together, although with a much lower efficiency.
B. Introduction of Recombinant DNA into E. coli host cells:
In order to isolated large amounts of DNA derived from a single recombinant molecule, the vector molecules containing foreign DNA must be introduced into a host cell so that replication and amplification can occur. E. coli is the most commonly used host for cloning recombinant DNA in the laboratory. Unlike certain other species of bacteria, E. coli cannot take up DNA from its environment and become genetically transformed by natural processes. Nonetheless, recombinant DNA can be introduced into E. coli by a artificial procedures. When cloning vectors derived from bacteriophage lambda are used, the recombinant molecules can be packaged into phage particles in the test tube by combining the DNA with a mixture of phage proteins. The packaged DNA can then be introduced into E. coli cells by a procedure called transfection. The synthetic phage attach to the lambda receptor on the cell surface and the recombinant DNA molecules are injected into the bacterium via the same mechanism as in normal infection. Depending upon the type of vector used, the recombinant phage can either grow lytically, lysing the infected cell and adjacent cells producing a clear plaque on a lawn of bacteria, or integrate into the bacterial chromosome to produce a lysogen. The recombinant molecules can be propagated either as free infectious phage or in a lysogenic strain of bacteria. Plasmid vectors containing recombinant DNA can be introduced into cells by either of two methods. The oldest procedure, which we will be using in this laboratory, involves simply mixing specially treated host cells with DNA under defined conditions. E. coli can be made competent for taking up foreign DNA (transformation) by prior treatment with compounds such as CaCl2 or DMSO. These treatments, permeablize the cell membrane to DNA molecules when followed by a mild exposure to elevated temperature. A more recent method used for introducing DNA into almost any type or cell is called electroporation. In this procedure, which requires special equipment, cell are exposed to DNA in a medium with high electrical resistance (ie., distilled water). Pores are induced to form in the cell membrane by a very short period of high voltage exposure. Under optimal conditions, DNA can be introduced into the majority of cells in the vessel by this procedure. CaCl2 treatment is less efficient than electroporation, but it is less expensive. CaCl2 competent cells are adequate for many purposes such as subcloning restriction fragments form larger vectors into small plasmids.
C. Screening Libraries and Isolation of DNA Sequences of Interest:
Even relatively simple cDNA libraries contain large numbers of different sequences. A single eukaryotic cell type contains approximately 20,000 different mRNA molecules. The abundance of individual mRNA species varies over several orders of magnitude. A few very abundant sequences such as actin and tubulin mRNAs make up as much as 3-6% of a typical mRNA population. Conversely, mRNAs encoding non-abundant proteins such as transcription factors and protein kinases involved in regulatory processes are present at only a few molecules per cell. Such rare mRNAs can comprise less than 0.0001% of the clones in a cDNA library. Consequently, large numbers of individual clones must be screened in order to have a reasonable chance of detecting a rare cDNA species.
Gene libraries are even more complex. A typical mammalian genome contains about 3 X 109 base pairs of DNA. The probability that a given clone will contain a target sequence of interest increases with the size of the cloned DNA fragment. Thus, fewer numbers of recombinants must be screened if very large DNA fragments can be cloned in each vector molecule. Large DNA molecules (>10,000 base pairs) are very difficult to clone in plasmid vectors since large DNA molecules are taken up poorly during the transformation process. For this reason, special phage derivatives and yeast artificial chromosomes have been developed specifically for cloning large genomic DNA fragments (20,000 - 100,000 base pairs). Nonetheless, in order to have a 95% chance of identifying a unique mammalian gene, more that 1,000,000 recombinant clones containing 20,000 base pair inserts must be screened.
Many techniques have been developed for rapidly screening large numbers of recombinant plasmid or bacteriophage clones. Colony or plaque hybridization involves growing large numbers of bacterial colonies or phage plaques on agar plates and making a blot of each plate onto a filter membrane. The filters are then hybridized with specific DNA or RNA probe sequences tagged with either a radioactive or colorimetric label. Individual colonies or plaques (possibly only 1 out of 1,000,000) that hybridize with the probe are then isolated with a sterile loop or tooth pick, re-grown, and screened repeatedly until pure. Related methods use antibodies to screen recombinant colonies or plaques for proteins expressed from target genes. This procedure require the use of special vectors designed to express recombinant genes as fusion products with bacterial proteins
In the present exercise, you will prepare gene libraries from the genome of brewer's yeast. The yeast genome contains only 3.1 X 107 base pairs of DNA and has been sequenced completely. Digestion of this chromosome with the restriction enzymes Hind III or Eco RI each produce thousands of discrete DNA fragments. Your lab group will prepare a library of either Hind III or Eco RI fragments cloned in the plasmid vector BlueScript KS. The host organism will be E. coli. You will then manually isolate 3 individual clones which will be grown up in liquid culture and presented to the Genomics Center for partial sequence analysis of the cloned DNA fragment. The sequence information that will be obtained will be used for the Bioinformatics exercise in Lab #6.
III. Procedure:
A. Digestion of Target and Vector DNA:
1. Obtain samples of yeast DNA and BlueScript KS plasmid DNA from your instructor. The DNA concentration should be 100 ug/ml in both cases. If the DNA is provided at different concentration, make the appropriate dilutions using TE buffer.
2. Assemble two restriction digestion reactions as follows and mix by vortexing:
Reaction #1 (vector DNA) Reaction #2 (target DNA)
10 ul BlueScript plasmid (1 ug) 20 ul yeast DNA (2 ug)
30 ul H2O 20 ul H20
10 ul 5X restriction buffer 10 ul 5X restriction buffer
------- -------
50 ul 50 ul
3. Add 1 ul (10 units) of either Hind III or Eco RI to each reaction, vortex gently, and incubate at 37oC for 1 hr. Prepare a 1% agarose gel and analyze 5 ul samples of the following:
a) Undigested plasmid DNA (provided by the instructor)
b) Undigested yeast DNA (provided by the instructor)
c) Digested plasmid DNA from part A
d) Digested yeast DNA from part A
B. Dephosphorylation of Vector DNA:
In order to reduce the number of self-ligated plasmids without recombinant DNA inserts, the 5'- phosphate groups at the ends of the plasmid vector will be removed by alkaline phosphatase treatment. Dephosphorylated vector molecules can only be circularized by DNA ligase if a DNA fragment containing 5'- phosphates is inserted between the ends of the plasmid molecule.
1. Remove 5 ul of each DNA sample and analyze for complete digestion on a 1% agarose gel.
2. Add 5 ul of shrimp alkaline phosphatase buffer (SAP buffer) to the remaining vector DNA, vortex, and add 0.5 ul (2.5 units) of shrimp alkaline phosphatase (SAP).
3. Vortex again and continue the digestion for 30 additional minutes at 37oC. You can allow the target DNA to continue digestion with the restriction enzyme for this additional time.
C. Deproteinization and Precipitation of Vector and Target DNA:
1. Remove both DNA samples from the water bath and add 50 ul of 1:1 phenol/chloroform to each tube. Vortex for 30 sec at high speed.
2. Centrifuge the samples for 2 min in the microcentrifuge to separate the phases. Carefully remove the upper aqueous phases to fresh, carefully labeled tubes. Do not carry over any organic phase! Discard the tubes containing the lower phases.
3. Add 2 ul of glycogen carrier (about 20 ug) to each sample. Add 25 ul of 7.5 M NH3OAc, mix, and add 150 ul of absolute ethanol.
4. Vortex again and hold the samples on ice for 2 min.
5. Collect the precipitated DNA by centrifugation for 10 min in the microcentrifuge.
6. Carefully remove the ethanol supernatant without resuspending the tiny DNA pellets. Rinse the excess salt from the tubes and pellets with 200 ul of 70% ethanol. Recentrifuge for 2 min and carefully remove as much ethanol from the pellets as possible.
7. Dry the DNA samples for 10 min in the SpeedVac.
8. Resuspend each DNA pellet in 20 ul of TE buffer.
D. DNA Ligation Reactions:
You will assemble three ligation reactions. The first will serve as a control for self-ligation and will contain only vector DNA. The other two reactions will contain different concentrations of target DNA. The formation of circular DNA molecules containing both vector and target DNA is highly sensitive to DNA concentration and to the size of the target fragments. We will use two different target DNA inputs to increase the likelihood of successful cloning.
1. Assemble the three ligation reactions as follows:
Ligation #1 (self-ligation) Ligation #2 (~200 ng target ) Ligation #3 (~400 ng target)
2 ul vector DNA 2 ul vector DNA 2 ul vector DNA
16 ul H20 11 ul H20 6 ul H20
0 ul yeast DNA 5 ul yeast DNA 10 ul yeast DNA
2 ul 10X ligase buffer 2 ul 10X ligase buffer 2 ul 10X ligase buffer
------- ------- -------
20 ul 20 ul 20 ul
2. Add 0.5 ul (0.5 U) of T4 DNA ligase to each tube.
3. Incubate overnight at 14oC. Your instructor will carry out this step and freeze your sample until the following lab meeting.
4. Save 5 ul of each reaction for gel analysis.
4. Freeze the reactions at -20oC until ready to perform the transformations during the next laboratory period.
E. Transformation of Competent E. coli: 2nd Lab Period
Competent E. coli cells can be prepared by the following procedure. Because of time constraints, your instructor may provide competent cells that have been stored frozen in medium containing 20% glycerol.
1. Start an exponential culture of the appropriate strain of E. coli by diluting an overnight culture 1:100 in pre-warmed L broth.
2. Chill the cells in an ice water bath when the A600 reaches 0.5. If the cells become denser than 0.6, dilute the culture with 5 vol of pre-warmed L broth and try again. Chill the cells at least 5 min.
3. Decant into 35 ml sterile Oak Ridge tubes and centrifuge at 2500 rpm for 10 min at 4oC.
4. Suspend the cells in 1/4 the original vol in ice cold 0.1 M MgCl 2. Suspend by gentle pipetting.
5. Centrifuge the cells for 10 min at 2500 rpm in the cold.
6. Suspend the cells as before in 1/4 the original vol of freshly prepared ice cold 0.1 M CaCl2. Hold on ice for 20 min.
7. Centrifuge the cells for 10 min at 2500 rpm in the cold.
8. Gently resuspend the cells in 1/20 the original vol of ice cold 0.1 M CaCl2 containing 7% glycerol. Freeze in dry ice-acetone bath and store at -70oC.
Transformation
1. Thaw out the ligation reactions and hold on ice. Remove 5 ul of each reaction and analyze for ligation efficiency by running the products on a 1% agarose gel. Use samples of your unligated digested vector and yeast DNA as markers.
2. Carefully thaw a vial of competent cells. As soon as the sample is completely thawed, place on ice. Try not to let the cells warm up as they do not retain viability for long at elevated temperatures in the CaCl2 media.
3. Gently suspend the cells by tapping the vial and pipette 100 ul of cells into each of three labeled 15 ml conical tubes. Pre-chill the tubes on ice before adding the cells.
4. Add 10 ul of each ligation reaction to the appropriate tubes. Mix by gently tapping the tubes and hold on ice for 30 min.
5. Heat tubes for exactly 2 min at 42oC. Do not shake the tubes.
6. Place on ice for 2 min.
7. Add 1 ml of SOC media (a rich broth that facilitates recovery) to each tube.
8. Place the tubes in a beaker and shake at 250 rpm at 37oC for 1 hr. This step allows the cells to express the ampicillin resistance gene before plating on medium containing the antibiotic. If this step were omitted, most of the transformed cells would be killed before they could develop resistance.
9. Spread 100 ul of each transformation on B-broth agar plates containing 125 ug/ml ampicillin and the b-galactosidase substrate X-gal. Use a glass spreader sterilized with 70% ethanol as demonstrated by your instructor.
10. Let the plates sit on the bench for 10-15 min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37oC.
11. Count and record the number of blue and white colonies found on each plates. The plates can be sealed with parafilm and stored until the following week if necessary.
F. Liquid Cultures for PCR/Sequencing Analysis of Cloned DNA Fragements:
1. Using a sterile pipette tip or tooth pick, pick 3 white colonies from the plates containing cells transformed with your 200 or 400 ng yeast DNA ligation reactions. Pick 2 blue plaques from any plate. Touch the tip or tooth pick to the surface of the liquid medium and replace the cap. Be sure to label your tubes carefully.
2. Grow up you cultures overnight at 37oC with the shaker incubator. Your instructor will place the culture on ice the following morning.
3. Present your cultures to the Nevada Genomics Center (FA 328).
IV. Final Evaluation:
1) Tabulate the number of blue and white colonies obtained with each transformation reaction. Why are some blue colonies still obtained despite alkaline phosphatase treatment of the vector? Are there any white colonies on the plate containing the self-ligated control plasmids? What are possible explanations for this type of result?
V. References:
Asubel, F.M. et al., 1993, Current Protocols in Molecular Biology, Wiley & Sons, Inc., pp. 1.0.1-1.15.8.
Watson, J.D. et al., 1992, Recombinant DNA, Scientific American Books, pp. 63-127.
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