• Day 1
• Day 2
• Day 3
• Day 4
• Day 5
• Day 6

Resources

• *LAB SAFETY**
• Laboratory notebook
• Graphing tutorial
• Molecular biol tips
• Writing assignment
• Bios 211 web site
• Searching literature

Other links

• Bios 111 honor code
• Graphing quiz (rice only)

Bios 111 Day 3

DNA Ligation and Bacterial Transformation

Introduction

Today you will conduct a DNA ligation, transform bacteria, and streak LB-antibiotic plates to obtain single colonies of transformants.

Background

DNA LIGATION

T4 DNA ligase is an enzyme encoded by the T4 bacteriophage that "ligates" DNA molecules by covalently joining a 3'-OH to an adjacent 5'-phosphate group. The joined ends may be from a single DNA molecule or from different molecules. Molecules with protruding single strand ends can be ligated together if the ends are compatible (i.e. complementary), so that they can anneal to each other. It is also possible to ligate any two blunt-ended DNA molecules together, although this is considerably less efficient since there is nothing to hold the DNA molecules next to each other. Ligations are used in our experiment to create stable recombinant DNA molecules for use in transformations.

BACTERIAL TRANSFORMATION

Foreign DNA can be placed in cells by several methods. If the foreign DNA is introduced into the cell in a form acceptable to the host, genes on that DNA can be expressed and the DNA can be propagated by the cells. In many cases this is done by attaching the foreign DNA to a piece of DNA that is capable of replicating within the host. For bacteria and some eukaryote species, plasmids or phages represent suitable vectors. Plasmids can carry only relatively small segments of DNA (<15 kb) but phage or cosmid vectors can carry up to 50 kb. Yeast artificial chromosomes (YAC) can be used to propagate very large foreign DNA fragments (>200 kb) in yeast. Phage or virus particles are available to transfer DNA into almost any type of cell. In other cases, foreign DNA can be introduced without any attached vector, and can sometimes integrate itself into the host chromosome where it is replicated as part of the host genome.

The overall process of changing the phenotype of a bacterium by introducing a plasmid into it is called transformation.  Bacteria may be transformed with plasmids by several techniques. The simplest is merely incubating the plasmid with bacteria whose cell wall has been weakened. Treating the bacteria with calcium or rubidium makes the membrane permeable to DNA through an unknown mechanism; these chemically treated cells are referred to as "competent" because they are now ready to take up foreign DNA.  Organic solvents (DMSO) and polyethylene glycol (PEG 8000) may be used in transformation procedures; these methods may have slightly lower efficiencies but are more rapid to perform. Less natural methods of placing DNA into cells are also used. DNA attached to microscopic particles can be physically "shot" into cells (Ballistic transformation) or soluble DNA can enter by blasting holes in the cell membrane by a high-voltage electric discharge (electroporation). The method of choice depends on the type of cell and the instrumentation available.

For every transformation, one or more controls should be performed:

• Positive Control -- transform competent cells with plasmid DNA (not digested); provides measure of the efficiency of transformation and serves as a standard for comparison with other transformations
• Negative Controls
• NO DNA -- transform competent cells, without added DNA, and plate on selection media; if high background, may indicate a problem with the antibiotic in the agar plates
• Digestion efficiency -- transform competent cells with digested plasmid DNA / NO ligase treatment; if high background, may indicate that the digestion did not go to completion
• Vector recircularization -- transform competent cells with digested vector DNA that was treated with alkaline phosphatase before adding ligase (NO insert is present); if high background, may indicate that the dephosphorylation reaction was incomplete

Agarose gel purification of digested plasmid DNA is one way to decrease background from inefficient digestion by restriction enzymes. Using restriction enzymes that generate "sticky" ends decreases background from vector recircularization

References

• Chung, C.T. and R.H. Miller. (1988) A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucl. Acid. Res. 16: 3580.
• Cohen, S.N., A.C.Y. Chang, and L. Hsu. (1972) Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coliby R-factor DNA. Proc. Natl. Acad. Sci., USA 69: 21102114.
• Cohen, S.N., A.C.Y. Chang, H.W. Boyer, and R.B. Helling. (1973) Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. USA 70: 3240-3244.
• Tsong, T.Y. (1991) electroporation of cell membranes. Biophys. J. 60: 297-306.
• Weaver, J.C. (1993) Electroporation: A general phenomenon for manipulating cells and tissues. J. Cell. Biochem. 51: 426-435.

Experimental Overview

• DNA ligation (pair)
• Bacterial transformation (pair)
• Evaluation of streak plate (individual)

SPECIAL NOTE:  Record enough procedure details in your notebook during lab today so that you can repeat these procedures using your notebook as the ONLY resource.  Write the methods in your own words (i.e., do not just “copy” the steps from the web page or handouts).

A) DNA ligation

PROTOCOL (Promega’s Liga-Fast™ Rapid DNA Ligation System)

1. Thaw Rapid Ligation Buffer, 2X RLB (60 mM Tris-HCl, pH 7.8; 20 mM MgCl2; 20 mM DTT; 2 mM ATP; 10% PEG), on ice; the ligase buffer contains ATP and Mg2+ necessary for the reaction
2. Thaw PstI-digested/gel purified DNA (from lab day 2) at room temp. and pulse spin to collect contents at bottom of tube
3. Add 4 µl of PstI-digested/gel purified plasmid DNA to a 1.5 ml tube
4. Add 5 µl of Rapid Ligation Buffer (2x RLB) followed by 1 µl of T4 DNA ligase (lig) (3u/µl)
5. Gently flick tube to mix and pulse spin samples
6. Incubate the reaction at room temperature for 5 min.
7. Pulse spin the ligation reaction and place on ice (in a Styrofoam container)

B) Bacterial transformation (Chemical transformation of E. coli)

We are going to transform bacteria with the ligation reaction. You will have one negative control transformation and one ligated DNA transformation. The cells we are using are Subcloning Efficiency™DH5a™ Chemically Competent E. coli (Invitrogen, Catalog no. 18265-017; for genotype, see manufacturer's specs). The particular bacterial strain used depends on not only the type of DNA in the transformation but also the kinds of experiments you want to perform. Bacteria with specific genes mutated, deleted, inserted, fused, etc. are commercially available; it is very important to maintain a complete description of the strains used in experiments and to confirm the phenotype.

1. Obtain two tubes of 50 µl competent cells (one for each transformation) and place tubes on ice
2. Label each transformation
• (-) DNA: NEGATIVE control (NO DNA is added!)
• (+) lig. DNA: ligation reaction
3. Add 10 µl ligation reaction directly into the cells in the tube labeled (+) lig. DNA; mix by tapping gently
4. Incubate on ice for 30 minutes
5. Heat-shock transformations in 42°C water bath for exactly 20 seconds
6. Place transformations on ice for 2 minutes
7. Add 950 µl of prewarmed sterile LB medium to each transformation
8. Incubate the sample at 37°C for 1 hour with shaking at 225 rpm (large incubator at front of lab)

 ATTENTION:  “Writing a Materials & Methods Section” and “Avoiding Plagiarism” lectures will be given during this incubation period

9. Label LB-Kan plates.             

NOTES: each student will “spread” at least one plate; dispose of used pipet tips and tubes containing transformed cells in the clear biohazard bag

10. Pipet 200 µl of the negative control transformation onto the center of a LB-Kan plate
11. Pour 10 - 20 sterile solid glass beads onto the plate and "shake" plate in a perpendicular motion; invert plate to pour off beads (collect in your hand and put in a 50 ml centrifuge tube -- these can be cleaned, autoclaved, and reused)
12. Pipet 200 µl from transformation with ligated DNA onto the center of a 2nd LB-Kan plate and spread as in step #13 (use “clean” beads for each plate)
13. Pipet 50 µl from the ligated DNA transformation onto the center of a 3rd LB-Kan plate; add 150 µl LB to the cells and spread as in step #13 (use “clean” beads)
14. Let the plates sit 5 minutes at room temperature so that the liquid absorbs into the agar
15. Incubate the plates upside down overnight at 37°C (the next day, the instructor will move the plates to 4°C for storage)

C) Evaulation of streak plate

Examine your “streak” plates and record your observations in your notebook.  Did you get single, well-isolated colonies?  If not, how might you modify your technique next time to obtain single colonies? 

 Homework Assignment

"Materials & Methods" Draft:  Use the guidelines on the course web site and from the lecture to write a draft of a "Materials & Methods" section for lab days 1-3. Bring a typed copy to lab day 4; please double space with 1 inch margins and use either Times or Times New Roman font (12 point).