Home

Character may be manifested in the great moments,
but it is made in the small ones.
Phillips Brooks

Background Topics

• DNA ligation
• Bacterial transformation

Experimental Procedures

• Analytical gel of digested PCR product
• Ligation with GUS vector
• Transformation

Overview of Procedures

You are ligating the PCR product of the 5'-regulatory region to the GUS vector. After ligation, chemically competent E. coli will be transformed with the reporter gene construct.

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.

Ligations require planning. Restriction fragments with protruding ends to be ligated must contain compatible complementary sequences. If orientation of an insert is important, two different ends will increase the probability of the correct orientation. The joining of a blunt end to a sticky end can be achieved by converting the sticky end to a blunt end, either by filling in the missing bases of a 5' protruding end using the Klenow fragment or by chewing off a 3' overhang with T4 DNA polymerase (the polymerase has a 3'-5' exonuclease activity used to correct misincorporation of nucleotides).

The components to be ligated are mixed in a ratio determined by the desired product. If recircularization (intramolecular ligation) is the goal, the concentration of fragments is kept low to decrease the probability of two different molecules contacting each other. If a product is to be inserted, such as in a cloning procedure, an excess of insert of 2 to 3 x the vector concentration is used, and the concentration of DNA is higher to increase the occurrences of intermolecular ligation. Treating the vector with alkaline phosphatase decreases recircularization by removing both of the 5' phosphates required for ligation, so that only a molecule with a 5' phosphate at each end (untreated fragment) will be inserted by ligation.

Introduction of Foreign DNA into Cells

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 (there are other processes that are also called transformation but we will not concern ourselves with these). 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. Special strains are also available with genetic alterations that limit the formation of the polysaccharide layer making the transformation of these cells 2 to 3 orders of magnitude more efficient. 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 and insert 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.

Growth and Check of Bacterial Strains

Bacteria can be propagated on liquid or solid media. The use of liquid allows large quantities of bacteria to be harvested but does not permit easy selection or determination of phenotype of single cells. The technique of "streaking" cells onto a solid media provides simple isolation of colonies arising from single cells. Colonies selected for the desired phenotype are then used to inoculate liquid broth. A single colony inoculum is preferred because bacteria can undergo many types of mutations naturally. The instability of some of the mutations, especially transposons and phages, can allow some cells to lose characteristics important to the selection scheme and may complicate the analysis. It is always wise to check the parent strain for proper phenotype that reflects the genotype and then use "picks" from single colonies to start liquid cultures.

The phenotype of drug resistance is fairly easily diagnosed. The GUS insert is present if the cells can grow on kanamycin (Km) plates.

Strain Descriptors

Strains are described by six indicators.

1. Individual genes - The listing of a genetic locus or operon functionality associated with the genome of the strain denotes a lack of the identified activity unless a "+" accompanies the description. Each locus is described by a three letter code and may contain more than one gene (e.g. lac indicates lactose utilization and encompasses an operator region and three catalytic enzymes). A capital letter following the code specifies the individual gene, as in the case of lacZ, which indicates that the mutation occurs in the b-galactosidase gene. The addition of a number specifies the allele involved. For emphasis of a wild-type gene or locus containing no mutation a superscript of "+" may be placed after the identification (e.g. lac+ denotes a fully functional lacoperon). An example of a complete strain description is: MC4100 - F- araD139 d(argF-lac)U169 rpsL150 relA1 flbB3501 deoC1 ptsF25 rbsR. Each mutant locus listed is not functional and the deficiency causes the phenotype described in the description list.
   Note: The following descriptions frequently cause confusion because they are NOT locus descriptions:
   Drug resistance is indicated by the presence of a two or three letter code and sometimes a superscript "r" for resistance and "s" for sensitive (e.g., kanamycin, Kmr)>
   F' [present and functional genes] - loci descriptions listed in square brackets after episome or plasmid designations are present and functional.
2. Deletions are indicated by the D symbol and the deleted genes or loci are given in parenthesis. As in the MC4100 description above, the deletion occurs from argF to the lacgene and the particular allele of this mutation is U169.
3. Fusions are denoted in a similar manner as deletions but the f symbol is used. A " ' " indicates the fusion product is an incomplete gene. The position of the mark preceding the code indicates a portion of the 5' end is missing and a post code mark indicates a 3' deletion.
4. Insertions are identified by what is inserted and where. A double colon, " :: ," separates the position of insertion from the insert. This is demonstrated in the description of GNB824 by the denotation hns-24::Tn10 (Tcr). The transposon, Tn10 containing tetracycline resistance gene, is located in the hnslocus. This particular insert is designated 24, but this number does not denote the position within the gene. It is an allele number to distinguish it from other insertions. If the insertion point is in an unknown gene the first letter is zfollowed by a two letter code giving the minute location on the chromosome (a-j for increments of 10 minutes followed by a-jfor minute interval, e.g., zfi=69 minutes).
5. Plasmids or lysogenic phages present in the strain are at the end of the genotype in brackets.
6. Fertility status is assumed to be F- unless indicated. F+ or Hfr strains are designated at the start of the description. F' status is listed at the end of the description and any functionalloci or genes contained on the F factor are given in square brackets.

Consult the photocopy provided of a description of specific genetic loci. It is very important to maintain a complete description of the strains used in experiments and to confirm the phenotype.

Experimental Procedures

Quantitation of PCR product

1. Prepare samples for analytical gel: add 3 µl digested/concentrated PCR product to 7 µl water; add 2 µl 6X loading buffer; load ALL of the sample.
2. Load 10 µl (0.5 µg) NEB Quick-Load 1 kb DNA ladder.
3. Run at 130 V for 20 minutes.  Refer to protocol for agarose gel electrophoresis.
4. Compare the size of the PCR product against the 1 kb ladder.
   Compare the intensity (e.g., 2X as bright) of the digest product to the 1 kb ladder to estimate the DNA concentration.
   

DNA ligation reaction

Chemical transformation of E. coli

In this lab, we use Z-Competent™ E. coli Transformation Kit & Buffer Set (Zymo Research Corp., Orange, CA) to transform bacteria.  [Z-Competent™ cells will be prepared by the instructor according to the manufacturer’s protocol.]

1. Pulse spin the ligation reaction and place on ice.
2. For each transformation, thaw 0.1 ml competent cells (prepared by instructor; see above) on ice
3. Add DNA and gently mix:
   • GUS construct transformation: add ALL of the ligation reaction directly into the cells
   • Positive control transformation: add 1 µl of undigested plasmid DNA (~100 ng)
   • Negative control transformation: use an untreated competent cell preparation (NO DNA added)
   
   You must be extremely gentle when working with competent cells. These cells are highly sensitive to temperature changes and/or mechanical lysis. Mix cells by gently tapping the tube or swirling with a pipet tip, not by pipetting up & down or vortexing.
   
   
4. Incubate samples on ice for 10 minutes.
5. Add 4 volumes of SOC medium (400 µl SOC to 100 µl transformation)
6. Incubate the samples at 37°C for 1 hour with shaking at 225 rpm
7. Pipet 100 µl of the negative control transformation onto the center of a LB-Kan plate (prewarmed to 37°C)
8. 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 a large beaker-- these can be cleaned, autoclaved, and reused)
9. Pipet 100 µl from the GUS transformation onto the center of a 2nd LB-Kan plate and spread as in step 8
10. Pellet the GUS transformation at 15,000 x g for 30 seconds; carefully remove most of the supernatant until there is ~ 50 µl left; gently resuspend the pellet and spread as in step 8 on a 3rd LB-Kan plate
11. Pipet 100 µl of the positive control transformation onto the center of a 4th LB-Kan plate and spread as in step 8
12. Let the plates sit 5 minutes at room temperature so that the liquid can be absorbed into the agar
13. Incubate the plates upside down overnight at 37°C
    NOTE: The next day, the instructor will move the plates to 4°C for storage

Growing Overnights

1. With your Sharpie, circle 3 well-isolated colonies on the plates.
2. Put 4 ml of LB-Kan in 3 sterile plastic tubes.
3. Gently swab a circled colony with a sterile pipet tip.
   Do not touch any other colonies.
4. Eject the tip into the appropriately labeled tube.
5. Slightly loosen the lids and put the tubes in the 37°C shaker overnight.
6. Store the plates at 4°C.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 23 July 2003
Updated 28 May 2010