• Agarose Gel Electrophoresis
• Bacterial Streak Plate
• Bacterial Transformation
• DNA Ligation
• PCR
• Pipettors
• Plasmid DNA Isolation and Restriction Enzyme Digests
• Preparation of Agar Plates

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Bacterial Transformation

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.

Bacterial cells can also be transformed by electroporation. Electroporation involves exposing a suspension of cells and DNA to a high-voltage electric discharge, which creates pores in the cell membrane. These pores are large enough to allow DNA entry into the cell. Physiologically, the cell membrane, which acts as a capacitor, is unable to pass current except through ion channels; subjecting the membrane to a high-voltage electric field causes a temporary breakdown in membrane integrity, and the reclosing of the holes is a natural decay process. Transformation using this method is very efficient (several orders of magnitude greater than traditional chemical methods).

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 noncompatible "sticky" ends decreases background from vector recircularization.

References:
• Andreason, G.L. and G.A. Evans. (1989) Optimization of electroporation for transfection of mammalian cell lines. Anal. Biochem.180 (2): 269.
• 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.
• Knutson, J.C. and D. Yee. (1987) Electroporation: parameters affecting transfer of DNA into mammalian cells. Anal. Biochem.164 (1): 44.
• Potter, H., L. Weir, and P. Leder. (1984) Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc. Natl. Acad. Sci.81: 7161.
• Sugar, I.P. and E. Neumann. (1984) Stochastic model for electric field-induced membrane pores: Electroporation. Biophys. Chem.19 (3): 211.
• 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.

Preparation of Competent Cells

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 desired insert is likely to be present if the cells can grow on antibiotic plates.

Picking a single colony: a well-isolated colony on the selection plate is lightly touched with a sterile toothpick or a sterile pipet tip; after twirling the tip in a 15 ml culture tube containing liquid media plus antibiotic, tubes are incubated with shaking at 37°C overnight.  NOTE: in a “real” lab situation, you would likely save some of an overnight culture for streaking a plate and preparing a glycerol stock.

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.

Two strains we frequently work with in our Biochemistry and Cell Biology teaching labs are

• Tet sensitive (TetS) strain:
  • GNB8385K - MC4100 cad::Mu dl 1734 (Kmr lac) = kanamycin (Kan) resistant/tetracycline (Tet) sensitive
    (MC4100 - F- araD139 d(argF-lac)U169 rpsL150 relA1 flbB3501 deoC1 ptsF25 rbsR)
  
  
• Tet resistant (TetR) strain:
  • GNB824 - GNB8385K hns-24::Tn10 (Tcr) = Kan and Tet resistant

GNB8385K and GNB824 strains were developed in the laboratory of George N. Bennett, Ph.D., Rice University:
Tet sensitive (TetS) strain GNB8385K was derived from E. coli MC4100: Mu transposon containing beta-galactosidase and kanamycin resistance was inserted into the arginine decarboxylase (adi) or lysine decarboxylase (cad) genes (reporter gene is expressed when bacteria are grown in acidic media = white at neutral pH; red at acidic on MacConkey agar).

Tet resistant (TetR) strain GNB824 was derived from strain GNB8385K: Tn10(Tcr) randomly inserted and strains were screened for loss of regulation of the decarboxylase loci (reporter gene is expressed under both neutral and acidic conditions = red colonies on MacConkey agar).

Efficiency of transformation (EOT) = # colonies / per µg DNA

EOT is calculated by counting the number of colonies that grow on selective media following transformation and dividing by the total µg DNA used in the transformation. Dilutions must be calculated to determine the amount of DNA present in the volume of transformed culture placed on each plate.

We would like to thank New England Biolabs for their generous support of our laboratory program