Edvotek Informational Video: Calculating Transformation Efficiency

Main Ideas

• Bacterial transformation occurs when a bacterium takes up free DNA from its surroundings. Some bacteria, such as Haemophilus influenzae, can do this naturally; E. coli can be made competent in the laboratory.
• Conjugation is different: DNA passes from a donor bacterium to a recipient through direct cell-to-cell contact and a bridge-like connection.
• In either process, acquired DNA can be stable and inherited by later generations. Although often not essential for survival, it may provide an advantage, such as ampicillin resistance from a gene encoding beta-lactamase.
• Bacteria often exchange extra genes on plasmids: small, circular, double-stranded DNA molecules that replicate independently of the bacterial chromosome.
• Engineered plasmids can give bacteria useful abilities. Transformed bacteria can act as “living factories” for products such as insulin, vitamins, and medications. A classroom example is giving E. coli a jellyfish gene so the cells glow green.

Laboratory Heat-Shock Transformation

Because E. coli is not naturally competent, laboratory methods are used to help it take up plasmid DNA. The video mentions electroporation, which uses electricity, and heat shock, which uses a rapid temperature change.

The heat-shock procedure described is:

1. Treat the cells with calcium chloride to make them competent.
2. Add plasmid DNA to the cells.
3. Heat shock the cells by moving them quickly between very different temperatures. The video says the calcium chloride and temperature change are believed to alter the permeability of the cell wall and membrane, allowing DNA to enter.
4. Add rich recovery medium and allow the cells to grow for 10–30 minutes. During recovery, cells repair their walls and membranes, copy the plasmid, and begin expressing its antibiotic-resistance gene.
5. Plate the cells on selective medium and allow them to grow overnight.
6. Count the resulting colonies. Each colony is treated as originating from one transformed cell. Marking counted colonies on the back of the plate can help prevent missed or double-counted colonies.

Calculating Transformation Efficiency

Transformation efficiency measures the number of transformed cells obtained per microgram of plasmid DNA.

Collect these four measurements:

• Plasmid DNA used, in micrograms. The video gives 10 nanograms, or 0.01 microgram, as a common amount.
• Total recovery volume: the competent-cell volume plus the recovery-broth volume. For example, 100 microliters of cells plus 250 microliters of broth gives 350 microliters (0.35 milliliters).
• Volume plated, since typically only part of the recovered suspension is placed on the plate. The video says researchers commonly plate 100–250 microliters.
• Number of colonies on the plate.

Because only part of the recovered cells may be plated, first estimate the total number of transformants in the recovery mixture:

Total transformants = colonies counted × (total recovery volume ÷ volume plated)

Then calculate:

Transformation efficiency = total transformants ÷ plasmid DNA used (in micrograms)

Example from the Video

• Plasmid used: 0.01 microgram
• Total mixture volume: 1 milliliter
• Volume plated: 0.1 milliliter
• Colonies counted: 100

The plated sample is one-tenth of the mixture, so the full mixture is estimated to contain 1,000 transformants. Dividing 1,000 by 0.01 microgram gives a transformation efficiency of 1 × 10⁵ transformed cells per microgram of plasmid DNA.

The video notes that transformation efficiency is generally around 1 × 10⁴ to 1 × 10⁸ cells per microgram. It suggests testing how heat-shock temperature or duration, or the amount of DNA, affects the result.

Speakers or Sources Featured

• Edvotek informational-video narrator — the only speaker identifiable from the subtitles; no individual narrator is named.
• The video mentions E. coli, Haemophilus influenzae, jellyfish, and the beta-lactamase/ampicillin-resistance example as scientific illustrations, not as speakers.