Bacterial Transformation: A Molecular Biology Technique

• Origin of Replication (ori): A specific DNA sequence that allows the plasmid to be copied by the host cell's machinery during replication.
• Multiple Cloning Site (MCS): A region containing several unique restriction sites that provides a specific location where a gene of interest can be inserted.
• Selectable Markers: These are essential for identifying which bacteria actually took up the plasmid.

Selection via Antibiotic Resistance

A common selectable marker is the gene for $\beta$-lactamase. This enzyme provides ampicillin resistance by destroying the structure of the antibiotic ampicillin. Since ampicillin normally prevents bacteria from synthesizing cell walls (thereby stopping replication), only the bacteria that have successfully taken up the plasmid containing the $\beta$-lactamase gene will be able to grow on agar plates containing ampicillin.

In many experiments, a reporter gene like Green Fluorescent Protein (GFP) is also included. This allows researchers to visually confirm the presence of the plasmid; when viewed under UV light, the colonies containing the GFP gene will glow green.

The Transformation Process

Most E. coli are not "naturally competent," meaning they cannot take up DNA from their environment on their own. To make them take up DNA, we must create chemically competent bacteria.

This is achieved through a process called heat-shock. The bacteria are treated with specific chemicals that modify the cell membrane. When the cells are suddenly moved from a cold environment to a warm one (typically $42^\circ\text{C}$) and then immediately back to ice, the temperature shift causes pores to open in the bacterial membrane. The foreign plasmid DNA enters the cell through these temporary pores, and the cells are then considered "transformed".

Calculating Transformation Efficiency

Not every cell will take up a plasmid. Because the process is not 100% efficient, we must calculate the transformation efficiency to determine how successful our procedure was. Efficiency is expressed as the number of transformants (colonies) produced per microgram ($\mu\text{g}$) of plasmid DNA used.

The Formula

To calculate efficiency, use the following equation:

$$\text{Transformation Efficiency} = \frac{\text{Number of Colonies (CFU)}}{\text{Amount of DNA added ($\mu\text{g}$)}}$$

Note: If you only plated a fraction of your total volume, you must multiply the result to account for the full volume.

[!TIP] Exam Tip: Counting Colonies When counting colonies on a plate, if the number exceeds 1,000, it is too crowded to count accurately. If there are between 200 and 1,000 colonies, divide the plate into sections (1/2, 1/4, or 1/8), count one section, and multiply by the corresponding factor to estimate the total Colony Forming Units (CFU).

Worked Example

Suppose you add $0.01\ \mu\text{g}$ of plasmid DNA to $1\text{ ml}$ of competent cells. You perform the transformation and plate $100\ \mu\text{l}$ of that total volume onto an ampicillin plate. After incubation, you count $120$ colonies.

Step 1: Calculate the initial efficiency based on the plate.

$$\begin{aligned} \text{Efficiency (plated)} &= \frac{120}{0.01} \\ &= 12,000 \text{ or } 1.2 \times 10^4 \text{ CFU/}\mu\text{g} \end{aligned}$$

Step 2: Account for the dilution factor. Since you only plated $100\ \mu\text{l}$ out of the total $1\text{ ml}$ ($1,000\ \mu\text{l}$), you plated $1/10$ of the culture. You must multiply your result by $10$ to find the efficiency of the entire volume:

$$\begin{aligned} \text{Total Efficiency} &= 1.2 \times 10^4 \cdot 10 \\ &= 1.2 \times 10^5 \text{ CFU/}\mu\text{g} \end{aligned}$$

Typical laboratory efficiencies range from $1 \times 10^4$ to $1 \times 10^8$ transformants per $\mu\text{g}$ of DNA.