Lab 8: Bacterial Transformation - Comprehensive Notes

Important Considerations for Transformation:
- Sterile conditions are essential.
  - Use only autoclaved plastic-ware.
  - Work near a flame to maintain sterility.
- Bacteria are sensitive to high calcium concentrations.
  - Keep bacteria on ice to maintain viability.

Bacterial transformation is a fundamental technique in molecular biology used to:
- Introduce foreign plasmids into bacterial cells.
- Amplify plasmids to produce large quantities.
It relies on the natural function of plasmids to transfer genetic information vital for bacterial survival.

A plasmid is a small, circular DNA molecule (2,000 to 10,000 base pairs).
Plasmids contain crucial genetic information for bacterial growth, often genes encoding antibiotic resistance.
Origin of Plasmids:
- Evolved as a result of bacteria living in close proximity to other heterotrophs like molds and fungi.
- Molds and fungi produce toxins to kill bacteria for food competition.
- Bacteria evolved proteins to inactivate these toxins, sharing this information via plasmids.
Discovery and Use:
- Plasmids were discovered in the late 1960s.
- Recognized for their potential to amplify genes of interest.

A plasmid containing antibiotic resistance (e.g., ampicillin or kanamycin) is used as a vector.
The gene of interest is inserted into the vector plasmid.
The modified plasmid is introduced into E. coli that is sensitive to ampicillin.
Bacteria are then grown on a plate containing ampicillin.
- Ampicillin acts as a selective pressure, allowing only bacteria with the plasmid to grow.
- This ensures the bacteria continually replicate the plasmid along with the inserted gene of interest.

Selectable Marker:
- A gene that confers antibiotic resistance.
Origin of Replication:
- Used by the bacterial DNA replication machinery as the starting point for plasmid replication.
Multiple Cloning Site (MCS):
- Contains multiple restriction enzyme sites for inserting DNA of interest.
- Commonly located within a reporter gene like Lac Z.

The Challenge: DNA is hydrophilic and cannot easily pass through the bacterial cell membrane.
Solution: Bacteria must be made "competent" to take up DNA.
Method:
- Create small holes in the bacterial cells by suspending them in a solution with a high concentration of calcium.
- Incubate the cells and DNA together on ice.
- Apply a brief heat shock at 42°C.
- Return the cells to ice.
- This process facilitates DNA uptake.
Cells are then plated on media containing antibiotics.

Most bacteria require manipulation to become competent.
E. coli cells are made competent by:
- Adding calcium chloride $(CaCl_2)$ . It is suggested that $Ca^{2+}$ neutralizes the negative charges on the phosphate backbone of DNA, reducing repulsion from the cell membrane's phospholipids.
- Heat-shocking (42°C for 30 seconds) to increase cell membrane permeability.
The procedure can be tricky, as bacteria with compromised membranes die easily.

Competency is determined by calculating the number of E. coli colonies produced per microgram ( $10^{-6}$ grams) of DNA added.
Expected Ranges:
- Excellent preparation: ~$10^8$ colonies per microgram.
- Poor preparation: $10^4$ colonies per microgram or less.
- Typical preparation: $10^5$ to $10^6$ colonies per microgram.
In this experiment, competent cells will be made.

Most bacterial cells will not receive a plasmid during transformation.
It is essential to distinguish between cells that contain the plasmid and those that do not.
The E. coli cells used do not possess a natural antibiotic resistance gene.
The plasmid contains a gene that confers resistance to ampicillin.
Selective Agent:
- Ampicillin is used as a selective agent.
- Only cells containing the plasmid can grow in its presence.

The vector used does not contain the lacZ gene.
The lacZ gene codes for beta-galactosidase.
Beta-galactosidase metabolizes lactose into galactose and glucose.
X-Gal is a colorless sugar metabolized by beta-galactosidase, producing galactose and a blue product.
Functionality Test:
- X-Gal indicates whether the lacZ gene is functional.
- If a DNA insert disrupts the lacZ gene, beta-galactosidase becomes non-functional.
- Cells with an insert in their plasmid will be white because they cannot digest X-Gal.
- Cells without an insert will be blue due to functional beta-galactosidase.

Gene expression is regulated to adapt to different conditions and prevent overproduction of unneeded proteins.
Genes for breaking down food sources are highly regulated.
Example: Arabinose
- Arabinose, a plant sugar, serves as an energy and carbon source for bacteria.
- Bacterial genes for arabinose digestion are only expressed when arabinose is present.
- Arabinose initiates transcription by promoting RNA polymerase binding.
pGLO Plasmid:
- Genes involved in arabinose breakdown are replaced by the jellyfish gene coding for GFP (Green Fluorescent Protein).
- Bacteria transformed with pGLO and grown in arabinose will express GFP and glow green under UV light.
Central Molecular Framework:
- Demonstrates DNA➔RNA➔PROTEIN➔TRAIT.
- In the absence of arabinose, the GFP gene remains off, and colonies appear white.

Avoid contamination of bacteria or plates with external bacteria.
Plates without antibiotics are especially prone to contamination.
Precautions:
- Wear gloves.
- Keep lids on plates when not in use.
- Use sterilized solutions and plastics.

Although the bacteria used are not hazardous, avoid contaminating clothes or surfaces.
Disposal:
- Dispose of items that have contacted bacteria in biohazard bags or containers for sterilization.
- Wipe down the counter with alcohol after working with bacteria.
- Wash hands with soap before leaving the lab.

Agar: A gelatin-like substance from seaweed.
Luria Bertani broth (LB broth): Contains nutrients for bacterial growth.
Kanamycin: An antibiotic used as a selective agent.
- The plasmid contains a kanamycin-resistance gene so only transformed E. coli will grow on plates with kanamycin.
- Selection is needed to isolate the small percentage of cells that take up the plasmid.

Why use an antibiotic like ampicillin on plates? What differences are expected between plates with and without ampicillin?
What are satellite colonies? Are there any satellite colonies on your plates? Record the satellite colonies' morphology (size, shape, and color) compared with the transformed colonies and reasons for any difference.

Ampicillin is used as a selective agent: only cells containing the plasmid, which has the ampicillin resistance gene, can grow in its presence. On plates with ampicillin, only transformed bacteria (those with the plasmid) will grow, while on plates without ampicillin, both transformed and non-transformed bacteria can grow. The non-transformed bacteria will form a lawn if there are enough of them.
Satellite colonies are small colonies that sometimes appear around larger, transformed colonies on antibiotic-containing plates. They occur because the ampicillin is degraded by the enzyme beta-lactamase, which is produced by the transformed bacteria. The area around the transformed