Bacterial transformation

i will pass

What are the steps of bacterial transformation?

💛 Amplify gene by PCR →

💚 Create recombinant plasmid (with antibiotic resistance and reporter gene)

💜 Make bacteria competent with calcium chloride →

❤️ Use heat shock to transform bacteria →

🩵 Select successful transformants using antibiotics and reporter genes →

💙 Extract protein product for use.

What is a plasmid?

A small, circular, extrachromosomal DNA molecule naturally occurring in bacteria.

What do plasmids typically contain?

Antibiotic resistance genes, promoters, multiple restriction enzyme sites, and sometimes operons (if multiple genes must be expressed together).

What do antibiotic resistance genes on plasmids allow?

They allow selection of transformed bacteria by killing non-transformants with antibiotics.

Why is the small size of plasmids significant?

It makes them easier to isolate, cut with restriction enzymes, and reinsert into bacteria. Also, DNA is universal, so plasmids can express eukaryotic genes in bacteria.

Step 1 of bacterial transformation (gene amplification)

Amplify the gene of interest using PCR. Primers are designed to attach to the 5’ ends of the target gene.

Step 2 of bacterial transformation (creating recombinant plasmid)

Cut both the gene of interest and plasmid with the same restriction enzyme. Sticky ends anneal, and DNA ligase seals the sugar-phosphate backbone.

What occurs in bacterial transformation (steps 3 and 4)?

Competent bacteria uptake the recombinant plasmid via transformation (usually with calcium chloride and heat shock), then express and replicate the gene product via binary fission.

What does transformation mean?

Uptake of foreign DNA (often a plasmid) by a bacterial cell, enabling it to express the genes on that DNA.

Why do sticky ends improve the transformation process?

They improve efficiency by allowing complementary base pairing between the plasmid and gene insert.

How are competent bacteria created for (step 3 in bacterial) transformation?

calcium chloride binds to the membrane, which become positively charged (+ion).

- plasmids are negatively charged (because of DNA's backbone) so they're attracted to the positive charge and move and stick to the membrane of the bacteria

How are successful transformants selected and screened?

Antibiotics are added to agar plates. Only bacteria that took up plasmids with antibiotic resistance survive. Reporter genes can also be used to confirm gene expression.

Does having the plasmid mean the gene is expressed?

Not necessarily. A reporter gene is used to confirm gene expression by producing a visible change (like colour or fluorescence).

What do white colonies indicate in blue-white screening?

The gene of interest has interrupted the beta-galactosidase gene — indicating successful insertion and expression.

What do blue colonies indicate in blue-white screening?

The gene of interest is placed next to the beta-galactosidase gene as a fusion protein — indicating the gene was not inserted or not interrupting the original function.