Bacteriology: Cyanobacteria and Transposons

Photosynthetic bacteria that have major roles in global carbon and nitrogen cycles.

They helped create Earth's oxygen-rich atmosphere.

Biofuels, optogenetics, and biofertilizers.

Nostoc punctiforme.

Specialized cells that fix nitrogen.

Spore-like, dormant cells.

Motile cyanobacterial filaments that glide across solid surfaces.

A short piece of DNA that can move, or "hop," from one DNA location to another.

It cuts and re-ligates DNA, allowing the transposon to insert into a target site.

A DNA sequence recognized by transposase that helps define the transposon's insertion region.

It allows selection of cells in which the transposon inserted into the chromosome.

Neomycin resistance; the slides also mention kanamycin resistance.

Random insertion can disrupt a gene; researchers identify mutants with an altered phenotype and sequence the insertion site.

Conjugation → neomycin selection → screen for hypermotility → extract chromosome → digest → ligate → inverse PCR → gel purification → sequencing.

To find colonies with the desired phenotype, especially hypermotility.

Insertion into the middle of a gene can disrupt its normal function.

HpaII.

CCGG.

About once every 256 base pairs, assuming equal base frequencies.

Each base has an assumed probability of 1/4, so $(1/4)^4 = 1/256$.

It cuts more frequently, producing smaller DNA fragments.

The appropriate buffer and 37°C incubation for 30 minutes.

Heating to 80°C irreversibly denatures HpaII and prevents it from cutting DNA during ligation.

T4 DNA ligase.

To form circular DNA molecules containing the transposon and adjacent chromosomal DNA.

Their sticky ends anneal; T4 DNA ligase then covalently joins the ends.

ATP supplied in the reaction buffer.

16°C overnight.

To amplify a specific DNA region using primers, nucleotides, buffer, and heat-stable DNA polymerase.

Template DNA, primers, dNTPs, buffer/cofactors including Mg²⁺, and Taq DNA polymerase.

At about 94°C, double-stranded DNA separates into single strands.

At about 55°C, primers bind to complementary template sequences.

At about 72°C, Taq polymerase adds nucleotides to the primers' 3′ ends.

35 cycles.

They face inward toward one another, flanking the DNA region to be amplified.

They face outward from a known DNA region, so amplification proceeds into the unknown flanking DNA.

The primers point outward rather than inward as in standard PCR.

Restriction-digested DNA fragments are circularized by ligation before inverse PCR.

Outward-facing primers bind within known transposon DNA and amplify across the circle into unknown adjacent chromosomal DNA.

Amplify the chromosomal DNA flanking the transposon insertion so the insertion site can be identified by sequencing.

To confirm that PCR products are present and are the expected size.

Agarose forms a porous molecular sieve; smaller DNA fragments move through it faster.

DNA has a negatively charged phosphate backbone.

It binds DNA and fluoresces under UV light, allowing DNA bands to be visualized.

It provides DNA fragments of known sizes to estimate the sizes of sample fragments.

Cut the desired DNA band out of the agarose gel.

It disrupts hydrogen bonding in the agarose, helping dissolve the gel.

To help melt and dissolve the agarose gel.

The solution is applied to a silica-containing column.

Under high-salt conditions, DNA binds to the silica.

To remove contaminants while the DNA remains bound to the silica.

By elution with a low-salt, high-pH solution.

It is sent for DNA sequencing and analyzed to identify the transposon insertion site.