Cyanobacteria and Transposon Mutagenesis
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1
What are cyanobacteria?
Photosynthetic bacteria that produce oxygen.
2
Why are cyanobacteria important to study?
They help us understand photosynthesis, evolution, and environmental processes.
3
What model cyanobacterium are we using?
Anabaena (a filamentous cyanobacterium).
4
What is a vegetative cell?
A normal photosynthetic cell that grows and divides.
5
What is a heterocyst?
A specialized cell that fixes nitrogen.
6
When do heterocysts form?
When nitrogen is limited.
7
What is an akinete?
A thick-walled resting cell that survives harsh conditions.
8
What is a transposon?
A mobile DNA segment that can move to different locations in DNA.
9
What happens when a transposon inserts into a gene?
It can disrupt the gene and prevent it from working.
10
Why does our transposon contain an antibiotic resistance gene?
To identify and select bacteria that received the transposon.
11
What does antibiotic selection do?
Kills bacteria without resistance, leaving transposon-containing bacteria.
12
What is transposon mutagenesis?
Randomly inserting transposons to disrupt genes and study their functions.
13
What is the first step of transposon mutagenesis?
Introduce the transposon into bacterial cells.
14
What happens after transposon insertion?
Select bacteria using the antibiotic resistance marker.
15
How do we identify the disrupted gene?
Observe the mutant phenotype and identify where the transposon inserted.
16
What is a mutant phenotype?
An observable change caused by a disrupted gene.
17
How does transposon mutagenesis identify gene function?
Connect a disrupted gene to the biological function that changes.
18
What is chromosomal DNA extraction?
Isolating bacterial genomic DNA from the cell.
19
What is the first step of chromosomal DNA extraction?
Collect bacterial cells.
20
What does lysozyme do during DNA extraction?
Breaks down the bacterial cell wall.
21
What does proteinase K do?
Digests proteins, including DNA-associated proteins.
22
Why must bacterial cells be lysed?
To release their DNA.
23
Why are proteins removed during DNA extraction?
To prevent protein contamination of the DNA.
24
Why is DNA separated from cell debris?
To obtain a purified DNA sample.
25
What is the purpose of DNA washing?
Remove leftover salts and contaminants.
26
What is the final step of DNA extraction?
Elute or resuspend purified DNA in buffer or water.
27
How do we measure DNA concentration?
Using a spectrophotometer to measure absorbance at 260 nm.
28
What does A260 measure?
DNA and other nucleic acid absorbance.
29
How do we assess DNA purity?
Compare absorbance ratios at 260 nm and 280 nm.
30
What is the ideal A260/A280 ratio for pure DNA?
Approximately 1.8.
31
What does a low A260/A280 ratio suggest?
Protein contamination.
32
Why can't transposon mutagenesis study essential genes?
Disrupting an essential gene kills the cell, so the mutant cannot survive.
33
What are restriction enzymes?
Enzymes that cut DNA at specific recognition sequences.
34
What is a restriction site?
A specific DNA sequence recognized and cut by a restriction enzyme.
35
What is a restriction digest?
Using restriction enzymes to cut DNA into fragments.
36
What is a palindromic DNA sequence?
A sequence that reads the same 5' to 3' on both DNA strands.
37
How do you calculate restriction site frequency?
1 / 4^n, where n is the recognition sequence length.
38
How often does a 4-base restriction site occur?
About once every 256 base pairs (4^4).
39
How often does a 6-base restriction site occur?
About once every 4,096 base pairs (4^6).
40
What happens when a restriction enzyme cuts DNA?
It produces smaller DNA fragments with sticky or blunt ends.
41
What are sticky ends?
DNA ends with overhanging bases that can pair with complementary ends.
42
What are blunt ends?
DNA ends cut straight across with no overhangs.
43
Why do we heat-inactivate restriction enzymes?
To stop further DNA cutting before the ligation reaction.
44
Why must restriction enzymes be inactive before ligation?
Otherwise, they could cut the newly joined DNA again.
45
What is DNA ligation?
Joining DNA fragments together by forming bonds in the DNA backbone.
46
What enzyme performs DNA ligation?
T4 DNA ligase.
47
What does T4 DNA ligase do?
Seals breaks in the DNA sugar-phosphate backbone.
48
What type of bond does DNA ligase form?
Phosphodiester bonds.
49
What is DNA circularization?
Joining the two ends of a DNA fragment to form a circle.
50
Why must DNA fragments be circularized?
To create a circular DNA template for inverse PCR.
51
What is inverse PCR?
PCR used to amplify DNA sequences surrounding a known region.
52
Why is circular DNA needed for inverse PCR?
It allows primers facing outward to amplify across the unknown flanking DNA.
53
What happens if DNA fragments are not circularized?
Outward-facing primers cannot amplify across the DNA ends as intended.
54
What is the overall purpose of restriction digestion and ligation?
Prepare circular DNA fragments for inverse PCR.
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