Molecular Microbiology-2: Applications in research, diagnostics, Micro molecular lab expt

Question 1

Mutagenesis Studies

Answer 1

• discover if by mutating a gene, the protein function is disrupted and how?
• investigate active site in protein
• essential gene?
• make protein work in different way

Question 2

Plasmids

Answer 2

• plasmids can be made artificially-genetic engineering
• replicate independently of host chromosome
• double stranded DNA and circular

Question 3

Experiment mutagenesis of plasmid DNA containing ‘pink’ gene

Answer 3

Aims:

1. Transform plasmid DNA that has been mutated into E. coli
2. Examine colonies on LB plates containing antibiotic kanamycin - which colonies have been mutated?
3. Plate out single colonies from this plate

Question 4

Plasmid DNA

Answer 4

mCherry gene–> pink protein that makes bacterial colony pink

Question 5

E.coli mutator strain

Answer 5

• low mutation rate in microbes
• increased mutation rate due to mutation in DNA replication and repair systems
• example: Mutations in DnaQ gene-encodes subunit of DNA polymerase III-reduces proofreading capacity –> increased mutation
• use these bacterial mutator strains for random mutation of DNA- study function of gene products

Question 6

Study of mutagenised DNA with mCherry plasmid

Answer 6

E.coli mutator strain + mCherry plasmid
–>
mutated plasmids + wild type plasmids

Pink colonies = functional mCherry gene
White colonies = non-functional mCherry gene

Question 7

Transformation procedure:

Answer 7

CaCl2 -> E.coli + mutated plasmid vector

–> 42degrees –> 37degrees

Question 8

Selection using kanamycin

Answer 8

• kanamycin=is an aminoglycoside antibiotic, inhibits bacterial protein synthesis
• resistance gene inactivates kanamycin by modifying the structure
• only E.coli that contains plasmid with resistance gene will survive and grow into a colony

Question 9

Small subunit ribosomal RNA (SSUrRNA) gene analysis:

Answer 9

16S in prokaryotes

18S in eukaryotes

Question 10

PCR of 16S ribosomal RNA gene

Answer 10

• denaturation (95)
• primers stick to conserved regions
• Taq polymerase extends the sequence to make new template (72)
• 25-35 repeated cycles makes copies of original cDNA so there is enough to sequence

Question 11

LUCA

Answer 11

-last universal common ancestor

Question 12

Molecular diagnostics

Answer 12

-microbial identification
-what causes the disease and where did it come from
methods include:
-PCR
-real-time-quantitative PCR
-sequencing ribosomal RNA genes or species specific genes
-whole genome sequencing

Question 13

MRSA sequencing study

Answer 13

1/100 people carry MRSA on their skin without knowing

Question 14

Methods:

Answer 14

• cultures bacteria on selective medium
• antimicrobial susceptibility testing
• next generation sequencing (Illumina)
• bioinformatics : DNA sequence alignment alignment -SNPs

Question 15

MRSA sequencing study results

Answer 15

-contains genes for Panton-Valentine leukocidin-toxin causing necrotic lesions

Question 16

Molecular mechanisms of human infectivity of African trypanosomes

Answer 16

• what T.b gambiense genes are involved in human infectivity?
• what SNPs are present in field isolates of trypanosomes
• T.b.gambiense + T.b.rhodesiense = HAT (human infective)
• T.b.brucei = cannot survive human serum