Molecular Microbiology

Question 1

Sequencing microbes

Answer 1

• gene prediction

- genomic structure/mechanisms

Question 2

Bacteria

Answer 2

1 circular genetic molecule

plasmids

Question 3

A typical prokaryotic gene includes:

Answer 3

• promoter
• transcription start site
• 5’ untranslated region
• protein coding region
• 3’ untranslated region
• terminator sequence

Question 4

A typical eukaryotic gene:

Answer 4

introns and exons

Question 5

Transcription

Answer 5

• bacteria often have genes clustered together-operon
• these genes are transcribed all at once as a single mRNA
• termed ‘polycistronic mRNA’

-why? responds to environment allowing for metabolism of nutrients and replication

Question 6

Bacterial operon

Answer 6

• cluster of genes arranged in linear fashion with related function
• expression is under control of a single operator
• allows for expression of multiple genes to be coordinated

Question 7

Lac operon-induced gene expression

-negative control

Answer 7

• operator is located downstream of the promoter

- transcription is physically blocked when repressor binds to operator

Question 8

Plasmids:

Answer 8

• found in prokaryotes-additional genetic element
• replicate independently of the host chromosome
• double stranded DNA and circular
• variable abundance
• plasmids can be made artificially (genetic engineering)

Question 9

What info do plasmids carry?

Answer 9

• resistance plasmids

- cloning a gene

Question 10

How does a plasmid work?

Answer 10

-promoter, your gene, antibiotic, Ori –> cirular

Question 11

How can we clone a gene of interest?

STEP 1: DNA AMPLIFICATION

Answer 11

DNA–> PCR –> Agarose gel

Question 12

STEP 2: CLONING

Answer 12

DNA restriction enzymes

• evolved bacteria to protect against viral DNA infection
• enzymes recognise specific 4-8bp sequences
• cut with restriction enzyme
• add vector cut with same restriction enzyme
• add DNA ligase to form recombinant molecules

Question 13

STEP 3 : TRANSFORMATION

Answer 13

recombinant plasmid + E.coli host cell

=transformed cell

Question 14

Step 4: ANTIBIOTIC SELECTION

Answer 14

-transformed cell + non transformed cell –> Nutrient medium + ampicillin

overnight growth –> ampicillin-resistant colonies

Question 15

Why are plasmids useful in a lab?

Answer 15

• expression of proteins
• sequencing DNA
• Stability
• Transferring information (knock-ins and knock-outs)