Recombinant DNA Tech

Question 1

How can we engineer DNA basically?

Answer 1

All biochemically the same basis in life, so we can take human genes and put them in plasmids -> E. coli -> makes our insulin protein for us

Question 2

Where do we get the template?

Answer 2

• Genomic DNA (prok)
• cDNA (comp DNA from euk organisms)
• Gene synthesis

All can be amplified with PCR

Question 3

Multiple cloning site of plasmid

Answer 3

Where the gene of interest is inserted

• Tags (polyhistidine)
• Restriction enzyme cut sites

Question 4

What info does the multiple cloning site contain?

Answer 4

• T7 RNA pol
• mRNA shine-dalgarno
• Lac operator (control expression)
• Restriction enzyme cute sites
• Stop codon
• His tags

Question 5

Restriction Enzymes

Answer 5

Cleave phosphodiester backbone of DNA

- Creates sticky ends at the restriction sites, which are identified by H-bond acceptors/donors that stick out

Question 6

What is needed for PCR?

Answer 6

• DNA pol -> functions at high T
• Primers with restriction enzyme cute sites
• dNTPs
• Mg2+ (positions stuff)

Question 7

PCR Steps

Answer 7

1. Strand separation (95C for 15s)
2. Hybridization of primers (54C)
3. DNA synthesis (72C) by Taq DNA pol

Repeat steps 1-3 for ~30 cycles. After n cycles, sequence is amplified 2^n

Question 8

Lac Operator

Answer 8

• Controls transcription

- Inducer + ITPG -> bind to repressor and relives repression = transcription can occur

Question 9

When glucose is scarce, E. coli will metabolize

Answer 9

lactose

Question 10

Parts of Lac Operon

Answer 10

Repressor (i)
Promoter (p)
Operator (o)
z = beta-galactosidase
y
a

Question 11

When lactose is absent:

Answer 11

The inhibitor represses transcription of lac operon (z, y, a)
- Repressor is bound to o site

Question 12

When an inducer is present:

Answer 12

Inducer binds on repressor and it can’t bind on the operator, so transcription can now occur
Increased cAMP is pos regulation

Question 13

CRISPR/Cas System

Answer 13

• Specific target sequence: DNA-RNa hybrid

- Single guide RNA binds to nuclease protein (Cas9)

Question 14

Genome Editing: PAM

Answer 14

Protospacer-adjacent motif, short sequence recognized by Cas9

• Modifies target sequence of dsDNA gene
• REC lobe binds to NUC lobe = ds break in target sequence
• REC lob binds to nucleic acid and has sequence comp to gene of interest
• NUc lobe cleaves nucleic acid (nuclease)
• Both DNA strands are cleaved
• DNA repair pathways are activated
• An engineered DNA sequence can be used as a template for repair