Recombinant DNA Tech Flashcards
How can we engineer DNA basically?
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
Where do we get the template?
- Genomic DNA (prok)
- cDNA (comp DNA from euk organisms)
- Gene synthesis
All can be amplified with PCR
Multiple cloning site of plasmid
Where the gene of interest is inserted
- Tags (polyhistidine)
- Restriction enzyme cut sites
What info does the multiple cloning site contain?
- T7 RNA pol
- mRNA shine-dalgarno
- Lac operator (control expression)
- Restriction enzyme cute sites
- Stop codon
- His tags
Restriction Enzymes
Cleave phosphodiester backbone of DNA
- Creates sticky ends at the restriction sites, which are identified by H-bond acceptors/donors that stick out
What is needed for PCR?
- DNA pol -> functions at high T
- Primers with restriction enzyme cute sites
- dNTPs
- Mg2+ (positions stuff)
PCR Steps
- Strand separation (95C for 15s)
- Hybridization of primers (54C)
- DNA synthesis (72C) by Taq DNA pol
Repeat steps 1-3 for ~30 cycles. After n cycles, sequence is amplified 2^n
Lac Operator
- Controls transcription
- Inducer + ITPG -> bind to repressor and relives repression = transcription can occur
When glucose is scarce, E. coli will metabolize
lactose
Parts of Lac Operon
Repressor (i) Promoter (p) Operator (o) z = beta-galactosidase y a
When lactose is absent:
The inhibitor represses transcription of lac operon (z, y, a)
- Repressor is bound to o site
When an inducer is present:
Inducer binds on repressor and it can’t bind on the operator, so transcription can now occur
Increased cAMP is pos regulation
CRISPR/Cas System
- Specific target sequence: DNA-RNa hybrid
- Single guide RNA binds to nuclease protein (Cas9)
Genome Editing: PAM
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