Recombinant DNA Technology Recombinant DNA

Cloning – purify and propagate a piece of DNA 1. Cut the DNA (cut both plasmid and the
source of DNA
Application: if you want to study the gene use the
gene elsewhere *enzymes that could cut the DNA (DNAes)
*DNAes
-exonuclease – way of correcting the
reference by changing
-endonuclease
*Restriction endonuclease/ restriction
enzymes

*plasmids (extra chromosomal) *Palindromes (enzyme restriction)

-replicates independently 2. Mix plasmids & DNA and ligates

-some harbor genes that confer antibiotic

resistance

DNA Ligate
3. Transformation – culture cell
*you are trying to get the recombinant DNA

*can be done through heat shock

4. Screening
*put in a petri dish
RESULT:
*in the petri dish, choose which has
recombinant DNA
*SCREENING TECHNIQUES
a) Antibiotic Screening
b) Functional Compensation
• Auxotrophs – mutant (i.e
Arg auxotrophs – does not
produce Arginine)
(organisms fail to produce
essential nutrients)
• Prototrophs – able to
produce an essential
nutrients

c) Functional Complementation]
*control gene expression (turning
on/off gene)

*When do you want to turn on lac Z?

When lactose is present.
↓ 𝑔𝑙𝑢 ↑ 𝑙𝑎𝑐𝑡𝑜𝑠𝑒 – could remove
repressor by adding inducer
(allolactose)
→
𝑙𝑎𝑐𝑡𝑜𝑠𝑒 𝛽 − 𝑔𝑎𝑙𝑎𝑐𝑡𝑜𝑠𝑖𝑑𝑎𝑠𝑒 𝑔𝑙𝑢 + 𝑔𝑎𝑙

Blue-white screening

E-coli M15 string – has division in 11-41

(has la forming omega peptide)
Lac Z’ – plasmid lac Z’ codes α-peptide only