TEKNOLOGI DNA

REKOMBINAN
TDR
joining together ofDNAmolecules from two
differentspeciesthat are inserted into a host
organism to produce new genetic combinations
that are of value toscience, medicine, agriculture,
and industry. Since the focus of allgeneticsis the
gene, the fundamental goal of laboratory
geneticists is to isolate, characterize, and
manipulate genes
Sumber Gen
Sel eukariot, adanya intron, maka yang diambil adalah
mRNA
mRNA melalui PCR reverse transcriptase akan
didapatkan cDNA
Sel prokariot, apabila DNA rekombinan ditransformasi ke
sel prokariot juga maka dapat digunakan DNA nya
Gen yang diamplifikasi dengan menggunakan Taq DNA
Polimerase maka pada ujungnya akan ketambahan ujung
A (adenin)
Pemilihan Sel Inang

Karakter Protein Rekombinan

- Glikosilasi berperan pada aktivitas protein maka gunakan
host eukariot seperti Pichia Pastoris
- Perhatikan over glikosilasi
Vektor (Kloning dan Ekspresi)
Vektor Kloning (digunakan untuk memperbanyak DNA)
Komponen transkripsi (Promotor, terminator, RNA
Polimerase)
Transformasi
Transformasi
White-Blue screening X-gal
Restriction Enzyme
Expression Vector (pET 32b)
Syarat vektor ekspresi
Promotor Kuat : T7 promotor
Terminator Kuat : T7 terminator
rbs
Lac operon

The lac operon (lactose operon) is an operon required for the

transport and metabolism of lactose in Escherichia coli and many
other enteric bacteria. Although glucose is the preferred carbon
source for most bacteria, the lac operon allows for the effective
digestion of lactose when glucose is not available. Gene regulation
of the lac operon was the first genetic regulatory mechanism to be
understood clearly, so it has become a foremost example of
prokaryotic gene regulation. It is often discussed in introductory
molecular and cellular biology classes for this reason.
IPTG Induction
Isopropyl -D-1-thiogalactopyranoside (IPTG) is a
molecular biology reagent. This compound is a molecular
mimic of allolactose, a lactose metabolite that triggers
transcription of the lac operon, and it is therefore used to
induce protein expression where the gene is under the
control of the lac operator.