Rekayasa Metabolisme - BT6111 Sekolah Ilmu dan

Teknologi Hayati
(SITH)

Level One Metabolic

Engineering: Introduksi Dr. Rizkita Rachmi
Esyanti

gen
rizkita@sith.itb.ac.id

@sithitb @sithitb sith.itb.ac.id School of Life Sciences and Technology

 Level Metabolite Engineering

Satu Dua Tiga

 Penggunaan TF yang  Manipulasi dari flux dengan

• Penambahan gen baru atau
modifikasi biosintesis untuk
menghasilkan senyawa baru
atau meningkatkan jumlah
metabolit target
mengendalikan jalur
metabolisme khusus, TF dapat
digunakan untuk menginduksi
aktivitas jalur khusus dengan
meningkatkan transkripsi gen
yang mengkode enzim
biosintesis tertentu
meningkatkan pasokan
substrat, energi, dan daya
reduksi. Dengan memasok
lebih banyak, maka jalur
biosintesis hilir dapat dipenuhi
dan hasil maksimum dapat
dicapai.

@sithitb @sithitb sith.itb.ac.id School of Life Sciences and Technology

 Metode dan contoh level one/satu

Rekayasa metabolik melibatkan modifikasi suatu reaksi, tetapi ini bagian dalam network
metabolisme atau sistem

@sithitb @sithitb sith.itb.ac.id School of Life Sciences and Technology

Contoh pada tumbuhan : produksi Artemisin, pada yeast ?

• New antimalarial drug

Artemisinin • Derivatives have selective toxicity
to human breast cancer

Limited Supply :
relative low yield (0,01-0,6%)
Artemisin in A annua

Enhancing the production

Chemosynthesis, Regulation artemisinin biosynthesis
conventional breeding
Manipulation culture condition
(growth media, and hormone) by genetic engineering
Not successful
 Transformation of A. annua
Plant Material and tissue and optimazation of
Culture condition transformation procedure

Regeneration of A. annua
plants and the sensitifity of DNA isolation & PCR
A. annua to kan analysis
Method ?
Southern blotting analysis
Contruction of plant
expression vector and
transformation of A.
tumefaciens
Flourescence analysis
 Transformation of A. annua and optimization of
transformation procedure
A. tumefaciens strain : LB, Kan 50
LBA4404, EHA105 harboring pBI GFP mg/l,
YEB, Kan 50 mg/l, Pick monoclon,
Cultivated on MS in dark,
Rifamicycin 50mg/l liquid medium,
2-3 days, then MS shoot
3 with 500mg/l Cefo incubate 28°C 36 hr
Precultur
Controls : 2 days in
CK1, infected leaves on dark
MS shoot3 free kan Dilute 1 ml to 50ml
CK2, uninfected leaves YEB or LB, incubate
MS shoot3
on MS shoot3 to OD600 ≈ 0,5
then MS
infection
root3 Centrifuge
4°C,4000 rpm,
Investigate transformation efficiency : Resuspended and 10’
MS cocultivation 1 for cocultivation medium incubate 3h, infection
& liquid MS cocultivation 2 Co-cultivation
to leaves

leaves float on liquid

MS + log phase
Agrobacterium, cultivated in
dark 2 days, leaf CK2 without 60 ml 40 ml 20 ml 60 ml
Agrobacterium suspension MS MS LB LB
 DNA isolation and PCR analysis
Isolated Plant genomic
DNA (Geng et. al) 852 amplification fragment

Primer :
Forward design to sequence 35S Electrophoresed on 0,8% agarose
promoter gel
Reverse same with the construct

Taq DNA polimerase

Staining with Etbr and

observed with UV

@sithitb @sithitb sith.itb.ac.id School of Life Sciences and Technology

(Muynck et al. 2010)
 Contoh pada MikrobaEngineering Yeast to Glow Green in UV to Make Glowing
Beer/Wine
82.632 x ditonton•6 Okt 2018

@sithitb @sithitb sith.itb.ac.id School of Life Sciences and Technology

Contoh pada
Hewan :
MILKING
GOATS FOR
DRUGS
(ANTIBODIES)

@sithitb @sithitb sith.itb.ac.id School of Life Sciences and Technology

Rekayasa Metabolisme - BT6111 Sekolah Ilmu dan
Teknologi Hayati
(SITH)

TERIMA KASIH

@sithitb @sithitb sith.itb.ac.id School of Life Sciences and Technology