Kelompok 1

Sany Yulianati 11161051

Sofi Rifayani
Tysa Nurissalamah
11161053
11161056
TEKNIK DNA
REKOMBINAN
Andrey Prayudiansyah 11161067
Delia Nurfadillah 11161075
Fitriani Nurfajrin 11161082
Jajang Wiguna 111610
Mira Eka Putri 11161095
Pina Patonah P Elpana 11161100

3 FA 2
BIOTEKNOLOGI FARMASI
Out line
Pengertian Plasmid Sebagai Vektor
01 Dalam Rekayasa Genetik

Syarat Digunakan Plasmid Sebagai

02 Vektor

Penyisipan DNA Rekombinana

03 Pada Plasmid

Proses ligasi
04
Pengertian Plasmid
Sebagai Vektor dalam
Rekayasa Genetika
o Vektor adalah molekul DNA yang
berfungsi sebagai wahana atau
kendaraan yang akan membawa suatu
fragmen DNA masuk ke dalam sel
inang dan memungkinkan terjadinya
replikasi dan ekspresi fragmen DNA
asing tersebut.
o Vektor yang dapat digunakan pada sel
inang prokariot, khususnya E. coli,
adalah plasmid, bakteriofag, kosmid,
dan fasmid.
o Salah satu vektor yang bisa digunakan untuk
membawa molekul DNA asing masuk dalam sel
inang adalah plasmid.

o Plasmid digunakan untuk melakukan rekayasa pada

berbagai organisme yang tidak bisa diperoleh secara
alami. Rekayasa ini dilakukan pada tingkat genetik
sehingga disebut sebagai rekayasa genetika.
 Ciri Plasmid
Berbentuk lingkaran tertutup dan untaiannya
ganda (double stranded)
Plasmid terdapat
pada organisme
prokariot maupun Dapat melakukan replikasi sendiri di
eukariot. Plasmid luar kromosom inti
inilah yang
berfungsi sebagai
pembawa sifat Terdapat di luar kromosom
rekombinan pada
organisme yang
akan direkayasa.

Secara genetik dapat ditransfer secara stabil

Syarat plasmid menjadi vektor

Mempunyai sekurang-
kurangnya 2 gen marker
yang dapat menandai
masuk tidaknya plasmid
ke dalam sel inang.
Ukurannya relatif kecil
dibanding dengan pori
dinding sel inangnya.

Memiliki titik awal

replikasi sehingga dapat
melakukan replikasi
dalam sel inang.
Mempunyai tempat
pengenalan restriksi
sekurang-kurangnya di dalam
salah satu marker yang dapat
digunakan sebagai tempat
penyisipan fragmen DNA
asing
Plasmidd menurut tujuan pengunaan

kloning eukariot kloning prokariot

plasmid pUC 19 dan plasmid Ti

pBR 322

PLASMID
Gambar plasmid pBR
Plasmid pBR322 mengandung gen
322 dengan tempat
penyandi resistensi terhadap ampisilin
pengenalan pemotongan
dan tetrasiklin. Adanya gen resistensi
DNAnya
terhadap antibiotik yang di dalamnya
mengandung situs enzim restriksi yang
akan memudahan dalam menyeleksi
plasmid rekombinan, dengan adanya
enzim yang hanya memotong bagian
gen yang resisten terhadap antibiotik.
Misalnya, enzim BamHI yang akan
memotong plasmid pBR322 pada
bagian gen yang resistensi terhadap
tetrasiklin (gen TetR). Proses
pemotongan plasmid bukanlah sesuatu
yang mudah.
Gambar 1

Ada dua kemungkinan setelah pemotongan, yaitu:

a. Plasmid pBR322 yang tersambung kembali membentuk
lingkaran seperti semula, dan
b. Plasmid rekombinan yaitu plasmid pBR322 yang telah
disisipi dengan DNA asing.
Proses penyisipan dna recombinan

Plasmid pBR322 yang telah disisipi dengan fragmen dari Plasmid pBR322 ini kemudian
kehilangan bagian yang DNA asing dimasukkan ke dalam sel
resistensi terhadap tetrasiklin inangnya yaitu E. coli.

Koloni E. coli yang telah Pengujian lebih lanjut apakah plasmid ini
sedangkan pada telah tersisipi oleh fragmen DNA asing
kehilangan marker tetrasiklin
medium ampisilin, atau tidak adalah dengan
akan sensitif tetrasiklin,
koloni ini tetap tumbuh menbedahkannya pada medium
karena masih resistensi ampisilin, untuk kemudian
ampisilin. memplatingnya ke dalam medium
tetrasiklin dan ampisilin
Gambar plasmid pUC 19 dan tempat
pengenalan pemotongan DNAnya dapat dilihat
pada gambar2.

Plasmid pUC118 dan pUC119 adalah plasmid lain yang telah lama
digunakan sebagai vektor untuk mengklon gen. Plasmid
tersebut merupakan pengembangan dari pBR322. Plasmid pUC118
dan pUC119 mengandung gen lacZ yang menyandikan enzim β-
galaktosidase. Pada lacZ terdapat daerah yang disebut daerah
polikloning. Pada daerah ini terdapat banyak situs restriksi dari
berbagai enzim restriksi. Dalam hal ini, kita dapat menggunakan
berbagai enzim restriksi untuk memotong pUC118 dan pUC119 pada
bagian lacZ. Bila gen lacZ disisipi oleh DNA asing maka gen lacZ
tersebut tidak berfungsi (tidak menghasilkan β-galaktosidase). Bila
kita menggunakan pUC118 atau pUC119 sebagai vektor, maka
koloni yang membawa plasmid rekombinan dapat dideteksi dengan
menggunakan Xgal (5-bromo-4-chloro-indolyl-β-D-galactosida).
Enzim β-galaktosidase akan memecah Xgal menjadi galaktosa dan
5-bromo-4-chloroindigo yang berwarna biru. Koloni bakteri akan
berwarna putih bila pUC118 atau pUC119 telah disisipi DNA asing
pada bagian lacZ. Dalam hal ini sel bakteri tidak menghasilkan
enzim β-galaktosidase karena gen lacZ. Gen lacZ tidak berfungsi
karena disisipi oleh DNA asing.
Proses penggunaan plasmid di dalam rekayasa genetika
Penggunaan plasmid untuk rekayasa genetika harus disesuaikan kebutuhannya sebab ada berbagai
macam plasmid yang digunakan. Proses penggunaan plasmid dalam rekayasa genetika melalui langkah-
langkah sebagai berikut :

Penentuan terlebih dahulu jenis
plasmid yang hendak digunakan sebab
ada beberapa jenis plasmid seperti
pBR 322 dan pUC19 yang bisa
digunakan untuk prokariot dan plasmid
Ti yang bisa digunakan untuk
organisme eukariot
.
Bila plasmid telah ditentukan maka selanjutnya adalah
Contents
menentukan tempat pengenalan enzim restriksi
Contents (pemotongan) yang hendak digunakan sebagai tempat
penyisipan DNA asing dan marker untuk menandai masuk
tidaknya plasmid pada sel inang

Apabila telah diketahui tempat pengenalan restriksi dan Langkah selanjutnya adalah plasmid
Contents
markernya maka langkah selanjutnya adalah menyiapkan Contents dipotong dengan enzim restriksi yang
enzim restriksi sebagai pemotong plasmid. Enzim yang sesuai pada daerah potongannya
digunakan untuk memotong plasmid harus sama dengan
pemotong DNA asing sehingga nanti keduanya bisa bersatu
misal : EcoR1
.

Plasmid siap disambungkan dengan

DNA asing yang memiliki sifat tertentu, Contents
yang telah dipotong juga dengan enzim
restriksi yang sama dengan pemotong
plasmid.
 Enzim restriksi sebagai pemotong
plasmid
Untuk memotong plasmid digunakan
enzim restriksi. Enzim restriksi adalah
enzim yang digunakan untuk
memotong DNA secara spesifik. Prinsip kerja enzim restriksi adalah
Enzim restriksi disebut sebagai
gunting biologi. Enzim ini diisolasi
dari bakteri. Restriksi yang digunakan
untuk memotong plasmid harus sama
dengan pemotong DNA asing agar
urutan basanya bisa sesuai sehingga
antara plasmid dan DNA asing yang
disisipkan bisa bersatu.
Prinsip kerja enzim restriksi adalah:
Proses pemotongan DNA
Enzim Ligase sebagai penyembung plasmid dengan DNA asing

• Enzim ligase menyambung dua ujung

DNA yang semulanya terpotong
.

Enzim ligase adalah enzim yang berfungsi untuk

menyambung dua ujung potongan DNA Your Content Here
• Penyambungan dilakukan dengan cara
Enzim ligase yang sering digunakan adalah DNA ligase dari E. menyambung 2 ujung DNA melalui
ikatan kovalen antara ujung 3’OH dari
Coli dan DNA ligase dari Fage T4 utas satu dengan ujung 5’P dari utas
yang lain
Prinsip kerja enzim ligase sebagai
berikut

• Penggunaan ligasi DNA ini mengkatalis

ikatan fosfodiester antara kedua ujung
DNA sehingga kedua fragmen DNA
yang berupa potongan bisa bersatu
menjadi satu.
Proses penyambungan DNA ligasi pada
daerah pemotongan

Gambar 5. Penyambungan DNA ligase pada

potongan DNA
 Pemotongan dan
penyambungan
plasmid dan DNA asing
sehingga dihasilkan
Plasmid Rekombinan

Untuk menciptakan plasmid rekombinan yang

mengandung sifat DNA asing tertentu yang dilakukan
adalah dengan menyambung DNA asing tersebut
dengan plasmid yang ada. Plasmid rekombinan
terbentuk sebagai sambungan antara plasmid
dengan DNA asing, sehingga plasmid tersebut
mengandung sifat tertentu yang telah disesuaikan
dengan kebutuhan. Secara sederhana prosedur
untuk menciptakan plasmid rekombinan dapat
dilakukan sebagai berikut:
 Pemotongan plasmid yang akan digunakan
sebagai vektor dengan enzim restriksi yang
sama yaitu E. Coli

Menyiapkan bakteri yang mengandung DNA

asing dengan sifat tertentu Hasil potongan DNA dengan sifat tertentu
disambungkan pada plasmid dngan
menggunakan enzim penyambung yaitu
DNA ligase. DNA ligase akan mengikat
ujung 3’OH dengan ujung 5’P dan
membentuk ikatan fosfodiester sehingga
plasmid dan DNA asing dengan sifat
Menyiapkan plasmid yang akan digunakan tertentu bisa bersatu.
sebagai vektor

Terbentuklah plasmid rekombinan yang

membawa DNA asing dengan sifat tertentu
tersebut. Plasmid ini siap ditransfer ke
dalam sel inang untuk memperoleh
Pemotongan DNA asing dengan sifat yang organisme transgenik
dibutuhkan dengan enzim restriksi semisal
dari E. Coli
One Column
Style

Gambar proses terbentuknya Plasmid rekombinan sebagai hasil

penyambungan plasmid dengan DNA asing dengan sifat tertentu
dapat dilihat pada gambar 6 berikut ini.
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You can simply impress your audience and add a unique zing and appeal to your Presentations.
 Simple
Portfolio
Presentation

Text Here Text Here Text Here

You can simply impress You can simply impress You can simply impress
your audience and add a your audience and add a your audience and add a
unique zing and appeal unique zing and appeal unique zing and appeal
to your Presentations. to your Presentations. to your Presentations.
Easy to change colors, Easy to change colors, Easy to change colors,
photos and Text. photos and Text. photos and Text.
 Infographic Style

01 02 03 04
Your Text Here Your Text Here Your Text Here Your Text Here
You can simply impress You can simply impress You can simply impress You can simply impress
your audience and add a your audience and add a your audience and add a your audience and add a
unique zing and appeal to unique zing and appeal to unique zing and appeal to unique zing and appeal to
your Presentations. your Presentations. your Presentations. your Presentations.
 Infographic Style
1. Contents Here A
You can simply impress your audience and add a unique zing and
appeal to your Presentations. Easy to change colors, photos and Text.
Text A

2. Contents Here B
You can simply impress your audience and add a unique zing and
appeal to your Presentations. Easy to change colors, photos and Text.
Text B

3. Contents Here C
You can simply impress your audience and add a unique zing and
appeal to your Presentations. Easy to change colors, photos and Text.
Text C

4. Contents Here D
You can simply impress your audience and add a unique zing and
appeal to your Presentations. Easy to change colors, photos and Text.
Text D
Simple Portfolio
Presentation
Easy to change colors, photos and Text.

Get a modern PowerPoint Presentation that is beautifully designed. Easy to change

colors, photos and Text. You can simply impress your audience and add a unique zing
and appeal to your Presentations. I believe that this Template will your Time, Money and
Reputation. Get a modern PowerPoint Presentation that is beautifully designed. Easy to
change colors, photos and Text. Get a modern PowerPoint Presentation that is
beautifully designed.

You can simply impress your audience and add a unique zing and appeal to your
Presentations. I believe that this Template will your Time, Money and Reputation.

Your Text Here Your Text Here

Get a modern PowerPoint Get a modern PowerPoint
Presentation that is beautifully Presentation that is beautifully
designed. designed.
 Your Text Here Your Text Here
Two Column You can simply impress
your audience and add a
You can simply impress
your audience and add a

Style unique zing and appeal to

your Presentations. Easy
to change colors, photos
and Text. I hope and I
unique zing and appeal to
your Presentations. Easy
to change colors, photos
and Text. I hope and I
believe that this Template believe that this Template
will your Time, Money will your Time, Money
and Reputation. Get a and Reputation. Get a
modern PowerPoint modern PowerPoint
Presentation that is Presentation that is
beautifully designed. beautifully designed.
Easy to change colors, Easy to change colors,
photos and Text. You can photos and Text. You can
simply impress your simply impress your
audience and add a audience and add a
unique zing and appeal to unique zing and appeal to
your Presentations. your Presentations.

You can simply impress your audience

and add a unique zing and appeal to
your Presentations.
One Column Your Text Here
Get a modern PowerPoint Presentation that is beautifully
designed.

Style You can simply impress your audience and add a unique zing and
appeal to your Presentations. I hope and I believe that this
Template will your Time, Money and Reputation. You can simply
impress your audience and add a unique zing and appeal to your
Presentations. You can simply impress your audience and add a
unique zing and appeal to your Presentations. You can simply
impress your audience and add a unique zing and appeal to your
Presentations. I hope and I believe that this Template will your
Time, Money and Reputation.

I hope and I believe that this Template will your Time, Money and
Reputation. You can simply impress your audience and add a
unique zing and appeal to your Presentations.

Get a modern PowerPoint Presentation that is beautifully

designed.

You can simply impress your audience

and add a unique zing and appeal to
your Presentations.
 Thank You
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 Shapes IMAGES

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 Fully Editable Icon Sets: A
You can Resize without
losing quality
You can Change Fill
Color &
Line Color

FREE
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 Fully Editable Icon Sets: B
You can Resize without
losing quality
You can Change Fill
Color &
Line Color

FREE
PPT
TEMPLATES
www.allppt.com
 Fully Editable Icon Sets: C
You can Resize without
losing quality
You can Change Fill
Color &
Line Color

FREE
PPT
TEMPLATES
www.allppt.com