REKAYASA GENETIKA

GENETIC ENGINEERING
(DNA REKOMBINAN)

Suatu teknologi yang mempunyai kemampuan untuk melakukan manipulasi DNA. Rekayasa genetik dapat dimanfaatkan dalam berbagai bidang misal :  kedokteran, farmasi, pertanian, peternakan, industry. Pada bidang kedokeran : pembuatan insulin manusia, pembuatan hormon pertumbuhan dll. Pada bidang pertanian diciptakan tanaman transgenic. Pada bidang peternakan : pembuatan vaksin rekombinan misal untuk penyakit kuku dan mulut pada sapi, domba, babi

FAKTOR YANG TERLIBAT DALAM KLONING GEN

1. DNA asing
2. vektor
3. Enzim restriksi
4. Enzim ligase
5. Sel inang (host)

TAHAP KLONING GEN

1.  Isolasi dan fragmentasi DNA
2. Penyambungan ke suatu vektor kloning dengan enzim ligase
3. Penyisipan ke dalam sel inang
4. Deteksi dan pemurnian klon yang diinginkan

VEKTOR

1. PLASMID
2. KOSMID
3. BAKTERIOFAG

PLASMID

1. Unsur genetik ekstra kromosomal pada bakteri
2. DNA ganda sirkuler ukuran : 1-200 kb
3. Dapat replikasi sendiri
4. Berisi gen untuk mengkode enzim tertentu

    misal: - resistensi terhadap antibiotik

              - resistensi terhadap logam 

                berat

Contoh plasmid :

PBr 322  mengandung gen resisten thd amphisilin dan tetrasiklin

PMK 16 resisten thd kanamisin

Plasmid Ti

Plasmids are small circular molecules of extrachromosomal, double-stranded DNA.  They occur naturally in both bacteria and yeast where they replicate as independent units.  Unlike chromosomal DNA, plasmids usually occur as multiple copies.
 

KOSMID

Campuran dari DNA phage dan plasmid bakteri untuk kloning fragmen DNA eukariotik

BAKTERIOPHAGE

Virus yang menginfeksi bakteri terdiri molekul DNA/RNA diselebungi oleh kapsid

Sistem Kontrol Replikasi Plasmid

1.      Sistem Kontrol Ketat Replikasi di bawah kontrol yang sama dengan replikasi bakteri genom dan dalam setiap sel hanya terdapat 1-2 copy plasmid

2.      Sistem Kontrol Longgar multi copy plasmid 10-20 copy per sel

ENDONUKLEASE RESTRIKSI

•         Dihasilkan oleh bermacam-macam bakteri fungsi untuk merusak/memotong DNA asing (patogen)

•         Tiap enzim akan memotong DNA pada urutan spesifik tidakpada tempat lain, hasil potongan bisa dengan ujung tumpul atau ujung runcing

•         Mekanisme kerja enzim universal

ENZIM LIGASE

•         Menyambung jalin tunggal DNA

•         Diisolasi dari E coli atau bakteriophage T4

•         Dapat menggabungkan fragmen DNA secara invitro dan dilakukan terhadap ujung tumpul maupun runcing

SEL INANG

-Escheria coli

-Bacillus subtilis

-Sacharomyces cerevisiae

Persyaratan sel inang :

1. Tumbuh cepat
2. Dapat tumbuh pada medium yang murah
3. Tidak patogen
4. Stabil dalam kultur
5. Dapat ditransformasi dengan DNA
6. Plasmid: Plasmids are small circles of DNA found in bacteria cells, separate from the bacterial chromosome and smaller than it. They are able to pass readily from one cell to another, even when the cells are clearly from different species, far apart on the evolutionary scale. Consequently, plasmids can be used as vectors, permitting the reproduction of a foreign DNA by using the bacterial replicating system.
7. cDNA: Human genes composed of coding and non- coding sequences. The copy of the coding sequences is called cDNA. It can be obtained from the reverse transcription of messenger RNA.
   The transcription and translation of the insulin cDNA will allow the production of a functional insulin molecule.

Tahapan transfer gen insulin

1. Transfer of the Insulin gene into a plasmid vector.
2. The plasmid is cut across both strands by a restriction enzyme, leaving loose, sticky ends to which DNA can be attached.
3. Special linking sequences are added to the human cDNA so that it will fit precisely into the loose ends of the opened plasmid DNA ring.
4. The plasmid containing the human gene, also called a recombinant plasmid, is now ready to be inserted into another organism, such as a bacterial cell.
5. Cloning the Insulin gene
6. The recombinant plasmids and the bacterial cells are mixed up. Plasmids enters the bacteria in a process called transfection. With the recombinant DNA molecule successfully inserted into the bacterial host, another property of plasmids can be exploited - their capacity to replicate. Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several dozen copies.When the bacteria divide, the plasmids are divided between the two daughter cells and the plasmids continue to reproduce. With cells dividing rapidly (every 20 minutes), a bacterium containing human cDNA (encoding for insulin, for example) will shortly produce many millions of similar cells (clones) containing the same human gene.