You've used the restrictionĮnzymes to cut out your gene and then what you wannaĭo is you wanna paste it into what we'll call a plasmid. Left over on either side but essentially you have cut out the gene. And so now you would have, after you applied the restriction enzymes, you will have just that gene. These, those things right over there those are restriction enzymes. Sequence at the other side that we wanna cut. So that might be a restrictionĮnzyme right over there and then you might useĪnother restriction enzyme that identifies with the Sequence right over here and cuts right in the right place. We might use one restriction enzyme, Let me use a different color here, that latches on right over here and identifies the genetic They recognize specific sequences and then we can figure out well which restrictionĮnzyme should we use to cut out different pieces of DNA, but we have gotten to that Identify these enzymes and we know at what points Have gotten to the point that we can find and Restriction enzymes, and I personally find it fascinating that we as a civilization And the way we do that is using restriction enzymes. Well, the first thing we wanna do is we wanna cut this gene out some how. This is the double-stranded DNA and let's say that this part of this DNA has a gene that we want to clone. Actually, let me just draw, let me just try to draw the two strands just so we remind ourselves. I don't want to have to take the trouble of keep drawing the multiple strands. Strand of DNA right over here and I'm just drawing it as a long, but this is a double-stranded, and I'll just write it down, this is double stranded. So how do we do that? Well let's say that this is a But when we talk aboutĬloning and DNA cloning we're talking about somethingĪ little bit simpler. Or an organism, like a sheep, well then you are creating an animal that has the exact genetic Heard the term cloning in terms of the Clone Wars in Star Wars or Dolly the sheep and that is a related idea. And usually it's a piece of DNA that codes for something we care about, it is a gene that willĮxpress itself as a protein that we think is useful in some way. Which is all about making identical copies of a piece of DNA. Pustaka genom dengan sisipan fragmen ~40 Kb yang membawa gen MTGase telah berhasil dikonstruksi dengan menggunakan fosmid pCC1FOSTM di dalam E.coli EPI300-TIR.Little bit about DNA Cloning. Berdasarkan sekuen gen 16S rRNA, isolat TTA 02 SDS 14 memiliki kekerabatan terdekat dengan Streptomyces thioleteus dengan homologi 99%. Sekuen parsial gen MTGase dari isolat tersebut memiliki homologi 93 % dengan MTGase dari Streptomyces cinnamoneus. Analisis molekuler dengan PCR membuktikan bahwa isolat tersebut mengandung gen MTGase. Hasil penelitian menunjukkan bahwa penapisan terhadap bakteri Streptomyces spp penghasil MTGase yang diisolasi dari berbagai jenis tanah di Indonesia, mendapatkan satu isolat yang potensial yaitu TTA 02 SDS 14. Untuk itu, penelitian ini dilakukan melalui beberapa tahapan yaitu, penapisan bakteri Streptomyces spp penghasil MTGase, identifikasi isolat penghasil MTGase dan pembuatan pustaka genom dengan mengkloning fragmen DNA 40 kb yang mengandung gen penyandi MTGase. Penelitian ini bertujuan untuk mendapatkan bakteri yang menghasilkan MTGase dan mengklon fragmen DNA penyandinya. Pendekatan teknologi rekayasa genetika sangat diperlukan untuk mendapatkan hasil yang lebih baik sehingga dapat dikembangkan dalam skala industri. Selain itu proses pemurnian cukup sulit karena memerlukan beberapa tahap pemurnian. Produksi enzim Microbial Transglutaminase (MTGase) dari strain liar mempunyai beberapa kelemahan yaitu selain pertumbuhan selnya lambat, produk yang dihasilkan sedikit dan protein yang dihasilkan juga bercampur dengan protein lainnya. 2.3.2.13) merupakan enzim yang mengkatalisis reaksi perpindahan gugus asil antara residu glutamin (Gln) yang berfungsi sebagai donor asil dan residu lisin (Lys) sebagai aseptor yang membentuk ikatan silang (crosslinking) ε-(γ-glutamyl) lisin isopeptida yang menghasilkan ikatan kovalen inter atau intramolekuler yang berikatan silang dengan protein makanan.
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