Desain Primer Untuk Isolasi Gen L-LDH Berdasarkan Genom Streptococcus pluranimalium dan Aplikasinya

Daffa Muhammad Rifqi, Universitas Negeri Yogyakarta, Indonesia
Ixora Sartika Mercuriani, Universitas Negeri Yogyakarta, Indonesia

 10.21831/kingdom.v10i1.18581

Abstract


Asam laktat diketahui merupakan molekul dengan manfaat positif untuk manusia. Molekul tersebut dapat diproduksi oleh bakteri asam laktat. Produksi asam laktat seringkali terganggu karena beberapa spesies bakteri perlu penyesuaian khusus pada substrat, sehingga salah satu solusinya adalah kloning gen atau over-expression enzim L-LDH pada bakteri kompeten. Salah satu langkah prosedur tersebut adalah isolasi gen. Desain primer dilakukan menggunakan aplikasi Benchling pada subjek genom Streptococcus pluranimalium TH11417 dan seluruh primer diujikan secara in-silico menggunakan aplikasi UGENE dan in-vitro menggunakan metode Polymerase Chain Reaction (PCR). Empat pasang primer dihasilkan dengan rincian LSPai 1178, LSPbi 993, LSPad 765, dan LSPbd 846. Primer berhasil menempel dan menghasilkan amplikon yang sesuai pada uji in-silico menggunakan templat genom S. pluranimalium TH11417, namun hanya LSPai 1178 dan LSPad 765 yang dapat menempel pada templat genom S. pluranimalium 14A0014. Primer tidak dapat menempel pada uji in-vitro dikarenakan kontaminasi isolat bakteri templat DNA. Kontaminasi dikonfirmasi melalui analisis sekuen 16s rRNA dan didapatkan bahwa amplikon 16s rRNA isolat bakteri uji sesuai dengan sekuen 16s rRNA Bacillus cereus. Sekuensing basa amplikon uji in-vitro primer LSPai 1178 menunjukkan bahwa amplikon bukan merupakan bagian dari sekuen target.


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DOI: https://doi.org/10.21831/kingdom.v10i1.18581

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