STABILITAS DAN SPESIFISITAS SUBSTRAT KLON REKOMBINAN PENGHASIL PENISILIN ASLIIASE DARI PUSTAKA GENOM BACILLUS SP. BAC4
A research on the stability and substrate specificity of E. coli DHa/pMH3 has been done. E. coil DH5a4MH3 is a clone from the genomic library of Bacillus sp. pBAC4 predicted to contain pga gene in the cosmid vector HC79. Cosmid pHC79 contains two antibiotic resistant genes, i.e. tef and amp'. I...
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/3049 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | A research on the stability and substrate specificity of E. coli DHa/pMH3 has been done. E. coil DH5a4MH3 is a clone from the genomic library of Bacillus sp. pBAC4 predicted to contain pga gene in the cosmid vector HC79. Cosmid pHC79 contains two antibiotic resistant genes, i.e. tef and amp'. Insertion of Bacillus sp. BAC4 fragment into Pstl restriction site of amp` caused the inactivation of amp' gene. In the selection of E. coil DHa/pMH3 in medium containing antibiotics, 3 colonies were obtained which grew on tetracycline medium and died on ampicillin medium. These colonies, are clones 3.6, 3.7, and 3.18 and the cosmids of these clones are named pMH36, pMH37, and pMH318 respectively. Penicillin G acylase (PGA) activity assay of these three clones showed positive results. The cosmids isolated from these clones showed the same migration pattern. The results of the PGA activity assay and DNA migration of gel electrophoresis concluded that these three colonies were derived from E. coil DHa/pMH3. Characterization was continued with clone 3.7. Cosmid pMH37, digested with restriction enzymes EcoRI and EcoRV, produced one band of 5,8 kb and this DNA size was smaller than that of pHC79 (6,5kb). Restriction digest of cosmid pMH37 with Pstl was unsuccessful. The transformation product of a new host cell with pMH37 had no PGA activity. Cosmid curing of clone 3.7 followed with PGA, PVA, and ampicillin acylase activity assays of the cosmidless cells showed positive results. This meant that the genetic material from the cell with no cosmid contained pga gene and therefore there was a DNA transfer from the recombinant cosmid into the host cell chromosome. This transfer of DNA included part of the pHC79 cosmid DNA which contained the Pstl restriction site. PGA, PVA and ampicillin acylase activity assay of the purified enzyme of Bacillus sp. BAC4 showed that the substrate specificity of this purified enzyme was similar to that of clone 3.7. These results meant that the penicillin acylase gene of clone 3 originated from Bacillus sp. BAC4 |
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