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Home > Mutagenesis for Hyper Production of Streptokinase from Streptococcus Equisimilis and Comparative Sequence Analysis of Sk Gene

Mutagenesis for Hyper Production of Streptokinase from Streptococcus Equisimilis and Comparative Sequence Analysis of Sk Gene

Thesis Info

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Author

Naeem, Muhammad

Program

PhD

Institute

University of Agriculture

City

Faisalabad

Province

Punjab

Country

Pakistan

Thesis Completing Year

2019

Thesis Completion Status

Completed

Subject

Biotechnology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/10497/1/Muhammad%20Naeem%20%28CABB%29%2c%20UAF.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726737769

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Streptokinase (SK) is protein excreted by strains of β-hemolytic Streptococci. It is applied as treatment of life-threatening diseases thrombosed arteriovenous shunts, and thromboembolism. Streptokinase is naturally produced by several bacterial strains especially Streptococci. In connection with its broad application in the field of medicine, there is an urgent need to search for methods of its hyper production. Present study aimed at SK hyper production by mutagenesis of S. equisimilis strain. The wild bacterial strain S. equisimilis was subjected to random mutagenesis (UV irradiation and Ethidium bromide) for enhanced streptokinase production. The modified strain was assessed for the activity of streptokinase. The mutant S. equisimilis UV6 showed the maximum SK production and activity. By standardizing the effects of different factors like substrate (CSL), carbon source, salt concentration, temperature and pH an optimum fermentation protocol for SK production was established. The higher SK production was observed in mutant strain UV6 601 U mL-1 with increased activity up to 400 %. The SK was purified and its kinetics data indicated improved activity as its Km value was low as compared to wild SK. The SK gene from mutant UV6 was amplified and cloned in E. coli using pTZ57R/T vector. The gene was sequenced and compared with the wild SK gene to check the genetic difference between wild and mutant strain. In our study, the comparative sequence analysis revealed 26 point mutations, among these 3 were transitions (A to G) while the remaining 23 were transversions (A/G to C/T) in coding region of sk gene. Moreover, 3 amino acid deletions at the C terminal region of SK protein were predicted due to non-sense mutation in coding region (resulting in a stop codon after residue 437). The activity of any protein is direcly linked with amino acid composision. in our studies, the in-silico analyses predicted that amino acid residues at position 22, 62, 64 and 80 could be accountable in SK hyperactivity.
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خلاصہ بحث

ایمان اسلام کو جزو لاینفک ہے اس کے بغیر اسلام قابل قبول نہیں کوئی بھی شخص مکمل ایمان لائے بغیر دائرہ اسلام میں داخل نہیں ہو سکتا ۔ فصل اول میں ایمانیات کے بارے میں قرآن مجید کی درج ذیل سورتوں کی ۲۰ آیات سے وضاحت کی گئی ہے۔

سورة آل عمران آیت نمبر ۱۶۰، سورة النساء آیت نمبر۸۲، سورة الاعراف آیت نمبر۱۸۵، سورة يونس آیت نمبر۳۴، سورة بنى اسرائیل آیت نمبر ۹۹، سورة الانبياء آیت نمبر ۴۲، سورة الحج آیت نمبر۷۰، سورة المؤمنون آیت نمبر ۸۵،۸۶،۸۷،۸۸،۸۹، سورة النور آیت نمبر ۵۰، سورة الروم آیت نمبر ۳۵،۳۷ ،سورةالزمر آیت نمبر ۴۳، سورة المؤمن آیت نمبر ۸۱،۸۲، سورة المرسلات آیت نمبر۲۰،۲۵، اس فصل میں ان آیات میں اللہ تبارک وتعالیٰ نے اپنے بندوں سے سوال فرمایا ہے۔

اس فصل میں ہر آیت کو بیان کرنے کے بعد اس کی تفسیر پر روشنی ڈالی گئی ہے اور اس آیت میں استفہام کے استعمال کی وضاحت کی گئی ہے کہ ہر آیت میں اللہ تبارک و تعالیٰ کا سوال کرنے کے پیشِ نظر جو مقصد کارفرما تھا اس کو واضح کیا گیا ہے۔

PENGARUH STRATEGI KONFLIK KOGNITIF BERBANTUAN MEDIA SEJARAH SAINS TERHADAP SIKAP ILMIAH DAN PERUBAHAN KONSEPTUAL PADA MATERI GERAK

Penelitian ini merupakan penelitian kuantitatif dan kualitatif. Tujuan penelitian ini untuk melihat pengaruh strategi  pembelajaran konflik kognitif berbantuan media sejarah sains terhadap sikap ilmiah dan perubahan konseptual siswa pada materi gerak. Rancangan penelitian yang digunakan adalah quasi eksperimen. Sampel penelitian ini adalah kelas XB dan XC yang terdiri atas 66 sisiwa, 33 siswa pada kelas eksperimen dan 33 siswa pada kelas kontrol. Data pada penelitian ini diambil dengan menggunakan: angket, tes pemahaman konsep dan wawancara. Angket digunakan untuk mengambil data sikap ilmiah, tes pemahaman konsep untuk mengambil data perubahan konseptual siswa, dan wawancara sebagai pelengkap dan pendukung data perubahan konseptual. Data di analisisn dengan statistik uji-t pada taraf signifikan 5% dan uji N-Gain. Hasil penelitian menunjukkan dengan menggunakan aplikasi SPSS 20  diperoleh untuk sikap ilmiah thitung > ttabel (4,637 >2,042 atau sig.2-tailed (0,000) < (0, 05) dan  thitung > ttabel (6.974 > 2.024)  sig.2-tailed (0,000) < (0, 05) untuk perubahan konseptual, yang berarti hipotesis (H0) ditolak dan (H1) diterima dan yang diperoleh pada kelas eksperimen lebih tinggi dari kelas kontrol. Kesimpulan dari penelitian ini adalah stategi pembelajaran konflik kognitif berbantuan media sejarah sains berpengaruh secara nyata meningkatkan sikap ilmiah, perubahan konseptual siswa kelas X SMA pada materi gerak.  Kata Kunci: Strategi Konflik Kognitif, Media Sejarah Sains, Sikap Ilmiah dan Perubahan Konseptual.

Hemoglobin Alpha 1 Hba1 Gene Sequence Analysis in the Thalassemia Affected Patients

Alpha-thalassemia is an inherited blood disorder which is an autosomal recessive type disorder characterized by a microcytic hypochromic anemia and hemolytic anemia. The a-thalassemias involve the genes?HBA1?and?HBA2.The aim of this research was to determine the mutations in hemoglobin alpha 1 (HBA1) in Thalassemia affected patients and in silico analysis of identified mutations to predict the functional effect. In this study, genomic DNA was extracted from 40 Patients affected with Thalassemia (n=40) disease. Blood samples were collected in vacutainers with EDTA as an anticoagulant from the patients and relatives. Blood samples with anticoagulant were used for leukocytes based DNA extraction. Standard organic method was used for DNA extraction. DNA samples were quantified using agarose gel and DNA ladder. Primers were designed using gene sequence from NCBI gene bank. Primer3 software was used for primer designing. PCR conditions will be optimized for amplification and PCR was performed to determine the SNPs. A 382 base pair fragment of DNA of HBA1gene of exon 3 was amplified using polymerize chain reaction (PCR) technique. Sanger sequencing of the selected samples was done to identify polymorphisms. A total of 24 samples out of 40 samples of DNA were sequenced and these SNPs were confirmed by alignment. We were unable to find the mutations in the HBA1 gene but two heterozygous variations were found in HBA1exon 3.Two heterozygous variations were confirmed in exonic area of HBA1 gene of Patients affected with Thalassemia. The findings of this research revealed no mutations were found in HBA1 gene. Two heterozygous variants were confirmed at the position of c.514 on amplified fragment from G> C and second change at the position of c.470 on amplified fragment G > C in 3?UTR.. Variations were further subjected for splice site analysis. The splicing site analysis was done by using an online tool (Human splicing finder).A variation which were present at c.514 G>C were found in the potential splice site and its consensus value is 88.39 and the second one is not in the target region of splicing.