Anotasi gen-gen terkait respon terhadap heat stress dan analisis ekspresi gen hsp pada Lactiplantibacillus plantarum strain SU-KC1a = Heat stress response related gene annotation and analysis of gene expression in Lactiplantibacillus plantarum strain SU-KC1a

Pang, Clara Meirinzha (2022) Anotasi gen-gen terkait respon terhadap heat stress dan analisis ekspresi gen hsp pada Lactiplantibacillus plantarum strain SU-KC1a = Heat stress response related gene annotation and analysis of gene expression in Lactiplantibacillus plantarum strain SU-KC1a. Bachelor thesis, Universitas Pelita Harapan.

Preview	Text (Title) Title.pdf Available under License Creative Commons Attribution Non-commercial Share Alike. Download (101kB) | Preview
Preview	Text (Abstract) Abstract.pdf Available under License Creative Commons Attribution Non-commercial Share Alike. Download (214kB) | Preview
Preview	Text (ToC) ToC.pdf Available under License Creative Commons Attribution Non-commercial Share Alike. Download (135kB) | Preview
Preview	Text (Chapter1) Chapter1.pdf Available under License Creative Commons Attribution Non-commercial Share Alike. Download (267kB) | Preview
	Text (Chapter2) Chapter2.pdf Restricted to Registered users only Available under License Creative Commons Attribution Non-commercial Share Alike. Download (369kB)
	Text (Chapter2) Chapter3.pdf Restricted to Registered users only Available under License Creative Commons Attribution Non-commercial Share Alike. Download (493kB)
	Text (Chapter4) Chapter4.pdf Restricted to Registered users only Available under License Creative Commons Attribution Non-commercial Share Alike. Download (729kB)
	Text (Chapter5) Chapter5.pdf Restricted to Registered users only Available under License Creative Commons Attribution Non-commercial Share Alike. Download (227kB)
Preview	Text (Bibliography) Bibliography.pdf Available under License Creative Commons Attribution Non-commercial Share Alike. Download (269kB) | Preview
	Text (Appendices) Appendices.pdf Restricted to Repository staff only Available under License Creative Commons Attribution Non-commercial Share Alike. Download (1MB)

Abstract

Lactiplantibacillus (Lpb) plantarum adalah bakteri asam laktat yang sering digunakan sebagai probiotik. Aplikasi sebagai probiotik menyebabkan Lpb. plantarum sering berhadapan dengan berbagai stres akibat proses manufaktur dan penanganan seperti stres suhu tinggi yang dapat berpengaruh terhadap viabilitas dan fungsionalitas probiotik. Penelitian ini bertujuan: (1) Uji ketahanan Lpb. plantarum strain SU-KC1a pada suhu tinggi berdasarkan viabilitas (OD dan CFU). (2) Analisis molekular terhadap ekspresi gen terkait stres suhu tinggi (gen hsp3). (3) Analisis hasil anotasi gen terkait stres suhu tinggi pada SU-KC1a. Metode yang digunakan dalam penelitian ini adalah: (1) Inkubasi kultur pada suhu 37℃, 42℃, 47℃ dan 52℃ selama satu jam untuk menentukan viabilitas sel dengan enumerasi koloni yang terbentuk. Dari hasil sebelumnya, dilakukan pertumbuhan pada suhu 37, 40 dan 42. Hasil pertumbuhan diamati melalui nilai OD. (2) RNA total dari SU-KC1a yang telah diinkubasi pada suhu 37℃, 40℃ dan 42℃ selama satu jam diekstraksi kemudian dikonversi menjadi cDNA. Sampel cDNA diamplifikasi menggunakan primer spesifik F(5’-TGTCAACATCAACGTCGGGTAA-3’) R(5’-CGTAGTCATGGGTCGGTCCT-3’) gen hsp3. Amplikon divisualisasi pada gel agarose 1,2%. (3) Data anotasi whole genome sequence SU-KC1a dibandingkan terhadap data anotasi tiga strain Lpb. plantarum lain (SK151, F75 dan WCFS1) untuk melihat perbandingan keberadaan gen terkait stres suhu tinggi. Hasil uji ketahanan pada suhu tinggi menunjukkan temperatur maksimal SU-KC1a adalah sekitar 42℃ dengan jumlah pertumbuhan sel dari 15,8 CFU/mL menjadi 19,1 CFU/mL setelah inkubasi satu jam. Hasil visualisasi amplikon gen hsp3 pada agarose menunjukkan penebalan pita pemisahan DNA seiring dengan peningkatan suhu. Berdasarkan hasil perbandingan gen terkait stres suhu tinggi pada keempat strain Lpb. plantarum (SU-KC1a, F75, WCFS1 dan SK151), terlihat bahwa gen-gen terkait stres suhu tinggi antara lain gen groES, groEL, hsp1, hsp2 dan hsp3 terkonservasi pada Lpb. plantarum karena bersifat esensial. / Lactiplantibacillus (Lpb) plantarum is a lactic acid bacteria that commonly used as probiotics. Lpb. plantarum will encounter many stresses due to utilization in functional food industry as a probiotic during manufacturing and handling process such as heat stress thus will affect cell’s viability and functionality. The aims of this study are: (1) Examination of SU-KC1a heat stress resistance (2) Molecular analysis of hsp3 gene expression. (3) Analysis of heat stress related genes based on Whole Genome Sequencing annotation data. Methods used in this research were: (1) Examination of SU-KC1a heat resistance was done by incubating cultures at 37℃, 42℃, 47℃ and 52℃ for one hour to determine cell’s viability with enumeration of colonies formed on agar plates. Based on previous result, optical density value also observed every one hour from cultures incubated at 37℃, 40℃ and 42℃ for three hours in total to determine growth rate of SU-KC1a during heat stress. (2) Total RNA of treated SU-KC1a extracted. Previously, SU-KC1a cultures treated by incubating at 37℃, 40℃ and 42℃ for one hour. After extraction, RNA samples were converted into cDNA and amplified by PCR reaction with hsp3 gene specific primers F(5’-TGTCAACATCAACGTCGGGTAA-3’) R(5’-CGTAGTCATGGGTCGGTCCT-3’). PCR amplicons visualized on 1,2% agarose gel. (3) SU-KC1a Whole Genome Sequence annotation data was compared to Lpb. plantarum SK151, F75 and WCFS1 annotation to identify presence and absence of heat stress related genes. Result of SU-KC1a resistance towards heat stress shows that maximum temperature of SU-KC1a is around 42℃ with increased cell’s viability from 15,8 CFU/mL to 19,1 CFU/mL after incubated for one hour. Growth rate of SU-KC1a incubated at higher temperature exhibit slower rate. Visualization of hsp3 amplicon appear clearer at higher temperature indicating increase of hsp3 gene expression. According to comparative analysis of stress related genes, heat stress response genes are conserved by reason of it is essential for survival capability.

Actions (login required)

View Item