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Prediction of small RNAs in selected bacterial producers of bioplastics
Heřmánková, Kristýna ; Vítková, Helena (referee) ; Sedlář, Karel (advisor)
Využití bakterií v biotechnologii vyžaduje důkladné pochopení schopností použitých bakterií pro maximalizaci výtěžku. To platí například pro výrobu bioplastů, kde konkurenceschopnost s levnějšími plasty na bázi fosilních paliv závisí také na ceně procesu pro získání požadovaného výtěžku. Nahlédnutí do regulačních mechanismů bakterií, jako je regulace pomocí malých RNA, která zajišťuje rychlou reakci především na stresové podmínky, může pomoct odhalit jejich vlastnosti a posoudit tak jejich využitelnost v biotechnologiích. Tato diplomová práce se zabývá zpracováním bakteriálních transkriptomických dat, které byly sekvenovány pomocí RNA-Seq. Práce je zaměřena zejména na predikci malých RNA u poskytnuté bakterie, slibného kandidáta pro využití v biotechnologii. Cílem je získat přehled o malých RNA v této bakterii, zejména analyzovat potenciální regulaci syntézy PHA malými RNA, které by mohly rozšířit přehled o využitelnosti bakterie pro produkci PHA.
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A tool for prediction of small RNA in RNA-Seq data
Pomykalová, Barbora ; Čejková, Darina (referee) ; Jurečková, Kateřina (advisor)
This diploma thesis focuses on the detection of small RNA (sRNA) in the bacterial genome. sRNAs are short non-coding transcripts that play a key role in gene expression. To date, there are several algorithms focusing on the detection of sRNAs from RNA-Sequencing (RNA-Seq) data that can be obtained by some of the sequencing platforms. The most frequently used platforms are Illumina and Ion Torrent belonging to the next generation sequencing and PacBio with Oxford Nanopore belonging to the third generation of sequencing. In this work, the workflow of sRNA detection using freely available tools was described and then an own unique tool for sRNA detection – the SEARCHsRNA tool – was designed. Two open-source software tools – Rockhopper and DETR'PROK, together with newly created tool, were tested on RNA-Seq data for bacteria Vibrio atlanticus LGP32.
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A tool for prediction of small RNA in RNA-Seq data
Pomykalová, Barbora ; Čejková, Darina (referee) ; Jurečková, Kateřina (advisor)
This diploma thesis focuses on the detection of small RNA (sRNA) in the bacterial genome. sRNAs are short non-coding transcripts that play a key role in gene expression. To date, there are several algorithms focusing on the detection of sRNAs from RNA-Sequencing (RNA-Seq) data that can be obtained by some of the sequencing platforms. The most frequently used platforms are Illumina and Ion Torrent belonging to the next generation sequencing and PacBio with Oxford Nanopore belonging to the third generation of sequencing. In this work, the workflow of sRNA detection using freely available tools was described and then an own unique tool for sRNA detection – the SEARCHsRNA tool – was designed. Two open-source software tools – Rockhopper and DETR'PROK, together with newly created tool, were tested on RNA-Seq data for bacteria Vibrio atlanticus LGP32.
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Regulation of transcription in mycobacteria.
Páleníková, Petra ; Krásný, Libor (advisor) ; Mrvová, Silvia (referee)
The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far.
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Dynamics and variability of induced transgene silencing in tobacco cell line BY-2
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Pečinka, Aleš (referee) ; Lafon Placette, Clément (referee)
RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...
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Structural analysis of filamentous hemagglutinin (FhaB) from Bordetella pertusis
Jurnečka, David ; Kavan, Daniel (advisor) ; Man, Petr (referee)
: Filamentous hemagglutinin (FHA) is adhesive protein molecule that is secreted by Gram- negative bacterium Bordetella pertusis, the causative agent of whooping cough (pertussis). The C-terminal segment of FHA plays a crucial role in host-pathogen interaction, however, the structural features are still unknown. Here, we identified the C-terminal residue of FHA and processed form of FHA (FHA*) as alanine residues in position 2304 and 2228, respectively. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy demonstrated that the C-terminal segment of FHA(FHA 1995-2228) is characterized by alpha-helical contribution without any compact protein fold. Moreover, suppression of transcription of small regulatory RNA pairing to the 5'-end of fhaB transcript resulted in two- fold increase of FHA production. These data suggested that the C-terminal segment of FHA appear to be an unstructured protein and FHA secretion is negatively regulated by small regulatory RNA. (In Czech) Keywords: Bordetella pertussis, filamentous hemagglutinin, small RNA
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Regulation of transcription in mycobacteria.
Páleníková, Petra ; Krásný, Libor (advisor) ; Mrvová, Silvia (referee)
The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far.
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