Essential genes are involved in control of the basic metabolism of their host. These genes encode elements involved is such crucial processes as DNA replication, transcription, translation or biosynthesis of important molecules. What makes essential genes unique is the fact that they cannot be lost from the genome. If any of them becomes inactivated it would result in inevitable death of an organism. Because of their role they can be efficiently used to control the survival of genetically modified organisms. Specific regulatory mechanisms can be applied to modulate the activity of essential genes, which prevents an organism from growing at determined conditions. Such mechanisms are called “kill switches” and have been developed in recent years as a response to significant development in the field of molecular biology. Proteins encoded by psychrophilic organisms are characterized by decreased resistance to thermal denaturation. This is believed to be a result of adaptation to low-temperature environment, where mutations that destabilize the protein structure are not selected against. For these reasons they often cannot perform their functions at moderate temperatures, which are typical for mesophilic organisms. At the same time psychrophilic proteins do not display any inhibition at permissive conditions. Use of psychrophilic alleles of essential genes has been proposed as a method of rendering modified organisms incapable of surviving at elevated temperatures. This allows generation of attenuated strains of pathogenic bacteria or generally safe versions of laboratory organisms. A temperature-sensitive organism can be created by substituting a single essential gene in mesophilic organism with its psychrophilic homologue. This can be facilitated by using the host’s native recombination system or through the use of plasmid based allele shuffling mechanisms. The objective of this work was to analyze a number of psychrophilic alleles of various essential genes for their ability to cause temperature-sensitive phenotype in mesophilic bacterium Francisella novicida. The special attention has been placed on investigating psychrophilic alleles of bacterial DNA ligase. Furthermore a selected psychrophilic strain has been characterized as a potential source of multiple temperature-sensitive alleles of essential genes. Finally the secondary focus was to develop a simple and robust mechanism allowing efficient exchange of alleles of essential genes in the mesophilic host. / Graduate

Mycobacterium tuberculosis remains one of the most common worldwide causes of illness and death due to an infectious disease. The emergence of multiple and extreme-drug resistant strains has increased the need to find an effective vaccine for tuberculosis. The goal of our research group is to engineer a temperature-sensitive (TS) M. tuberculosis strain that can be used as a tool in vaccine development. One approach to create TS M. tuberculosis involves the integration of the essential gene ligA encoding a TS NAD+ dependent DNA ligase, which was taken from the psychrophilic organism Pseudoalteromonas haloplanktis. The integration and functioning of ligA was demonstrated in the fast-growing organism Mycobacterium smegmatis. This strain had a TS phenotype with growth limited to below 37°C. The strain was found to have a stable TS phenotype and did not mutate to a temperature-resistant form at a detectable level. Following experiments with the fast growing M. smegmatis, the integration of the ligA gene was attempted in slow-growing M. tuberculosis. Merodiploids of M. tuberculosis containing both the psychrophilic and the WT ligA gene in its chromosome were obtained. The second approach used for the development of TS M. tuberculosis was the directed evolution of native M. tuberculosis essential genes. An advantage of this approach is that the gene encoding the essential protein will resemble the native M. tuberculosis gene and thus will closely match the native transcriptional and translational rates. A system to screen and select for TS essential genes engineered by directed evolution was designed, where the essential gene on the chromosome of E. coli was knocked out and this gene was supplied on a conditionally replicating plasmid. As a first step in developing this directed evolution approach, a family of conditionally replicating plasmids were created and tested in an essential gene knock-out strain of E. coli. / Graduate

<p>A metagenomic gene library prepared from soil found beneath a mummified seal carcass in the Miers Valley, Antarctica, suggests an environment rich in uncharacterised biodiversity including enzymes with possible application to industrial processes. A sequence based gene mining investigation was performed on a clone, which archives a metagenomic sequence from this environment. The sequence was annotated using de novo bioinformatics and molecular biology techniques. A predicted NAD+-dependent DNA ligase, ligDB1 was selected for further characterisation. LigDB1 encodes a gene product that contains all the sequence features of a functional ligase. The protein was overexpressed in a heterologous E. coli host and purified to homogeneity. LigDB1 did not exhibit nick sealing activity, but was able to perform AMP-dependent DNA relaxation in the presence of high concentrations of enzyme. DNA modifying enzymes from cold environments perform optimally at low temperatures and may be of use as molecular tools in biotechnology. Complete characterisation of this enzyme is subject to further investigations.</p>

<p>A metagenomic gene library prepared from soil found beneath a mummified seal carcass in the Miers Valley, Antarctica, suggests an environment rich in uncharacterised biodiversity including enzymes with possible application to industrial processes. A sequence based gene mining investigation was performed on a clone, which archives a metagenomic sequence from this environment. The sequence was annotated using de novo bioinformatics and molecular biology techniques. A predicted NAD+-dependent DNA ligase, ligDB1 was selected for further characterisation. LigDB1 encodes a gene product that contains all the sequence features of a functional ligase. The protein was overexpressed in a heterologous E. coli host and purified to homogeneity. LigDB1 did not exhibit nick sealing activity, but was able to perform AMP-dependent DNA relaxation in the presence of high concentrations of enzyme. DNA modifying enzymes from cold environments perform optimally at low temperatures and may be of use as molecular tools in biotechnology. Complete characterisation of this enzyme is subject to further investigations.</p>

A metagenomic gene library prepared from soil found beneath a mummified seal carcass in the Miers Valley, Antarctica, suggests an environment rich in uncharacterised biodiversity including enzymes with possible application to industrial processes. A sequence based gene mining investigation was performed on a clone, which archives a metagenomic sequence from this environment. The sequence was annotated using de novo bioinformatics and molecular biology techniques. A predicted NAD+-dependent DNA ligase, ligDB1 was selected for further characterisation. LigDB1 encodes a gene product that contains all the sequence features of a functional ligase. The protein was overexpressed in a heterologous E. coli host and purified to homogeneity. LigDB1 did not exhibit nick sealing activity, but was able to perform AMP-dependent DNA relaxation in the presence of high concentrations of enzyme. DNA modifying enzymes from cold environments perform optimally at low temperatures and may be of use as molecular tools in biotechnology. Complete characterisation of this enzyme is subject to further investigations. / Magister Scientiae - MSc

A metagenomic gene library prepared from soil found beneath a mummified seal carcass in the Miers Valley, Antarctica, suggests an environment rich in uncharacterised biodiversity including enzymes with possible application to industrial processes. A sequence based gene mining investigation was performed on a clone, which archives a metagenomic sequence from this environment. The sequence was annotated using de novo bioinformatics and molecular biology techniques. A predicted NAD+-dependent DNA ligase, ligDB1 was selected for further characterisation. LigDB1 encodes a gene product that contains all the sequence features of a functional ligase. The protein was overexpressed in a heterologous E.coli host and purified to homogeneity. LigDB1 did not exhibit nick sealing activity, but was able to perform AMP-dependent DNA relaxation in the presence of high concentrations of enzyme. DNA modifying enzymes from cold environments perform optimally at low temperatures and may be of use as molecular tools in biotechnology. Complete characterisation of this enzyme is subject to further investigations. / Magister Scientiae - MSc

>Magister Scientiae - MSc / A metagenomic gene library prepared from soil found beneath a mummified seal carcass in the Miers Valley, Antarctica, suggests an environment rich in uncharacterised biodiversity including enzymes with possible application to industrial processes. A sequence based gene mining investigation was performed on a clone, which archives a metagenomic sequence from this environment. The sequence was annotated using de novo bioinformatics and molecular biology techniques. A predicted NAD+-dependent DNA ligase, ligDB1 was selected for further characterisation. LigDB1 encodes a gene product that contains all the sequence features of a functional ligase. The protein was overexpressed in a heterologous E. coli host and purified to homogeneity. LigDB1 did not exhibit nick sealing activity, but was able to perform AMP-dependent DNA relaxation in the presence of high concentrations of enzyme. DNA modifying enzymes from cold environments perform optimally at low temperatures and may be of use as molecular tools in biotechnology. Complete characterisation of this enzyme is subject to further investigations