Diverse research into the model organism, Escherichia coli, has added substantial depth to our understanding of genome editing of bacteria. Recombineering using the λ Red system is the most disruptive molecular technology discovered thus far, and improved our ability to introduce targeted single nucleotide variants by ~1E4 fold. This discovery has catalyzed incredible progress and enabled ambitious genome/organism engineering projects such as high throughput metabolic engineering to genome-wide codon reassignment. While efforts in E. coli have since accelerated further, work in other bacterial model organisms has lacked this catalyst and continues to fall behind E. coli. To facilitate development of disruptive technologies for non-standard model organisms, we produced a library of homologs to the λ Red recombinase, λ β (NP_040617.1), to generate a toolbox for recombinase discovery in organisms with minimal tools. We demonstrated the recombinase discovery workflow, called Serial Evolutionary Enrichment for Recombinases (SEER), in E. coli and present a number of alternatives to using λ Red for genome editing. We then moved on to explore λ β-mediated recombination in vitro where we able to show that bet specifically unloads E. coli Ssb from Ssb-coated oligos to facilitate annealing. We hypothesized that ssb represents the minimal host interaction node that a recombinase must achieve to facilitate recombination in vivo, and demonstrated a gain-of-function phenotype when species-matched recombinase/ssb pairs are ported into foreign organisms, potentially opening up poorly understood organisms to recombineering using well understood recombinase/ssb pairs.

No description available.

VP16 initiates the HSV replication cycle by activating immediate early (IE) gene expression. It recruits the RNA pol II through an acidic C-terminal domain. The defective VP16 encoded by the V422 mutant of HSV-1 possesses a truncated C-terminal domain. Therefore, V422 replication is suppressed in most cell-lines, except U2OS osteosarcoma cells. The permissive phenotype of U2OS cells stems from a failure to express one or more inhibitory factors that are produced in restrictive cells. The initial project was designed to identify these host inhibitory factors in restrictive cells of V422, using siRNA silencing technology. To facilitate the siRNA screen, a GFP reporter gene has been inserted into the thymindine kinase (TK) gene of the V422 genome and the wild-type KOS genome. This thesis provides information about characterizing the kinetics of GFP expression from recombinant viruses at both protein and mRNA levels, during different infection times in HeLa and Vero cells. / Virology

Mospd3, a gene located on mouse chromosome 5, was identified in a gene trap screen in ES cells. The gene trap vector integration in multiple copies into the putative promoter of the gene, resulted in a loss of expression of Mospd3 at the trapped allele. In mice generated from ES cells carrying the vector integration it was found that the lack of Mospd3 expression resulted in the death of a proportion of the homozygote mutants within the first day after birth. Homozygote neonates exhibited a thinning of the right ventricular free heart wall which resembles other mouse mutant phenotypes as well as human congenital heart defects caused by a loss of desmosome and adherens junction mediated cell adhesion between cardiomyocytes. The protein encoded by Mospd3, contains an N-terminal Major Sperm Protein (MSP) domain implicated as a mediator of protein- protein interactions, as well as a two C-terminal transmembrane domains. Both, protein structure and phenotypic similarities with defects in desmosomal and adherens junction proteins suggests that Mospd proteins might play a role in cell adhesion and maintaining the structural integrity of the heart. The phenotype of Mospd3 mutants was highly dependent on genetic background, which led us to speculate that there might be genetic redundancy between Mospd3 and its closest family member the X-linked Mospd1. The aims of this thesis were to generate tools to better understand the function of the Mospd gene family in cardiac development as well as assessing genetic redundancy between Mospd1 and Mospd3. A conditional gene targeting strategy was designed for both Mospd genes. Large genomic regions of the Mospd1 and Mospd3 loci were subcloned from bacterial artificial chromosomes (BACs) and using a recombineering approach, loxP sites and a drug selection cassette (neomycin) were placed in precise locations surrounding the MSP domain of both genes. The conditional targeting vectors were electroporated into both CGR8 and E14 ES cells and homologous recombinant clones were identified at a frequency of 2% and 1.3% for Mospd1 and Mospd3 respectively. Five euploid targeted clones for both Mospd1 and Mospd3 have been generated. Transient expression of Cre recombinase in ES cells carrying the conditional Mospd1 allele was used to delete the one copy of this X-linked gene. Phenotypic characterisation of this null ES cell line revealed that Mospd1 is neither essential for ES cell viability and self-renewal, nor for the early differentiation of these cells towards a cardiac fate. In order to investigate the mechanism of action of Mospd proteins, specific polyclonal antibodies were generated to detect either Mospd1 or Mospd3. These antibodies were purified and tested by western blotting using COS7 cells overexpressing either Mospd protein as well as mouse tissue lysates. Whilst the antibodies were found to detect the proteins and differentiate between Mospd1 and Mospd3, they showed insufficient purification to be used in co-localisation and co-immunoprecipitation experiments to identify interacting proteins and determine whether Mospd proteins are involved in cell adhesion complexes. Monoclonal antibodies were subsequently generated and initial western blotting experiments showed promising results, indicating that these antibodies may be better suited for immunohistochemical analysis of Mospd proteins.

O diagnóstico da tuberculose por métodos tradicionais é lento e laborioso. Por outro lado, os testes moleculares são rápidos, mas com custo elevado para países em desenvolvimento. Este projeto teve o objetivo de inserir no micobacteriófago D29 o gene que codifica a proteína verde fluorescente (eGFP) e estudar o fago recombinante na detecção rápida de bacilos da tuberculose. Para tanto, foi inserido o cassete Hsp60- eGFP no genoma do fago D29 por recombinação. Micobacteriófagos recombinantes purificados foram utilizados para infectar M. smegmatis mc2 155 e M. tuberculosis H37Rv durante um período de 1- 6h nas temperaturas de 30°C, 37°C e 42°C. Bactérias fluorescentes foram observadas em um período de 2h, mas em número reduzido, indicando que o micobacteriófago lisou às células rapidamente, dificultando a expressão da eGFP e visualização em microscópio de fluorescência. A deleção do gene LysA, foi efetuada a fim de aumentar o período de latência do fago. Não foi possível a purificação de fagos recombinantes, devido à baixa quantidade de recombinantes nos halos de inibição. Será necessário a redução da atividade o gene LysA e, provavelmente, de outros genes associados a lise celular a fim de aumentar a concentração de eGFP no interior da célula. / Classical biochemical methods for Mycobacterium tuberculosis identification are lengthy and time-consuming. On the other hand, molecular assays are rapid but expensive for developing countries. This project aimed to insert into the mycobacteriophage D29, the gene coding for the green fluorescent protein (eGFP) and use the recombineered phage to detect Mycobacterium tuberculosis rapidly and less costly. For that, the Hsp-eGFP cassette was inserted into D29 genome. Recombineered mycobacteriophages was purified and used to infect M. smegmatis mc2 155 and M. tuberculosis H37Rv from 1-6 hs at 30°C, 37°C and 42°C. Observation of fluorescent bacteria was difficult and only a small number of them were seen at 2 hs of infection. This indicated that recombineered bacteriophages were lysing cells rapidly. Deletion of LysA gene, was carried out to increase the time needed for bacterial lysing. it was not possible to purify mutant mycobacteriophages due to the low concentration of recombinant phages. We conclude that might be necessary the deletion of other genes such as LysB, a gene also involved in cell lysis and reduction LysA activity to increase the concentration of eGFP inside cells.

Telomeres are the repetitive DNA sequence and associated proteins found at the ends of linear chromosomes. They have a role in biological processes including meiosis and aging as well as implications in a number of genomic instability disorders and cancers. Telomeres maintain genomic stability by protecting chromosome ends from terminal fusions and misidentification as DNA damage sites. Their wide range of functions has resulted in an increased interest in developing tools to study the dynamics of telomeres in live cells. To do this, current studies use the ubiquitously expressed protein Telomere Repeat Factor 1 (TRF1) tagged with a fluorescent protein. TRF1 is a negative regulator of telomere length that binds exclusively to telomere repeats. Over-expression of the fluorescent protein fused to TRF1 has been a useful tool to track telomere movement. The foci formed by the tagged TRF1 protein accurately represent the number of telomeres expected in the cells and the localization is maintained throughout the cell cycle. A caveat with this system is that over-expression of TRF1 leads to accelerated telomere shortening, as well as replication defects that can stall telomere replication. These caveats make it difficult to draw conclusions about telomere dynamics based solely on observations of cells over-expressing fluorescently tagged TRF1. To eliminate problems associated with protein over-expression, I have tried to develop knock-in embryonic stem (ES) cells expressing fluorescently tagged TRF1 from the endogenous Trf1 promoter. To do this, I have used a recombineering technique using Bacterial Artificial Chromosomes (BACs). BAC recombineering allows for the direct knock-in of a fluorescent tag into the mouse Trf1gene locus. Genetic constructs with the correct sequence inserts have been obtained and have been used for transfection of ES cells. While no correctly targeted ES cells have been identified so far, the expectation is that ES cell lines with correctly targeted fluorescently tagged TRF1 will be obtained in the near future. Such lines will be used to study telomere dynamics in ES cells, differentiated cells generated from ES cells, as well as to generate mice.

Telomeres are the repetitive DNA sequence and associated proteins found at the ends of linear chromosomes. They have a role in biological processes including meiosis and aging as well as implications in a number of genomic instability disorders and cancers. Telomeres maintain genomic stability by protecting chromosome ends from terminal fusions and misidentification as DNA damage sites. Their wide range of functions has resulted in an increased interest in developing tools to study the dynamics of telomeres in live cells. To do this, current studies use the ubiquitously expressed protein Telomere Repeat Factor 1 (TRF1) tagged with a fluorescent protein. TRF1 is a negative regulator of telomere length that binds exclusively to telomere repeats. Over-expression of the fluorescent protein fused to TRF1 has been a useful tool to track telomere movement. The foci formed by the tagged TRF1 protein accurately represent the number of telomeres expected in the cells and the localization is maintained throughout the cell cycle. A caveat with this system is that over-expression of TRF1 leads to accelerated telomere shortening, as well as replication defects that can stall telomere replication. These caveats make it difficult to draw conclusions about telomere dynamics based solely on observations of cells over-expressing fluorescently tagged TRF1. To eliminate problems associated with protein over-expression, I have tried to develop knock-in embryonic stem (ES) cells expressing fluorescently tagged TRF1 from the endogenous Trf1 promoter. To do this, I have used a recombineering technique using Bacterial Artificial Chromosomes (BACs). BAC recombineering allows for the direct knock-in of a fluorescent tag into the mouse Trf1gene locus. Genetic constructs with the correct sequence inserts have been obtained and have been used for transfection of ES cells. While no correctly targeted ES cells have been identified so far, the expectation is that ES cell lines with correctly targeted fluorescently tagged TRF1 will be obtained in the near future. Such lines will be used to study telomere dynamics in ES cells, differentiated cells generated from ES cells, as well as to generate mice.

O diagnóstico da tuberculose por métodos tradicionais é lento e laborioso. Por outro lado, os testes moleculares são rápidos, mas com custo elevado para países em desenvolvimento. Este projeto teve o objetivo de inserir no micobacteriófago D29 o gene que codifica a proteína verde fluorescente (eGFP) e estudar o fago recombinante na detecção rápida de bacilos da tuberculose. Para tanto, foi inserido o cassete Hsp60- eGFP no genoma do fago D29 por recombinação. Micobacteriófagos recombinantes purificados foram utilizados para infectar M. smegmatis mc2 155 e M. tuberculosis H37Rv durante um período de 1- 6h nas temperaturas de 30°C, 37°C e 42°C. Bactérias fluorescentes foram observadas em um período de 2h, mas em número reduzido, indicando que o micobacteriófago lisou às células rapidamente, dificultando a expressão da eGFP e visualização em microscópio de fluorescência. A deleção do gene LysA, foi efetuada a fim de aumentar o período de latência do fago. Não foi possível a purificação de fagos recombinantes, devido à baixa quantidade de recombinantes nos halos de inibição. Será necessário a redução da atividade o gene LysA e, provavelmente, de outros genes associados a lise celular a fim de aumentar a concentração de eGFP no interior da célula. / Classical biochemical methods for Mycobacterium tuberculosis identification are lengthy and time-consuming. On the other hand, molecular assays are rapid but expensive for developing countries. This project aimed to insert into the mycobacteriophage D29, the gene coding for the green fluorescent protein (eGFP) and use the recombineered phage to detect Mycobacterium tuberculosis rapidly and less costly. For that, the Hsp-eGFP cassette was inserted into D29 genome. Recombineered mycobacteriophages was purified and used to infect M. smegmatis mc2 155 and M. tuberculosis H37Rv from 1-6 hs at 30°C, 37°C and 42°C. Observation of fluorescent bacteria was difficult and only a small number of them were seen at 2 hs of infection. This indicated that recombineered bacteriophages were lysing cells rapidly. Deletion of LysA gene, was carried out to increase the time needed for bacterial lysing. it was not possible to purify mutant mycobacteriophages due to the low concentration of recombinant phages. We conclude that might be necessary the deletion of other genes such as LysB, a gene also involved in cell lysis and reduction LysA activity to increase the concentration of eGFP inside cells.

Telomeres are the repetitive DNA sequence and associated proteins found at the ends of linear chromosomes. They have a role in biological processes including meiosis and aging as well as implications in a number of genomic instability disorders and cancers. Telomeres maintain genomic stability by protecting chromosome ends from terminal fusions and misidentification as DNA damage sites. Their wide range of functions has resulted in an increased interest in developing tools to study the dynamics of telomeres in live cells. To do this, current studies use the ubiquitously expressed protein Telomere Repeat Factor 1 (TRF1) tagged with a fluorescent protein. TRF1 is a negative regulator of telomere length that binds exclusively to telomere repeats. Over-expression of the fluorescent protein fused to TRF1 has been a useful tool to track telomere movement. The foci formed by the tagged TRF1 protein accurately represent the number of telomeres expected in the cells and the localization is maintained throughout the cell cycle. A caveat with this system is that over-expression of TRF1 leads to accelerated telomere shortening, as well as replication defects that can stall telomere replication. These caveats make it difficult to draw conclusions about telomere dynamics based solely on observations of cells over-expressing fluorescently tagged TRF1. To eliminate problems associated with protein over-expression, I have tried to develop knock-in embryonic stem (ES) cells expressing fluorescently tagged TRF1 from the endogenous Trf1 promoter. To do this, I have used a recombineering technique using Bacterial Artificial Chromosomes (BACs). BAC recombineering allows for the direct knock-in of a fluorescent tag into the mouse Trf1gene locus. Genetic constructs with the correct sequence inserts have been obtained and have been used for transfection of ES cells. While no correctly targeted ES cells have been identified so far, the expectation is that ES cell lines with correctly targeted fluorescently tagged TRF1 will be obtained in the near future. Such lines will be used to study telomere dynamics in ES cells, differentiated cells generated from ES cells, as well as to generate mice. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

A systematic approach based on bacteriophage Lambda (Lambda Red) and flippase-flippase recognition targets (FLP-FRT) recombinations was proposed for genomic engineering of Escherichia coli. For demonstration purposes, DNA operons containing heterologous genes (i.e. pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e. Ptrc and ParaB), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e. lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e. kanamycin and chloramphenicol) under various genetic backgrounds (i.e. HB101 and DH5α). The expression of the inserted foreign genes was subject to regulation using appropriate inducers [Isopropyl β-D-1-thiogalactopyranoside (IPTG) and arabinose] at tuneable concentrations. The developed approach has paved an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.