In numerous Gram-negative and Gram-positive bacteria as well as in Archaea SL proteins form the outermost layer of the cell envelope. SL (glyco)monomers self-assemble with oblique (p2), tetragonal (p4), or hexagonal (p3, p6) symmetries [12]. SL subunits interact with each other and with the underlying cell surface by relatively weak non-covalent forces such as hydrogen-bonds, ionic bonds, salt-bridges or hydrophobic interactions. This makes them easy to isolate by applying chaotropic agents like urea and guanidine hydrochloride (GuHCl), chelating chemicals, or by changing the pH of the environment [10]. Upon dialysis in an ambient buffer monomers recrystallize into regular arrays that possess the forms of flat sheets, open ended cylinders, or spheres on solid substrates, at air-water intefaces and on lipid films, making them appealing for nanobiotechnological applications [3, 18]. The aim of this study was to investigate the structure, thermal stability, in vivo self-assembly process, recrystallization and metallization of three different recombinant SL proteins (SslA-eGFP, mSbsC-eGFP and S13240-eGFP) expressed in yeast S. cerevisiae BY4741 which could be further used in nanobiotechnological applications. In order to fulfill this aim, I investigated the in vivo expression of SL proteins (SslA, SbsC, S13240) tagged with eGFP (SL-eGFP) in the yeast S. cerevisiae BY4141. First, I characterized the heterologous expression of SL fusion constructs with growth and fluorescence measurements combined with Western blot analyses. Fluorescence microscopy investigations of overnight grown cultures showed that SslA-eGFP fusion protein was expressed as fluorescent patches, mSbsC-eGFP as tubular networks, and S13240-eGFP as hollow-like fibrillar network structures, while eGFP did not show any distinct structure Thermal stability of in vivo expressed SL-eGFP fusion proteins were investigated by fluorescence microscopy and immunodetection. In vivo self-assembly kinetics during mitosis and meiosis was the second main issue. In parallel, association of in vivo mSbsC-eGFP structures with the cellular components was of interest. A network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton was observed. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis surface layer structures were formed de novo in the buds. Surface layer assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point. In order to get these in vivo SL assemblies stably outside the cells (in situ), cell distruption experiments were conducted. The tubular structures formed by the protein in vivo were retained upon bursting the cells by osmotic shock; however their average length was decreased. During dialysis, monomers obtained by treatment with chaotropic agents recrystallized again to form tube-like structures. This process was strictly dependent on calcium ions, with an optimal concentration of 10 mM. Further increase of the Ca2+ concentration resulted in multiple non-productive nucleation points. It was further shown that the lengths of the S-layer assemblies increased with time and could be controlled by pH. After 48 hours the average length at pH 9.0 was 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications. For example, such metalized protein nanotubes could be used in conductive nanocircuit technologies as nanowires.

Results of recent studies indicate that dendritic cells are capable of transporting commensal intestinal bacteria into the mammary glands, which ultimately leads to their occurrence in breast milk. We have therefore decided to evaluate the phenotype of immunologically relevant antigen presenting cells (APCs) present in the mammary glands and the small intestine, respectively and perform a comparison study. We also studied plasticity of these populations during lactation. In situ immunodetection and flow cytometry methods were used to determine phenotype. We succeeded in optimising the methods for preparation of samples for flow cytometry and microscopy. We thoroughly tested protocols for 3D visualisation of APC populations and quantitative image analysis for correlation with flow cytometry, further optimization is nevertheless needed. We found out that during lactation large numbers of MHC II+ cells cluster around the alveoli and milk ducts. These cells are of a distinctly dendritic shape and their phenotype does not correspond to the APCs in the surrounding tissue. A pronounced increase of APC cells in the mammary glands between the fourth and sixth days of lactation was observed, with the majority of these cells expressing the CD103 antigen typical for cell populations of immune cells of the...

A baixa eficiência e a dificuldade de reprodução de resultados da técnica de transferência gênica mediada por espermatozoides (TGME) têm como possível explicação à ativação de endonucleases espermáticas. Assim, a inibição desta enzima poderia evitar a fragmentação de DNA (exógeno e genômico), possibilitando assim, o uso de maiores concentrações de DNA exógeno, aumentando a eficiência e garantindo a reprodutibilidade da técnica. O ácido aurintricarboxílico (ATA) é um inibidor geral de endonucleases (HALLICK et al., 1977), inclusive das endonucleases espermáticas (MAIONE et al., 1997; MAGNANO et al., 1998). Deste modo, o presente estudo objetivou avaliar a inibição das endonucleases espermáticas, pelo ácido aurintricarboxílico (ATA). Para isso, três experimentos foram realizados: 1) avaliar a inibição das endonucleases espermáticas pela adição do ácido aurintricarboxílico, após incubação com DNA exógeno; 2) verificar a eficiência do ATA na inibição de fragmentação de DNA genômico e 3) detectar o aumento nos índices de internalização após o uso de ATA. Para o primeiro experimento, um ensaio de digestão plasmidial com os plasmídeos PCX-EGFP e pmGENIE3 e três concentrações de ATA (10µM, 25µM e 50µM) foram testados. As digestões dos vetores plasmidiais ocorreram pela incubação dos plasmídeos PCX-EGFP e pmGENIE3, com e sem a presença de ATA, com extratos espermáticos. As incubações ocorreram durante 1 hora à 37ºC e os produtos foram analisados por eletroforese (2 horas, 100mV) em gel de agarose 0,7%. Os resultados foram avaliados em escala de cruzes, no qual 1 foi considerado digestão total dos plasmídeos e 3, a não digestão. Os resultados foram analisados em nível de significância de 5%. Os resultados demonstraram diferenças nas digestões dos dois vetores plasmidiais, sendo o pmGENIE3 mais susceptível à degradação pelo extrato espermático, demonstrando ausência de bandas em algumas replicatas (mediana=1). O PCX-EGFP apresentou inibição parcial da degradação já com 10µM de ATA. Já o pmGENIE só apresentou inibição da degradação com 25 ou 50µM de ATA. Assim a concentração utilizada nos experimentos consecutivos foi a de 50µM. Para o experimento 2, espermatozoides de camundongos da linhagem Bl-6/DBA (F1) foram incubados com duas concentrações (500 ou 1000ng) do plasmídeo PCX-EGFP, com ou sem pré-incubação com ATA. As incubações ocorreram durante 5 horas, em ar com 5% de CO2 à 37ºC. Assim, os espermatozoides foram submetidos ao teste de susceptibilidade à denaturação ácida e ao ensaio de cometa alcalino para verificar possível fragilidade da cromatina. Os dados demonstraram que o uso do ATA em espermatozoides murinos leva à fragilidade do genoma, independente de serem incubados com DNA exógeno e a concentração do mesmo. Além disso, foi possível verificar que pode existir um limiar de concentração ótima para que as endonucleases causem fragmentação do DNA cromossomal, o qual foi de 500ng. O uso de concentrações maiores, como 1000ng, pode ter agido como fator protetor ao DNA genômico, podendo este DNA exógeno ter sido o alvo primário das endonucleases, já que quando as amostras foram incubadas com essa concentração de plasmídeo, não houve altos índices de fragmentação no DNA endógeno. No experimento 3, espermatozoides de camundongos foram incubados com 500 ou 1000ng de PCX-EGFP/106 células, sendo ou não pré-incubados com ATA. O DNA genômico das células espermáticas foi extraído pelo método de fenol clorofórmio, diluído para concentração de 1ng/µl e submetidos à quantificação de DNA plasmidial pela técnica de quantificação absoluta em tempo real (qPCR). Os resultados demonstraram que o ATA não melhorou a eficiência de incorporação, já que tanto na concentração de 500 quanto na de 1000ng de DNA exógeno, a porcentagem foi menor (0,001% para os dois grupos) em relação aos grupos nos quais este não foi utilizado. Os grupos em que o ATA não foi utilizado não apresentaram diferença entre si demonstrando que a quantidade de 500ng de DNA exógeno foi suficiente na internalização deste ao espermatozoide, sendo a porcentagem de incorporação maior do que 1000ng (0,20% contra 0,10%). Contudo, o uso do inibidor de endonucleases, ao invés de aumentar os índices de incorporação apresentou resultados opostos, indicando que seu uso não trouxe melhorias para a técnica de TGME. / The low efficiency and low repeatability of sperm-mediated gene transfer (SMGT) could be due to the activation of sperm endonucleases. The inhibition of this enzyme would avoid genomic DNA fragmentation enabling the use of higher concentrations of exogenous DNA, increasing the efficiency and ensuring the reproducibility of this technique. Aurintricarboxilic acid (ATA) is a general inhibitor of endonucleases (HALLICK et al., 1977), including sperm endonucleases (MAIONE et al., 1997; MAGNANO et al., 1998). This study aimed to evaluate the inhibition of sperm endonucleases using the aurintricarboxilic acid (ATA). For that, three experiments were set: 1) evaluate the inhibition of sperm endonucleases by adding aurintricarboxilic acid after incubation with exogenous DNA, 2) study the inhibition efficiency of ATA in genomic DNA fragmentation and 3) detect exogenous DNA internalization after the use of ATA. For the first experiment, a plasmid digestion assay with pmGENIE3 and PCX-EGFP and three concentrations of ATA (10µM, 25µM and 50µM) were tested. The digestion of plasmid vector occurred by incubation of PCX-EGFP and pmGENIE3 with and without the presence of ATA with sperm extracts. Incubations took place for 1 hour at 37°C and the products were analyzed by electrophoresis (2 hours, 100mV) in 0,7% agarose gel. The results were evaluated on a cross scale whereas 1 was considered a total plasmid digestion and 3 no digestion. The results were analyzed with a significance level of 5%. The results show differences in the digestion of the two plasmid vectors, being pmGENIE3 more susceptible to degradation by sperm extract, demonstrating absence of bands in some replicates (median = 1). The PCX-EGFP showed a parcial inhibition of the degradation using 10µM ATA. PmGENIE3 presented inhibiting of degradation only using 25 or 50µM of ATA. Thus, the concentration used in the consecutives experiments was 50µM. For experiment 2, sperm from Bl-6/DBA (F1) mice strain were incubated with two concentrations (500 or 1000ng) of the PCX-EGFP plasmid, with and without pre-incubation with ATA. Incubation took place for 5 hours, with 5% CO2 in air, at 37°C. Sperm samples were subjected to acid denaturation susceptibility test and alkaline comet assay to check for possible chromatin fragility. The data showed that the use of ATA in murine sperm leads to a fragility of their genome, independently of the incubation with exogenous DNA and its concentration. Result showed that there might be a threshold concentration for chromosomal DNA fragmentation caused by endonucleases, which was 500ng of plasmid. The use of higher concentrations, as 1000ng, may be a protective factor for genomic DNA integrity, since exogenous DNA seems to be the primary target of endonucleases, showed by, lower DNA fragmentation levels. In experiment 3, sperm were incubated with 500 or 1000ng PCX-EGFP/106 cells, pre-incubated or not with ATA. Genomic DNA was extracted by phenol chloroform method, diluted to concentrations of 1ng/µl and subjected to quantification of plasmid DNA insertions by absolute quantification in real-time PCR (qPCR). The results showed that ATA did not improve the efficiency of DNA internalization, whereas both concentration of 500 and 1000ng presented a lower percentage of exogenous DNA integration (0.001% in both groups) compared with the groups in which ATA was not used. The groups without ATA did not differ indicating that the amount of 500ng of DNA was able to integrate exogenous DNA to sperm, and have higher percentage of incorporation compared to 1000ng (0,20% versus 0,10%). Thereby, the use of an endonuclease inhibitor instead of increasing integration indexes showed opposite results, indicating that its use did not bring improvements to the SMGT technique.

In numerous Gram-negative and Gram-positive bacteria as well as in Archaea SL proteins form the outermost layer of the cell envelope. SL (glyco)monomers self-assemble with oblique (p2), tetragonal (p4), or hexagonal (p3, p6) symmetries [12]. SL subunits interact with each other and with the underlying cell surface by relatively weak non-covalent forces such as hydrogen-bonds, ionic bonds, salt-bridges or hydrophobic interactions. This makes them easy to isolate by applying chaotropic agents like urea and guanidine hydrochloride (GuHCl), chelating chemicals, or by changing the pH of the environment [10]. Upon dialysis in an ambient buffer monomers recrystallize into regular arrays that possess the forms of flat sheets, open ended cylinders, or spheres on solid substrates, at air-water intefaces and on lipid films, making them appealing for nanobiotechnological applications [3, 18]. The aim of this study was to investigate the structure, thermal stability, in vivo self-assembly process, recrystallization and metallization of three different recombinant SL proteins (SslA-eGFP, mSbsC-eGFP and S13240-eGFP) expressed in yeast S. cerevisiae BY4741 which could be further used in nanobiotechnological applications. In order to fulfill this aim, I investigated the in vivo expression of SL proteins (SslA, SbsC, S13240) tagged with eGFP (SL-eGFP) in the yeast S. cerevisiae BY4141. First, I characterized the heterologous expression of SL fusion constructs with growth and fluorescence measurements combined with Western blot analyses. Fluorescence microscopy investigations of overnight grown cultures showed that SslA-eGFP fusion protein was expressed as fluorescent patches, mSbsC-eGFP as tubular networks, and S13240-eGFP as hollow-like fibrillar network structures, while eGFP did not show any distinct structure Thermal stability of in vivo expressed SL-eGFP fusion proteins were investigated by fluorescence microscopy and immunodetection. In vivo self-assembly kinetics during mitosis and meiosis was the second main issue. In parallel, association of in vivo mSbsC-eGFP structures with the cellular components was of interest. A network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton was observed. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis surface layer structures were formed de novo in the buds. Surface layer assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point. In order to get these in vivo SL assemblies stably outside the cells (in situ), cell distruption experiments were conducted. The tubular structures formed by the protein in vivo were retained upon bursting the cells by osmotic shock; however their average length was decreased. During dialysis, monomers obtained by treatment with chaotropic agents recrystallized again to form tube-like structures. This process was strictly dependent on calcium ions, with an optimal concentration of 10 mM. Further increase of the Ca2+ concentration resulted in multiple non-productive nucleation points. It was further shown that the lengths of the S-layer assemblies increased with time and could be controlled by pH. After 48 hours the average length at pH 9.0 was 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications. For example, such metalized protein nanotubes could be used in conductive nanocircuit technologies as nanowires.

La dystrophine est une protéine cytoplasmique qui lie physiquement le cytosquelette à la matrice extracellulaire par le biais du complexe dystrophine-protéines associées (DAPC), assurant ainsi la stabilité du sarcolemme. Des mutations dans le gène DMD codant pour la dystrophine, conduisant à l’absence de la protéine, sont à l’origine de la dystrophie musculaire de Duchenne qui est une maladie liée au chromosome X. Pour mes travaux de thèse, j’ai généré et caractérisé un nouveau modèle de souris transgéniques rapportrices, dénommé DmdEGFP, qui exprime une protéine dystrophine endogène fusionnée avec la protéine fluorescente EGFP. La protéine dystrophine est liée dans sa région C-terminale qui est présente dans la majorité des isoformes. Dans le modèle, une expression forte et naturelle de l’EGFP était observée dans les muscles squelettiques, lisses, le cœur, le cerveau et l’œil, ce qui suggère un étiquetage correct de tous les isoformes de la dystrophine. La fluorescence de l’EGFP co-localisait exactement avec la dystrophine dans tous les sites. Dans le muscle squelettique, la dystrophine ainsi que d’autres protéines de la DAPC étaient exprimées dans des quantités normales et dans la bonne localisation subsarcolemmale. L’architecture du tissu musculaire squelettique était normale, suggérant que la fonction de la protéine de fusion était maintenue. In vitro, l’EGFP est également exprimée dans les fibres musculaires isolées, ainsi que dans les myotubes dérivés des cellules satellites. Par conséquent, cette nouvelle souris rapportrice de la dystrophine devient un outil important pour la visualisation directe et in vivo de l’expression de la dystrophine. / Dystrophin is a rod-shaped cytoplasmic protein that physically links the cytoskeleton to the ECM through the dystrophin-associated protein complex (DAPC), thereby providing sarcolemmal stability. Mutations in the dystrophin encoding DMD gene cause the severe X-linked disorder Duchenne muscular dystrophy. In this work a novel DmdEGFP reporter mouse that expresses a fluorescently labelled endogenous dystrophin – EGFP fusion protein was generated and characterized. The protein was tagged at the C-terminus that is present in the most dystrophin isoforms. To date, no dystrophin reporter mice exist, thus imaging is only possible by indirect antibody-mediated processing ex vivo. In DmdEGFP mice strong natural EGFP expression was observed in skeletal, smooth muscles, heart, brain and the eye and EGFP fluorescence co-localized with dystrophin at all sites suggesting proper tagging of the major dystrophin isoforms. In skeletal muscle, dystrophin as well as other proteins of the DAPC were expressed in normal quantity at correct sarcolemmal/subsarcolemmal localization. Skeletal muscle maintained normal tissue architecture, suggesting a correct function of the fusion protein. Isolated myofibers as well as satellite-cell derived myotubes expressed EGFP in vitro. Thus, the novel dystrophin reporter mouse provides a valuable tool for direct visualization of dystrophin expression.

Proteases are essential for regulating a wide range of physiological and pathological processes. The imbalance of protease activation and inhibition will result in a number of major diseases including cancers, atherosclerosis, and neurodegenerative diseases. Although fluorescence resonance energy transfer (FRET)-based protease probes, a small molecular dye and other methods are powerful, they still have drawbacks or limitations for providing significant information about the dynamics and pattern of endogenous protease activation and inhibition in a single living cell or in vivo. Currently protease sensors capable of quantitatively measuring specific protease activity in real time and monitoring activation and inhibition of enzymatic activity in various cellular compartments are highly desired. In this dissertation, we report a novel strategy to create protease sensors by grafting an enzymatic cleavage linker into a sensitive location for changing chromophore properties of enhanced green fluorescent protein (EGFP) following protease cleavage, which can be used to determine protease activity and track protease activation and inhibition with a ratiometric measurement mode in living cells. Our designed protease sensors exhibit large relative ratiometric optical signal change in both absorbance and fluorescence, and fast response to proteases. Meanwhile, these protease sensors exhibiting high enzymatic selectivity and kinetic responses are comparable or better than current small peptide probes and FRET-based protease probes. Additionally, our protease sensors can be utilized for real-time monitoring of cellular enzymogen activation and effects of inhibitors in living cells. This novel strategy opens a new avenue for developing specific protease sensors to investigate enzymatic activity in real time, to probe disease mechanisms corresponding to proteases in vitro and in vivo, and to screen protease inhibitors with therapeutic effects. Strong fluorescence was still retained in the cleaved EGFP-based protease sensors, which stimulated us to identify the EGFP fragment with fluorescence properties for further understanding chromophore formation mechanisms and investigating protein-protein interactions through fluorescence complementation of split EGFP fragments. Through fusing EF-hand motifs from calbindin D9k to split EGFP fragments, a novel molecular probe was developed to simultaneously track the calcium change or calcium signaling pathways and calcium-dependent protein-protein interaction in living cells in real time.

Bacteriophage Lambda (λ) has played a historical role as an essential model contributing to our current understanding of molecular genetics. Lambda’s major capsid protein “gpD” occurs on each capsid at 405 to 420 copies per phage in homotrimeric form and functions to stabilize the head and likely to compact the genomic DNA. The interesting conformation of this protein allows for its exploitation through the genetic fusion of peptides or proteins to either the amino or carboxy terminal end of gpD, while retaining phage assembly functionality and viability. The lytic nature of λ and the conformation of gpD in capsid assembly makes this display system superior to other display options. Despite previous reports of λ as a phage display candidate, decorative control of the phage remains an elusive concept. The primary goal of this study was to design and construct a highly controllable head decoration system governed by two genetic conditional regulation systems; plasmid-mediated temperature sensitive repressor expression and bacterial conditional amber mutation suppression. The historical λ Dam15 conditional allele results in a truncated gpD fragment when translated in nonsuppressor, wild-type E. coli cells, resulting in unassembled, nonviable progeny. I sequenced the Dam15 allele, identifying an amber (UAG) translational stop at the 68th codon. Employing this mutant in combination with a newly created isogenic cellular background utilizing the amber suppressors SupD (Serine), SupE (Glutamine), SupF (Tyrosine) and Sup— (wild type), we sought to control the level of incorporation of undecorated gpD products. As a second dimension, I constructed two separate temperature-inducile plasmids whereby expression of either D or D::eGFP was governed by the λ strong λ CI[Ts]857 temperature-sensitive repressor and expressed from the λ PL strong promoter. Our aim was to measure the decoration of the λ capsid by a D::gfp fusion under varying conditions regulated by both temperature and presence of suppression. This was achieved utilizing this controllable system, enabling the measurement of a variable number of fusions per phage based on diverse genetic and physical environments without significantly compromising phage viability. Surprisingly, both SupE and SupF showed similar levels of Dam15 suppression, even though sequencing data indicated that only SupE could restore the native gpD sequence at amino acid 68 (Q). In contrast, SupD (S), conferred very weak levels of suppression, but imparted an environment for very high decoration of gpD::eGFP per capsid, even at lower (repressed) temperatures. The presence of albeit few wild-type gpD molecules allowed for an even greater display than that of the perceived “100%” decoration scenario provided by the nonsuppressor strain. It appears that the lack of wild-type gpD does not allow for the space required to display the maximum number of fusions and in turn creates an environment that affects both phage assembly and therefore phage viability. Finally, the use of Western blotting, confirmed the presence of gpD::eGFP fusion decoration by employing a polyclonal anti-eGFP antibody. The significance of this work relates to the unique structure of λ’s capsid and its ability to exploit gpD in the design of controlled expression, which is guiding future research examining the fusion of different therapeutic peptides and proteins. Furthermore this approach has important implications specifically for the design of novel vaccines and delivery vehicles for targeted gene therapy in which steric hindrance and avidity are important concerns. The execution of this project employed basic bacterial genetics, phage biology and molecular biology techniques in the construction of bacterial strains and plasmids and the characterization of the phage display system.

NMDA (N-methyl D-aspartate) Receptor is a ligand and voltage gated ion channel and involved in many processes such as synaptic plasticity, memory formation, behavioral responses and cell survival. In the sense of functional activity, cellular localization of NMDAR is important since this receptor shows its activity on the membrane. Although NMDA receptor is intensely studied there are no satisfying study showing its localization with microscobic methods. Besides, the effect of florescent protein labelling of NMDA receptor on its distribution is not shown. It is expected to provide basis for further interaction and distribution studies with this comparison. Contrary to literature, in this study it is shown that NMDA receptor does not localize only in ER and membrane instead has a cytosolic pattern and this pattern is compatible with the distribution of wild type NMDA receptor. In addition, florescent protein labelling of NMDA receptor does not interrupt cellular distribution of NMDAR. Moreover, this study shows that N-terminal domain of NR1 subunit is sufficient to prevent degradation of NR2B in the cell. In consideration of this study it can be concluded that EGFP and mCherry labelled NMDA receptors can be used in interaction studies such as FRET and other studies, making use of fluorescent labelling of NMDA receptors, in terms of cellular distribution.

The largest class of cell surface receptors in mammalian genomes is the superfamily of G protein-coupled receptors (GPCRs) which are activated by a wide range of extracellular responses such as hormones, pheromones, odorants, and neurotransmitters. Drugs which have therapeutic effects on a wide range of diseases are act on GPCRs. In contrast to traditional idea, it is recently getting accepted that G-protein coupled receptors can form homo- and hetero-dimers and this interaction could have important role on maturation, internalization, function or/and pharmacology. Bimolecular fluorescence complementation technique (BiFC) / is an innovative approach based on the reassembly of protein fragments which directly report interactions. In our study we implemented this technique for detecting and visualizing the GPCR interactions in yeast cells. The enhanced green fluorescent protein (EGFP) fractionated into two fragments at genetic level which does not possess fluorescent function. The target proteins which are going to be tested in terms of interaction are modified with the non-functional fragments, to produce the fusion proteins. The interaction between two target proteins, in this study Ste2p receptors which are alpha pheromone receptors from Saccharomyces cerevisiae, enable the fragments to come in a close proximity and reassemble. After reassembly, EGFP regains its fluorescent function which provides a direct read-out for the detection of interaction. Further studies are required to determine subcellular localization of the interaction. Moreover, by using the fusion protein partners constructed in this study, effects of agonist/antagonist binding and post-translational modifications such as glycosylation and phosphorylation can be examined. Apart from all, optimized conditions for BiFC technique will guide for revealing new protein-protein interactions.

Bacteriophage Lambda (λ) has played a historical role as an essential model contributing to our current understanding of molecular genetics. Lambda’s major capsid protein “gpD” occurs on each capsid at 405 to 420 copies per phage in homotrimeric form and functions to stabilize the head and likely to compact the genomic DNA. The interesting conformation of this protein allows for its exploitation through the genetic fusion of peptides or proteins to either the amino or carboxy terminal end of gpD, while retaining phage assembly functionality and viability. The lytic nature of λ and the conformation of gpD in capsid assembly makes this display system superior to other display options. Despite previous reports of λ as a phage display candidate, decorative control of the phage remains an elusive concept. The primary goal of this study was to design and construct a highly controllable head decoration system governed by two genetic conditional regulation systems; plasmid-mediated temperature sensitive repressor expression and bacterial conditional amber mutation suppression. The historical λ Dam15 conditional allele results in a truncated gpD fragment when translated in nonsuppressor, wild-type E. coli cells, resulting in unassembled, nonviable progeny. I sequenced the Dam15 allele, identifying an amber (UAG) translational stop at the 68th codon. Employing this mutant in combination with a newly created isogenic cellular background utilizing the amber suppressors SupD (Serine), SupE (Glutamine), SupF (Tyrosine) and Sup— (wild type), we sought to control the level of incorporation of undecorated gpD products. As a second dimension, I constructed two separate temperature-inducile plasmids whereby expression of either D or D::eGFP was governed by the λ strong λ CI[Ts]857 temperature-sensitive repressor and expressed from the λ PL strong promoter. Our aim was to measure the decoration of the λ capsid by a D::gfp fusion under varying conditions regulated by both temperature and presence of suppression. This was achieved utilizing this controllable system, enabling the measurement of a variable number of fusions per phage based on diverse genetic and physical environments without significantly compromising phage viability. Surprisingly, both SupE and SupF showed similar levels of Dam15 suppression, even though sequencing data indicated that only SupE could restore the native gpD sequence at amino acid 68 (Q). In contrast, SupD (S), conferred very weak levels of suppression, but imparted an environment for very high decoration of gpD::eGFP per capsid, even at lower (repressed) temperatures. The presence of albeit few wild-type gpD molecules allowed for an even greater display than that of the perceived “100%” decoration scenario provided by the nonsuppressor strain. It appears that the lack of wild-type gpD does not allow for the space required to display the maximum number of fusions and in turn creates an environment that affects both phage assembly and therefore phage viability. Finally, the use of Western blotting, confirmed the presence of gpD::eGFP fusion decoration by employing a polyclonal anti-eGFP antibody. The significance of this work relates to the unique structure of λ’s capsid and its ability to exploit gpD in the design of controlled expression, which is guiding future research examining the fusion of different therapeutic peptides and proteins. Furthermore this approach has important implications specifically for the design of novel vaccines and delivery vehicles for targeted gene therapy in which steric hindrance and avidity are important concerns. The execution of this project employed basic bacterial genetics, phage biology and molecular biology techniques in the construction of bacterial strains and plasmids and the characterization of the phage display system.