O desenvolvimento de novas ferramentas para manipulação genética é necessário para um melhor entendimento da biologia de protozoários que causam doenças sérias e negligenciadas. Neste contexto, apresentamos uma nova aplicação do sistema Cre recombinase em Leishmania major, utilizado para a expressão condicional de genes de interesse. Como prova de conceito demonstramos por ensaios de PCR, western blotting e imunofluorescência que o gene da Proteína Fluorescente Verde (Green Fluorescent Protein, GFP) é condicionalmente expresso em função do tempo e dose de rapamicina necessários para a ativação da recombinase. A aplicação do sistema diCre em L. major é feita com o estudo da proteína Rad9, que participa na sinalização e resposta a danos ao DNA. A Rad9 de L. major possui um domínio C-terminal desestruturado, que no homólogo humano não é essencial para a formação do complexo 911 (Rad9-Hus1-Rad1), mas possui sítios de fosforilação importantes para a cascata de sinalização que mantém a integridade do DNA. Usando predições da estrutura de Rad9, a proteína foi dividida em domínios e foram geradas linhagens que expressam, condicionalmente, versões truncadas de Rad9. Por análises de PCR, western blotting, citometria de fluxo e imunofluorescência, acessamos alguns dos papeis que o domínio C-terminal de Rad9 pode desempenhar em L. major. / The development of new tools for genetic manipulation is necessary for a better understanding of the biology of protozoa that cause severe and neglected diseases. In this context, we present a new application of the Cre recombinase system in Leishmania major, using it for the conditional expression of genes of interest. As proof of concept we show by PCR, western blotting and immunofluorescence assays that the Green Fluorescent Protein (GFP) gene can be conditionally expressed depending on the time and dose of the rapamycin treatment required for the recombinase activation. The application of the diCre system in L. major was tested with the study of the Rad9 protein, which participates in the parasite\'s DNA damage response. Rad9 from L. major presents an unstructured C-terminal domain, which in the human homolog is not essential for the 911 (Rad9-Hus1-Rad1) complex formation, but has phosphorylation sites that are important in the signaling cascade that maintains the DNA integrity. Using structure predictions of Rad9, the protein was divided into specific domains and cell lines were generated conditionally expressing truncated versions of Rad9. By PCR, western blotting, flow cytometry and immunofluorescence assays, we assessed some of the roles that the C-terminal domain of Rad9 can perform in L. major.

L’un des objectifs principal de la biologie synthétique est de reprogrammer les organismes vivants pour résoudre des challenges mondiaux actuelles dans le domaine industriel, environnemental et de la santé. Tandis que de nombreux types de portes logiques génétiques ont été conçus, leur extensibilité reste limitée. Effectivement, la conception de portes logiques reste en grande partie un processus fastidieux et repose soit sur l’intuition humaine, soit sur des méthodes computationnelles de force brute. De plus, les circuits conçus sont généralement de grande taille et ne sont donc pas faciles à implémenter dans les organismes vivants.Durant ma thèse, mon objectif a été d’augmenter la puissance de calcul des circuits logiques utilisant des intégrases tout en permettant aux chercheurs d’implémenter simplement ces circuits à un large éventail d'organismes et d’entrées.Tout d’abord, j’ai développé un cadre extensible et composable pour le design systématique de systèmes multicellulaires implémentant de la logique Booléenne et histoire dépendent. Ce design est basé sur l'utilisation de sérine intégrases et peut intégrer un nombre arbitraire d’entrée. J’ai implémenté dans Escherichia coli des circuits logiques Booléens multicellulaires jusqu’à quatre entrées et des circuits histoire-dépendent jusqu’à 3 entrées. En raison de son extensibilité et de sa composabilité, ce design permet une implémentation simple et directe de circuits logiques dans des systèmes multicellulaires.J’ai également poussé le compactage des circuits logiques biologiques. Pour cela, j’ai généré une base de données complète de tous les circuits logiques unicellulaires possibles pour l’implémentation de fonctions booléennes à deux, trois et quatre entrées. La caractérisation d’un ensemble réduit des circuits de cette base de données devra être effectuée pour prouver la faisabilité de leur implémentation.Je pense que ces différentes stratégies de conception et les différents outils distribués (pièces biologiques et interface web) aideront les chercheurs et les ingénieurs à reprogrammer le comportement cellulaire de manière simple pour diverses applications. / A major goal of synthetic biology is to reprogram living organisms to solve pressing challenges in manufacturing, environmental remediation, or healthcare. While many types of genetic logic gates have been engineered, their scalability remains limited. Indeed, gate design remains largely a tedious process and relies either on human intuition or on brute-force computational methods. Additionally, designed circuits are usually large and therefore not straightforward to implement in living organisms.Here, I aimed at increasing the computation power of integrase-based logic circuits while permitting researchers to simply implement these circuits to a large range of organisms and of inputs.First, I developed a scalable composition framework for the systematic design of multicellular systems performing integrase-based Boolean and history-dependent logic and integrating an arbitrary number of inputs. I designed multicell Boolean logic circuits in Escherichia coli to up to 4 inputs and History-dependent circuits to 3 inputs. Due to its scalability and composability, this design framework permits a simple and straightforward implementation of logic circuits in multicellular systems.I also pushed forward the compaction of biological logic circuits. I generated a complete database of single-cell integrase-based logic circuits to obtain all possible designs for the implementation of up to 4-input Boolean functions. Characterization of a reduced set of circuits will have to be performed to prove the feasibility of the implementation of these circuits.All these design strategies can be implemented via easily accessible web interfaces, and open collections of biological components that are made available to the scientific community. These tools will enable researchers and engineers to reprogram cellular behavior for various applications in a streamlined manner.

O desenvolvimento de novas ferramentas para manipulação genética é necessário para um melhor entendimento da biologia de protozoários que causam doenças sérias e negligenciadas. Neste contexto, apresentamos uma nova aplicação do sistema Cre recombinase em Leishmania major, utilizado para a expressão condicional de genes de interesse. Como prova de conceito demonstramos por ensaios de PCR, western blotting e imunofluorescência que o gene da Proteína Fluorescente Verde (Green Fluorescent Protein, GFP) é condicionalmente expresso em função do tempo e dose de rapamicina necessários para a ativação da recombinase. A aplicação do sistema diCre em L. major é feita com o estudo da proteína Rad9, que participa na sinalização e resposta a danos ao DNA. A Rad9 de L. major possui um domínio C-terminal desestruturado, que no homólogo humano não é essencial para a formação do complexo 911 (Rad9-Hus1-Rad1), mas possui sítios de fosforilação importantes para a cascata de sinalização que mantém a integridade do DNA. Usando predições da estrutura de Rad9, a proteína foi dividida em domínios e foram geradas linhagens que expressam, condicionalmente, versões truncadas de Rad9. Por análises de PCR, western blotting, citometria de fluxo e imunofluorescência, acessamos alguns dos papeis que o domínio C-terminal de Rad9 pode desempenhar em L. major. / The development of new tools for genetic manipulation is necessary for a better understanding of the biology of protozoa that cause severe and neglected diseases. In this context, we present a new application of the Cre recombinase system in Leishmania major, using it for the conditional expression of genes of interest. As proof of concept we show by PCR, western blotting and immunofluorescence assays that the Green Fluorescent Protein (GFP) gene can be conditionally expressed depending on the time and dose of the rapamycin treatment required for the recombinase activation. The application of the diCre system in L. major was tested with the study of the Rad9 protein, which participates in the parasite\'s DNA damage response. Rad9 from L. major presents an unstructured C-terminal domain, which in the human homolog is not essential for the 911 (Rad9-Hus1-Rad1) complex formation, but has phosphorylation sites that are important in the signaling cascade that maintains the DNA integrity. Using structure predictions of Rad9, the protein was divided into specific domains and cell lines were generated conditionally expressing truncated versions of Rad9. By PCR, western blotting, flow cytometry and immunofluorescence assays, we assessed some of the roles that the C-terminal domain of Rad9 can perform in L. major.

L'objectif principal de mon projet de doctorat est d'étudier l'évolution génomique de l'ordre des Archaea Thermococcales. Je me suis intéressé à comprendre les mécanismes des éléments mobiles génétiques (MGE) pouvant influencer l'évolution des génomes. En utilisant une approche multidisciplinaire, nous avons pu explorer les différents aspects de ce phénomène in silico, in vitro et in vivo. Grâce à des analyses in silico de tous les génomes de Thermococcales complètement séquencés disponibles, nous avons montré que cet ordre affiche un niveau élevé de réarrangements pouvant perturber les modèles d'expression génique. Dans une première approche, nous avons étudié l'existence de l'organisation chromosomique. L'inefficacité dans la prédiction de l'origine et de la terminaison de la réplication sur la seule base de la composition de l'ADN chromosomique ou skew, nous a motivé à utiliser une approche différente basée sur des séquences biologiquement pertinentes. Nous avons donc déterminé la position de l'origine de la réplication (oriC) dans tous les 21 génomes séquencés Thermococcales. La position potentielle de la terminaison a été prédite dans 19 génomes à ou près du site dif, où les dimères chromosomiques sont résolus avant la ségrégation de l'ADN. Le calcul du génome central a révélé un certain nombre de grappes de gènes essentiels avec une position chromosomique remarquablement stable à travers les espèces, en utilisant oriC comme référence. D'autre part, les régions core-free semblent correspondre à des éléments mobiles intégrés putatifs. Ces observations indiquent qu'un degré remarquable d ' «ordre» a été maintenu à travers les Thermococcales, même s'ils présentent des chromosomes fortement brouillés, les inversions étant particulièrement fréquentes. La découverte et la caractérisation d'un nouvel organisme, Thermococcus nautili nous ont permis de mieux comprendre le mécanisme sous-jacent causant ces inversions. En effet, le séquençage et l'analyse in silico de son génome ont fortement suggéré l'implication d'une nouvelle classe de tyrosine recombinases dans la plasticité génomique. Le plasmide pTN3 de T. nautili, qui est intégré dans le chromosome et auto-réplicable, code une intégrase appartenant à la classe des tyrosine recombinases. Des plasmides similaires ont également été trouvés intégrés dans le chromosome d'autres séquences de Thermococcales (par exemple TKV4 dans T. kodakarensis). Afin de tester son activité enzymatique, l’integrase codée par le plasmide pTN3 a été surproduite et purifiée. Les expériences in vitro ont d'abord permis de déterminer le segment de séquence minimal requis pour l'activité de l'intégrase et optimisé la réaction enzymatique in vitro. Ces résultats nous ont permis, en suite, de démontrer la réaction d'excision / d'intégration observée avec d'autres recombinases de tyrosine. De plus, l'excision in vivo d'un élément intégré apparenté (TKV4 de T. kodakarensis) par l'intégrase pTN3 a été réalisée au cours de cette étude. Pour cela, le gène IntpTN3 a été clone dans un vecteur de la bactérie E. coli / Thermococcus pour la transformation et l'expression dans T. kodakarensis. Après incubation, les cellules ont montré la présence de l'élément TKV4-intégré dans la forme circulaire libre. Enfin, nous avons pu imiter l'inversion chromosomique in vitro en utilisant des substrats synthétiques contenant des séquences cibles d'intégration. Nous avons également pu montrer que l'intégrase pTN3 possède une activité qui peut intervenir sur des inversions génomiques à grande échelle en utilisant différents sites et donc expliquer les réarrangements observés dans Thermococcales (Cossu et al, in prep). / The main goal of my PhD project is to investigate the genomic evolution of the Archaea Thermococcales order. I am interested in understanding how mobile genetic elements (MGE) can influence the evolution of genomes. Using a multidisciplinary approach, we were able to explore the different aspects of this phenomenon in silico, in vitro and in vivo. Through in silico analyses of all available completely sequenced Thermococcales genomes, we showed that this order displays a characteristic high level of rearrangements potentially disrupting gene expression patterns. In a first approach, we investigated the existence of chromosomal organization. The inefficiency in predicting origin and termination of replication on the sole basis of chromosomal DNA composition or skew, motivated us to use a different approach based on biologically relevant sequences. We determined the position of the origin of replication (oriC) in all 21 sequenced Thermococcales genomes. The potential position of the termination was predicted in 19 genomes at or near the dif site, where chromosome dimers are resolved before DNA segregation. Computation of the core genome uncovered a number of essential gene clusters with a remarkably stable chromosomal position across species, using oriC as reference. On the other hand, core-free regions appear to correspond to putative integrated mobile elements. These observations indicate that a remarkable degree of “order” has been maintained across Thermococcales even if they display highly scrambled chromosomes, with inversions being especially frequent. The discovery and characterization of a new organism, Thermococcus nautili allowed us to better understand the underlying mechanism causing these inversions. The sequencing and in silico analysis of its genome strongly suggested the involvement of a new class of tyrosine recombinases in genomic plasticity. T. nautili pTN3 plasmid, which is found integrated into the chromosome and also self-replicating encodes an integrase belonging to this class. Similar plasmids have also been found integrated in the chromosome of other sequenced Thermococcales (e.g. TKV4 in T. kodakarensis). In order to test its enzymatic activity, we overproduced and purified the integrase encoded by pTN3. In vitro experiments first determined the minimal sequence segment required for integrase activity and optimized the enzymatic reaction in vitro. Due to this early results, we were able to demonstrate the excision/integration reaction observed with other tyrosine recombinases. Additionally, the in vivo excision of a related integrated element (TKV4 from T. kodakarensis) by the pTN3 integrase was performed during this study. The IntpTN3 gene has been cloned into an E. coli/Thermococcus shuttle vector for transformation and expression in T. kodakarensis. After incubation, cells showed the presence of the TKV4-integrated element in free circular form. Finally, we were able to mimic in vitro chromosomal inversion using synthetic substrates containing integration target sequences. We were also able to show that pTN3 integrase possesses an activity which can mediate large scale genomic inversions using different sites and therefore explain the rearrangements observed in Thermococcales).

Förster resonance energy transfer (FRET) has become an important tool for studying biochemical reactions at the single-molecule level, despite its increasing maturity there is an on-going effort to improve and expand the technique. This thesis presents methods for extending conventional two-colour single-molecule FRET measurements; by expanding the range and applicability of single-molecule fluorescence methods a greater variety of biological reactions can be studied, in greater detail than previously possible. To circumvent the complexities of multi-colour FRET measurements and extend the range of observable distances I developed and characterised a new single-molecule fluorescence method termed tethered fluorophore motion (TFM). TFM is based on the existing technique of tethered particle motion (TPM) which relies on Brownian motion of a particle, attached to a surface by DNA, to probe the effective length of the DNA tether. TFM takes this concept and applies it at the single-fluorophore level, allowing simultaneous measurement of other fluorescence observables such as FRET and protein induced fluorescence enhancement (PIFE). Having developed TFM I combined it with FRET to study site-specific recombinase proteins at the single-molecule level, in greater detail than possible by either technique alone. Studying the model tyrosine recombinase Cre, I extend and clarify previous ensemble observations regarding the order of DNA strand exchange, as well as uncovering a previously unobserved complex conformation and molecular heterogeneity. Finally, I used TFM-FRET to study the more complex XerCD recombination system and its interaction with the DNA translocase FtsK. I made observations, for the first time, of synaptic complex formation and of recombination at the single-molecule level, and these suggest intriguing and unexpected intermediates in the recombination reaction. I also combine TFM with PIFE to investigate the mechanism of DNA looping by FtsK. The introduction of TFM, and its combination with other fluorescence techniques, allows observation of complex protein-DNA interactions from a variety of perspectives and will help expand the repertoire and applicability of single-molecule biophysical experiments.

Plasmodium falciparum est un parasite protozoaire responsable de la forme la plus sévère de la malaria. Depuis quelques années, les cas de résistance aux antipaludiques sont devenus de plus en plus fréquents et de plus en plus répandus. En plus de sa résistance aux drogues actuellement disponibles, ce parasite reste jusqu' à aujourd'hui réfractaire aux vaccinations. L’identification de nouvelles approches basées sur l`inhibition spécifique de certaines de ses cibles moléculaires vitales est devenue une nécessité. La recombinase à site spécifique de P. falciparum (Pf-Int) est un enzyme qui a été récemment identifié dans le laboratoire à partir de PlasmoDB. Cette recombinase à site spécifique joue potentiellement un rôle clé dans le système de recombinaison nécessaire à la viabilité du parasite. Cette protéine de 490 acides aminés, soit ~57 kDa, contient une région C-terminale qui porte les résidus conservés du site catalytique des recombinases à tyrosine R-H-K-R-(H/W)-Y. La prédiction montre une région N-terminale qui ressemble à celle de l’intégrase du phage lambda avec un mélange de structures secondaires α et β.Lors de ces travaux, nous avons d’abord montré par RT-PCR que le gène (MAL13P1.42) qui code pour PF-Int est transcrit pendant le cycle intra-érythrocytaire avec un maximum pendant la phase schizont. Nous avons ensuite essayé de montrer l`implication de Pf-Int dans le cycle parasitaire. Ceci a été réalisé grâce à un parasite (KO: knock-out) dont le gène Pf-Int a été invalidé. Ces analyses montrent que Pf-Int n'a aucun impact apparent sur le cycle de développement intra-érythrocytaire du parasite, en particulier sur la durée du cycle et le taux de croissance. Au niveau moléculaire, nous avons également procédé à la production d'anticorps anti-Pf-Int en utilisant le fragment C-162 (Résidus 162-490). La comparaison des profils de marquage, par cet anticorps, des extraits protéiques du KO et du parasite sauvage par la technique de Western blot n'a pas permis d'identifier la protéine endogène dans le parasite sauvage. Dans le but de déterminer la localisation sub-cellulaire de Pf-Int, nous avons réalisé des essais de sur-expression de différentes protéines de fusion dans le parasite. Nous avons essayé de déterminer l’impact de trois codons d’initiation différents ainsi que l’impact de la présence de la région N-terminale (1-190aa) de Pf-Int sur sa localisation subcellulaire en utilisant une chimère entre la partie N-terminale et la protéine GFP. Lors de ces travaux, nous avons réussi à sur-exprimer différentes régions de Pf-Int sous forme recombinante dans E. coli. Nous l’avons d’abord caractérisé par des études biophysiques. Ainsi nous avons pu déterminer, par dichroïsme circulaire (CD), le contenu en structures secondaires de Pf-Int, qui est proche de celui des autres membres de la même famille. Nous avons également démontré sa stabilité par CD couplé à la dénaturation thermique. Le spectre RMN-1D a aussi pu être enregistré. La troisième partie de nos travaux a concerné l’identification des cibles ADN de Pf-Int. Deux stratégies de recherche de cibles par affinité ont été utilisées au laboratoire en utilisant une première bibliothèque de séquences synthétisées chimiquement et une deuxième bibliothèque formée de fragments d’ADN génomique de P. falciparum. Ces deux approches ont permis l’identification de deux séries de cibles ADN. Grace aux cibles ADN identifiées, nous avons pu démontrer l’interaction de différents fragments de Pf-Int avec ces cibles par des expériences de retard sur gel natif (EMSA). Nous avons aussi pu démontrer que les protéines recombinantes sont actives in vitro. En effet, ces dernières sont capables de former des complexes covalents en présence de l’ADN cible. La conservation de la protéine, ainsi que son expression différentielle nous laisse à penser que son rôle est certes loin d’être élucidé, mais que Pf-Int reste une cible potentielle pour P. falciparum. / Plasmodium falciparum is a protozoan parasite responsible for the most severe form of malaria. In recent years, cases of resistance to antimalarial drugs have become increasingly frequent and common. In addition to its resistance to drugs currently available, there is no vaccine available against this parasite till now. The identification of new approaches based on the specific inhibition of some of its molecular targets has become vital.The identification of the Pf-Int site specific recombinase in Plasmodium falciparum by analysis of PlasmoDB is a new opportunity to study the role of genetic variation in this parasite as it needs to adapt to its hosts. This ~ 57 kDa protein contains a C-terminal domain carrying the putative tyrosine recombinase conserved active site residues R-H-K-R-(H/W)-Y, an N-terminus with a predicted alpha-helical bundle and a mixed alpha-beta domain resembling Lambda-Int. Here, we show that the sequence is highly conserved among members of the Plasmodia. It is expressed differentially during distinct life stages as estimated by RT-PCR, namely with a peak in the schizont phase. We then tried to show the involvement of Pf-Int in the parasitic cycle. We were able to create a parasite where the Pf-Int gene was knocked-out. The comparison test showed that Pf-Int has apparently no impact on the intraerythrocytic developmental cycle of the parasite, particularly in the cycle length and the growth rate.At the molecular level, we produced two sets of anti-Pf-Int antibodies using the purified recombinant fragment C-162 (residues 162-490). Comparison of protein extracts from KO and wild parasite by Western blot technique using our antibody has failed to identify the endogenous protein in the wild type parasite.We also tried to determine the subcellular localization of Pf-Int and the role of possible alternate initiation codons by over-expressing different constructs in the parasite Plasmodium falciparum. In order to determine the impact of the N-terminal region (1-190aa) of Pf-Int on its subcellular localization, we also created a chimeric protein using a fusion of Pf-Int(1-190aa) with the GFP. We successfully expressed a variety of the recombinant form of Pf-Int in E. coli. We have first determined its secondary structure content by circular dichroism (CD) and its solution stability by thermal denaturation-CD. An 1-D NMR spectrum was also recorded. The third part of our work has involved the identification of the DNA targets of Pf-Int. Two search strategies conducted in the laboratory using a library of chemically synthesized sequences and a second library made of fragments of genomic DNA of P. falciparum. Both approaches have allowed the identification of two sets of target DNA. Secondly, electrophoretic mobility shift assays (EMSA) were used to show its affinity and specificity for DNA. The recombinant proteins were shown to be functional as they form a covalent complex with DNA. Thus Pf-Int could be a potential agent that binds to and alters DNA, either in a specific or in random fashion. Its conservation and differential expression leads us to conclude that although its role is far from being understood, Pf-Int remains a key target for P. falciparum.

<i>Escherichia coli </i>RecA is a versatile protein that is involved in homologous recombination, and coordination of both the DNA damage response and translesion synthesis. Single-stranded DNA (ssDNA) that is generated at the site of double-stranded breaks serves as a signal to activate RecA. This allows RecA to form a long helical filament on the ssDNA, which is required in recombination, hydrolysis of ATP, and mediating the self-cleavage of some ser-lys dyad proteins such as the LexA repressor. In this thesis, the formation of the RecA/LexA complex did not require preactivation by ssDNA, instead a volume excluding agent in the presence of LexA was able to stimulate its formation. These preliminary results led to a hypothesis that the formation of the RecA/LexA complex is a thermodynamic process that involves three steps: (1) a change in RecAs conformation towards the active form, (2) a change in LexAs conformation towards the cleavable form (i.e. burial of the ser-lys dyad catalytic residues), and (3) the binding between the active form of RecA and the cleavable form of LexA. Evidence for this model was shown by the ability of either NaCl, LexA K156A, an ATP substrate, or a volume excluding agent to enhance the stability of the RecA/LexA complex, which was detected by both the ATPase and coprotease assays. Hyper-active RecA mutants, isolated form the yeast two-hybrid screen, were also tested, however they did not enhance the stability of the complex. Additionally, RecAs binding preference for the monomer or dimer form of LexA was examined, since it is unknown which species of LexA is able to enhance the stability of the complex. To generate the monomer form of LexA, single point mutations were introduced at the dimer interface of the protein such that its dimerization was disrupted by charge-charge repulsions. Based on the inhibition assay, RecA was found to bind preferentially to dimer form and not the monomer form of LexA, possible reasons for these results are discussed.

<i>Escherichia coli </i>RecA is a versatile protein that is involved in homologous recombination, and coordination of both the DNA damage response and translesion synthesis. Single-stranded DNA (ssDNA) that is generated at the site of double-stranded breaks serves as a signal to activate RecA. This allows RecA to form a long helical filament on the ssDNA, which is required in recombination, hydrolysis of ATP, and mediating the self-cleavage of some ser-lys dyad proteins such as the LexA repressor. In this thesis, the formation of the RecA/LexA complex did not require preactivation by ssDNA, instead a volume excluding agent in the presence of LexA was able to stimulate its formation. These preliminary results led to a hypothesis that the formation of the RecA/LexA complex is a thermodynamic process that involves three steps: (1) a change in RecAs conformation towards the active form, (2) a change in LexAs conformation towards the cleavable form (i.e. burial of the ser-lys dyad catalytic residues), and (3) the binding between the active form of RecA and the cleavable form of LexA. Evidence for this model was shown by the ability of either NaCl, LexA K156A, an ATP substrate, or a volume excluding agent to enhance the stability of the RecA/LexA complex, which was detected by both the ATPase and coprotease assays. Hyper-active RecA mutants, isolated form the yeast two-hybrid screen, were also tested, however they did not enhance the stability of the complex. Additionally, RecAs binding preference for the monomer or dimer form of LexA was examined, since it is unknown which species of LexA is able to enhance the stability of the complex. To generate the monomer form of LexA, single point mutations were introduced at the dimer interface of the protein such that its dimerization was disrupted by charge-charge repulsions. Based on the inhibition assay, RecA was found to bind preferentially to dimer form and not the monomer form of LexA, possible reasons for these results are discussed.

Genomic complexity in non-Hodgkin’s Diffuse Large B-cell Lymphoma (DLBCL) leads to a treatment failure in ~40% of patients. Activation-Induced Cytosine Deaminase (AID), one of the enzymes involved in generating antibody diversity via class switching recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes in activated B-cells is one mechanism for the introduction of genomic lesions. In previous studies, AID was shown to preferentially bind to super-enhancer (SE) regions within the genome, but 26% of AID targets were not within the SE regions. The mechanism by which AID interacts with SE elements and its off-target interactions still remains a mystery. Recent evidence suggests that AID may cause genomic lesions in DLBCL via interaction with oncogenes such as MYC and BCL2 resulting in mutations and translocations. Sequences within the MYC promoter contain the four-nucleotide AID target sequence (WRCY) and highly G-rich sequences known to form G-quadruplex DNA secondary structures. We hypothesize that key DNA secondary structures act as recruiting elements for aberrant AID activity at promoters and SEs of key genes involved in the development of DLBCL. Here, we first sought to determine whether known AID DNA targets have the potential to form G-quadruplex DNA secondary structures. The data collected from activated mouse B-cells showed 90% of the AID targets contained sequences that could potentially form G-quadruplexes and the data collected from the human Ramos cell line showed 100% of the sequences had the potential to form G-quadruplexes. To further study our hypothesis we used the techniques circular dichroism (CD) and the electrophoresis motility shift assay (EMSA) to explore the potential interaction between AID and the BCL2 and MYC G-quadruplexes. We observed no significant interactions between AID and these two G-quadruplexes, however further experimentation with different conditions and molecular techniques may show interaction. Additional studies will not only provide key insight into the genomic instability within DLBCL, but will also provide a potential mechanism by which AID is recruited to its DNA targets.

Synthetic genetic circuits have revolutionized numerous fields, ranging from academic research and point-of-care diagnostics to disease therapeutics and industrial biomanufacturing. These circuits provide a powerful tool for precise spatiotemporal control over biological and biochemical interactions, thereby enhancing our understanding of these complex systems and expanding their applicability. The last few decades have witnessed a surge in research efforts, both in cell-free and cellular systems. These endeavors include those to improve the sensitivity and specificity of diagnostics and optimize the safety, efficacy, and tunability of existing treatments. This dissertation delves into the exploration of Boolean logic gates in the cell-free realm: the development of a 'Cell-Free Recombinase Integrated Boolean Operating System' (CRIBOS) for expanding the capabilities of cell-free sensing applications. Applications of Boolean logic gates have flourished within cellular systems and animal models. However, a persisting gap in the field is in their exploration within the cell-free system. This deficiency has resulted in a constrained toolkit for studying and applying Boolean logic gates in cell-free settings. Recognizing this limitation in the field and aiming to extend the frontiers of genetic circuits beyond traditional boundaries, I introduce CRIBOS, leveraging the advantages of recombinase, known for its high orthogonality, efficiency, and sensitivity. I designed more than 20 multi-input-multi-output recombinase Boolean logic gates in a cell-free context, from which a set of critical rules crucial for building genetic circuits in the cell-free environment was also established. In addition, integrating allosteric transcription factor (aTF)-based sensors with CRIBOS enabled multiplex environmental sensing within the cell-free environment. Moreover, the CRIBOS system showcased its versatility by facilitating the creation of a biological memory storage device, demonstrating robust functionality with high stability over four months. Implementing CRIBOS not only expands the application of multiplex Boolean logic gates from cellular systems to the cell-free environment but also expands their overall versatility, opening new avenues for the design and application of sophisticated genetic circuits.