Murine Zfp106 is an 1,888 amino acid protein with two N-terminal and two C-terminal zinc finger domains with a beta-propeller upstream of the C-terminal zinc fingers. The transcription of the protein was found to be controlled through two promoter regions, leading to two isoform families, P1 and P2 Zfp106. The splice variants from each promoter are thought to have distinct starting exons and n-terminal regions. However, the isoforms are not well studied. Since its identification, Zfp106 has been implicated in RNA metabolism, transcription control, immune response, and muscle and testis development. It has been found to be capable of binding C9ORF72 repeats as well as being associated with TDP43 and FUS. However, its function is unknown. The aim of this study is to understand the role of Zfp106 in vivo through the use and development of various mouse models targeting exons specific to either the P1 or P2 family of isoforms. To begin with, we studied the Zfp106LacZ mouse model whose homozygous mice showed severe muscle atrophy beginning at 4 weeks leading to a premature death by 16 weeks. Research has supported the theory that the muscle atrophy is due to a motor neuron dysfunction potentially stemming from perturbed mitochondrial and spliceosome function. We, along with other researchers, found that this mouse model is not a complete disruption of Zfp106 through qPCR and RNAseq. We then found that this mouse model is an effective depletion of Zfp106 exon 2 and 3 which are exclusive to the P1 Zfp106 isoform family. Additionally, the Zfp106LacZ mouse model has an increased amount of the 1b exon associated with P2-Zfp106 in the skeletal muscle. Next, we established a CRISPR mouse line (ΔZfp106) targeting an exon common to the full-length splice variants of both the P1 and P2 family of isoforms, exon 5. This was in an attempt to dissect whether or not the muscle atrophy in the Zfp106LacZ mice was due to the interruption of exons 2 and 3 or from the increase in the P2 Zfp106 isoforms. Motor neurons derived from homozygous ΔZfp106 mouse embryonic stem cells, were found to be susceptible to CPA-induced endoplasmic reticulum stress and rotenone induced mitochondrial stress. Interestingly, the in vivo penetration rate of the muscle atrophy phenotype of homozygous ΔZfp106 mice is 60% for male and 12.5% for female mice. This is in stark contrast to the 100% penetration rate of the Zfp106LacZ mice. The reason behind this is currently unclear but may be due to either the incomplete backcrossing of the mouse model, a difference in the splice variants affected by the Zfp106 targeting, or because the muscle atrophy in the Zfp106LacZ mouse model is caused in part by the increase in the expression the P2 Zfp106 family of isoforms. These two mouse models show that affecting the expression of the full-length isoforms of P1-Zfp106 can lead to muscle atrophy. In an attempt to see if the Zfp106LacZ muscle atrophy was due to a lack of Zfp106 in the skeletal muscle, spinal cord, or necessitated its depletion in both, we derived a mouse line from the Zfp106LacZ that conditionally depletes exon 3 which impairs the expression of full length P1 Zfp106. This was used to target exon 3 removal to the skeletal muscle (Myf5), cholinergic neurons (ChAT), simultaneously (Myf5/ChAT), or a whole body depletion (Ella2). Surprisingly, the whole body depletion of Zfp106 exon 3 did not lead to muscle atrophy even though its removal leads to a frame shift and premature stop codon. The lack of a muscle atrophy phenotype may be because of the expression of a splice variant without exon 3, thereby rescuing the neuromuscular pathology. Lastly, to better understand the role of the P2 Zfp106 in vivo, we created a mouse line with a CRISPR mediated knockout of exon 1b (ΔP2). Exon 1b is the start exon of the P2 Zfp106 isoform family and the introduction of a destructive INDEL should independently affect the P2 isoform family. Interestingly, this mouse model showed no observed neuromuscular dysfunction or metabolic disorder, responding to a glucose bolus similarly with controls. The lack of a phenotype may be due to compensation by other Zfp106 isoforms or that the P2 isoform family is important in other biological roles.

Submitted in partial fulfillment of the academic requirements for the degree of Master of Technology: Biotechnology, 2009. / Production of numerous biotechnologically-important products such as threonine is based on cultivation of bacterial cultures. Infection of these bacterial cultures by bacteriophages has a detrimental effect in the production of these bioproducts. Despite this, most people controlling these bioprocesses do not recognize the early signs of bacteriophage infection. SA Bioproducts (Ply) Ltd was no exception and has suffered tremendous loss of production time after bacteriophages infected threonine producing E. coli strain B. This study was aimed at developing assays to control and prevent bacteriophage infection at this company. These included determining the source of phages by monitoring the process plant environment, optimising the detection and enumeration methods so as to monitor the levels of bacteriophages in the environment, identification of bacteriophages in order to determine the number of bacteriophages capable of infection threonine producing E. coli strain B, treatment and of phages, and possible prevention of phage infection. Adam's DAL method was very efficient at detecting phages in the samples collected at various areas (sumps, odour scrubber, process water, and soil) around the plant for 16 weeks. High levels of phages were found in the sumps and this was identified as the source of infection. Samples collected were grouped together according to their source. The samples were enriched and purified in order to characterise them. The prevalent phage in all samples was identified as a T1-like phage. Bacterial strains that grew on the plate in the presence of phages were assumed to be resistant to phages or contained lysogenic phages which would explain the new lytic cycles that were observed whenever these resistant strains were used for production. UV light, green v indicator plates, and a mutagen (Mitomycin C) were used to detect Iysogens. Mitomycin C at 1 IJg/ml was found to be most effective in detecting lysogenic phages. This was shown by new plaque forming units that were visible on the DAL plates. Temperature (heat), chemicals, and inhibitors (vitamins) were investigated as strategies for prevention and treatment of bacteriophage infection. Bacteriophage samples were exposed to 70, 80, 100, and 120°C. At these temperatures pfu counts in the samples were reduced significantly. At 120°C there was a complete inactivation of bacteriophages within 30 minutes. Chemicals investigated such as sodium hydroxide and Albrom 100T were capable of complete deactivation of bacteriophages at a very low concentration (0.1%). Therefore, these chemicals can be used to clean the plant area and sumps. Vitamins C, K and E solutions were investigated to determine their inhibitory effect on bacteriophages. Vitamin C, K and E reduced pfu counts by 3, 2, and 4 logs, respectively. Therefore vitamin C and E solutions were mixed and to determine if mixing them would enhance their inactivation capabilities. This resulted in a reduction greater than 9 logs of phage in the sample (from 7.7 x 109 to 3 pfu/ml). The host bacterium was also exposed to this mixture to determine effect of the vitamin mixture on its growth. It was found that there was no effect exerted by this mixture on the host bacteria. This proved to be an ideal mixture for combating phages during fermentation. However, vitamin E is not cost effective for co-feeding in 200 m' fermenters, and therefore vitamin C solution was a cost-effective alternative. It was concluded that bacteriophage contaminated bioprocessing plant should be properly cleaned using a combination of heat and chemicals. Bacteriophage infection should be prevented by employing inhibitors.

Submitted in partial fulfillment of the academic requirements for the degree of Master of Technology: Biotechnology, 2009. / Production of numerous biotechnologically-important products such as threonine is based on cultivation of bacterial cultures. Infection of these bacterial cultures by bacteriophages has a detrimental effect in the production of these bioproducts. Despite this, most people controlling these bioprocesses do not recognize the early signs of bacteriophage infection. SA Bioproducts (Ply) Ltd was no exception and has suffered tremendous loss of production time after bacteriophages infected threonine producing E. coli strain B. This study was aimed at developing assays to control and prevent bacteriophage infection at this company. These included determining the source of phages by monitoring the process plant environment, optimising the detection and enumeration methods so as to monitor the levels of bacteriophages in the environment, identification of bacteriophages in order to determine the number of bacteriophages capable of infection threonine producing E. coli strain B, treatment and of phages, and possible prevention of phage infection. Adam's DAL method was very efficient at detecting phages in the samples collected at various areas (sumps, odour scrubber, process water, and soil) around the plant for 16 weeks. High levels of phages were found in the sumps and this was identified as the source of infection. Samples collected were grouped together according to their source. The samples were enriched and purified in order to characterise them. The prevalent phage in all samples was identified as a T1-like phage. Bacterial strains that grew on the plate in the presence of phages were assumed to be resistant to phages or contained lysogenic phages which would explain the new lytic cycles that were observed whenever these resistant strains were used for production. UV light, green v indicator plates, and a mutagen (Mitomycin C) were used to detect Iysogens. Mitomycin C at 1 IJg/ml was found to be most effective in detecting lysogenic phages. This was shown by new plaque forming units that were visible on the DAL plates. Temperature (heat), chemicals, and inhibitors (vitamins) were investigated as strategies for prevention and treatment of bacteriophage infection. Bacteriophage samples were exposed to 70, 80, 100, and 120°C. At these temperatures pfu counts in the samples were reduced significantly. At 120°C there was a complete inactivation of bacteriophages within 30 minutes. Chemicals investigated such as sodium hydroxide and Albrom 100T were capable of complete deactivation of bacteriophages at a very low concentration (0.1%). Therefore, these chemicals can be used to clean the plant area and sumps. Vitamins C, K and E solutions were investigated to determine their inhibitory effect on bacteriophages. Vitamin C, K and E reduced pfu counts by 3, 2, and 4 logs, respectively. Therefore vitamin C and E solutions were mixed and to determine if mixing them would enhance their inactivation capabilities. This resulted in a reduction greater than 9 logs of phage in the sample (from 7.7 x 109 to 3 pfu/ml). The host bacterium was also exposed to this mixture to determine effect of the vitamin mixture on its growth. It was found that there was no effect exerted by this mixture on the host bacteria. This proved to be an ideal mixture for combating phages during fermentation. However, vitamin E is not cost effective for co-feeding in 200 m' fermenters, and therefore vitamin C solution was a cost-effective alternative. It was concluded that bacteriophage contaminated bioprocessing plant should be properly cleaned using a combination of heat and chemicals. Bacteriophage infection should be prevented by employing inhibitors.

Synthetic biology, which aims to enable the design and assembly of customized biological systems, holds great promise for delivering solutions to numerous modern day challenges in agriculture, sustainable energy production, and medicine. However, at its current stage, synthetic biology is not yet equipped with the necessary tools and understanding to reprogram the immensely complex molecular environment of the cell beyond simple proof of concept demonstrations. One current objective within synthetic biology is to create robust tools that can be used to manipulate biological systems in a predictable and reliable manner. While many transcription-based control devices have been reported, little consideration has been given to the eukaryotic protein translation apparatus as a target for engineering gene-regulatory tools. In this work, we explore the potential for reprogramming the protein synthesis machinery for cell engineering. We begin in Chapter 1 by reviewing canonical protein synthesis and survey the assortment of translation reprogramming mechanisms that exist in nature, focusing on the role of RNA in these processes. We then cover previous efforts to engineer the protein synthesis machinery and discuss their methodological approaches. Lastly, we examine potential opportunities for engineering protein synthesis that have not yet been explored. RNA’s prominent role in protein synthesis and its amenability to high-throughput in vitro selection approaches raises the possibility that the translation apparatus could be engineered through in vitro directed evolution of its RNA components. In Chapter 2, we develop an experimental framework for identifying mRNA sequence elements that reprogram protein synthesis, focusing on stop codon readthrough. By adapting a previously developed in vitro selection technology called mRNA display, we demonstrate that molecules of RNA derived from expansive libraries of random sequences can be enriched as a result of their translation reprogramming activity. We then analyze these stop codon readthrough signals and propose the use of these sequences for enhanced unnatural amino acid incorporation technologies. In Chapter 3, we apply this very same selection principle for the in vitro directed evolution of RNA sequences that stimulate -1 programmed ribosomal frameshifting. Then, using previously reported RNA aptamers, we rationally engineer RNA switches that regulate translation reading frame in response to small molecule inputs. To further optimize switch performance, an in vivo directed evolution platform was established. We explore the utility of these RNA switches, particularly their ability to regulate multi-protein stoichiometry, for performing cellular logic operations and controlling cell fate. A major focus of translation engineering has been the incorporation of unnatural amino acids for fluorescent labeling of proteins in living cells. The successful achievement of this goal will require small molecule fluorophores with desirable biological properties, as well as robust synthetic methods for their production. In Chapter 4, we present a scalable approach to oxazine and xanthene fluorophores that utilizes a general diaryl ether synthetic intermediate. Finally, in Chapter 5, we describe a photoactivatable oxazine fluorophore and demonstrate its utility as a live-cell imaging reagent with applicability to advanced microscopy techniques.