Date of Award
Master of Science
Bacterial promoter systems have been widely used as biosynthetic engineering tool for the research and study of biomedical engineering concepts through out the last few decades. In a recent publication by Bonnet et al, they designed the Rewritable Addressable Data (RAD) module which uses a tet promoter and ara promoter to have digital computation output to store memory as "1" or "0" within a live bacterial cell. The RAD module accepts inputs as either anhydrotetracyline or arabinose based on combinatory logic that allows interaction with the promoters to produce two recombinase enzymes known as Bxb1 integrase and excisionase. Consequently, this RAD system uses the recombination properties of these enzymes to invert or revert host DNA containing either attP/attB or attL/attR recognition sites on the host DH5alphaZ1 chromosome of Escherichia coli, to produce the desired output to store 1 bit of information. Furthermore, the RAD module is able to hold two stable states and allows effective switching between the two states. This makes the RAD modules an effective tool for aging and cancer studies. Although, both the tet and ara promoters have been shown to robustly control the functions of the RAD module, this thesis focuses on switching the tet promoter to lac promoter on the RAD module to increase the application of the RAD module as a tool in many other biosynthetic engineering applications. The lac promoter uses a gene repressor mechanism to enable its bi-stable characteristic and also works with a non-hydrolysable inducer known as isopropyl-D-thio-&beta-galactopysonoside (IPTG). In addition, the host genome has a lacI gene, which produces the LacI repressor protein, therefore increasing the tightness of regulation of the RAD storage device. Lastly, the lac promoter is a highly characterized promoter with many applications. Consequently, we hypothesize that we can have tighter control of the RAD system with the lac promoter and its inducer (isopropyl-D-thio-&beta-galactopysonoside (IPTG)) than the initial tet promoter. In addition, an alternative promoter will allow creation of a device capable of responding to multiple signals.
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