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Genetically modified organisms (GMOs) are confined physically in order to contain their spread in nature and to minimise chances of horizontal gene transfer. However, with the potential that GMOs hold as cheap, reliable and efficient micro-machines, their eventual uncontrolled release into the wider space is becoming more likely. Indeed, their application as environmental sensors is largely increasing. Nevertheless, the field of synthetic biology may also afford solutions to the problem. A major potential application of GMOs is the delivery of antigens to human and animal hosts, through the utilization of live, engineered microbes. Recombinant technology is promising for several reasons including their capacity to be less reactogenic, more potent, safer and genetically definable. Also, they have the potential to provide protection against multiple targets simultaneously, are relatively inexpensive and can be eradicated with antibiotics, as the need arises. Besides, delivery of vaccines to mucosal surfaces is more efficient. Mutant Salmonella expressing heterologous antigens have been shown to induce protection against a variety of pathogens. Nevertheless, limited containment systems are available that can be applicable for bacterial antigen carriers. This project aims to design safeguards for the bacterial antigen delivery systems that limit ORF translatability and self-inactivates/destructs upon exit from the host. In this work, double quadruplet codons were suppressed by orthogonal tRNAs, providing a barrier for gene translation in the recipient cells when antigen is horizontally transferred. Furthermore, three kill switches were designed that are activated by a decrease in temperature from 37 °C. First, Sau3AI endonuclease was activated by protein self-splicing at low temperature mediated by Mtu recA intein. The activation of the endonuclease led to three-fold logarithmic decrease in the number of viable cells within two hours of gene expression. Second, RNA-dependent activation of RNase 7 showed a reduction in the number of viable cells at low temperature of three logarithmic folds. RNase 7 was controlled by the cspA 5’UTR, which sequesters ribosome binding site at 37 °C and allows translation at low temperature. Third, CspA 5’UTR was shown to regulate expression of TEV protease at 37 °C and low temperature. This led to bacterial cellular inhibition within two hours of TEV induction and five-fold logarithmic reduction in the number of viable cells at low temperature. In addition, for the first time and contrary to previous studies, the TEV protease was shown to inhibit cellular growth. It was also shown that biofilm formation was drastically impaired by the TEV activity. The three killing switches and the quadruplet translation system are poised to function as robust safeguards for bacterial antigen delivery systems.

Our understanding of the folding of membrane proteins lags behind that of soluble proteins due to the challenges posed by the exposure of hydrophobic regions during in vitro chemical denaturation and refolding experiments. While different folding models are accepted for soluble proteins, only the two-stage model and the long-range interactions model have been proposed so far for helical membrane proteins. To address our knowledge gap on how different membrane proteins traverse their folding landscapes, Chapter 2 investigates the structural features of SDS-denatured states and the kinetics for reversible unfolding of sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis. pSRII is difficult to denature, and only SDS can dislodge the retinal chromophore without rapid aggregation. Even in 30% SDS (0.998 $\mathit{\Chi}_{SDS}$), pSRII retains the equivalent of six out of seven transmembrane helices, while the retinal binding pocket is disrupted, with transmembrane residues becoming more solvent-exposed. Folding of pSRII from an SDS-denatured state harbouring a covalently-bound retinal chromophore shows deviations from an apparent two-state behaviour. SDS denaturation to form the sensory opsin apo-protein is reversible. This chapter establishes pSRII as a new model protein which is suitable for membrane protein folding studies and has a unique folding mechanism that differs from those of bacteriorhodopsin and bovine rhodopsin. In Chapter 3, SDS-denatured pSRII, acid-denatured pSRII and sensory opsin obtained by hydroxylamine-mediated bleaching of pSRII were characterised by solution state NMR. 1D $^1$H and $^{19}$F NMR were first used to characterise global changes in backbone amide protons and tryptophan side-chains. Residue-specific changes in backbone amide chemical shifts and peak intensities in 2D [$^1$H,$^{15}$N]-correlation spectra were analysed. While only small changes in the chemical environment of backbone amides were detected, changes in backbone amide dynamics were identified as an important feature of SDS- and acid-denatured pSRII and sensory opsin. $^{15}$N relaxation experiments were performed to study the backbone amide dynamics of SDS-denatured pSRII, reflecting motions on different timescales, including fast fluctuations of NH bond vectors on the ps-ns timescale and the lack of exchange contributions on the µs timescale. These studies shed insight on differences in the unfolding pathways under different denaturing conditions and the crucial role of the retinal chromophore in governing the structural integrity and dynamics of the pSRII helical bundle. Hydrogen bonds play fundamental roles in stabilising protein secondary and tertiary structure, and regulating protein function. Successful detection of hydrogen bonds in denatured states and during protein folding would contribute towards our understanding on the unfolding and folding pathways of the protein. Previous studies have demonstrated residue-specific detection of stable and transient hydrogen bonds in small globular proteins by measuring $^1{\it J}_{NH}$ scalar coupling constants using NMR. In Chapter 4, different methods for measuring $^1{\it J}_{NH}$ scalar coupling were explored using RalA, a small GTPase with a mixed alpha/beta fold, as proof-of-concept. Detection of hydrogen bonds was then attempted with OmpX, a beta-barrel membrane protein, both in its folded state in DPC micelles and in the urea-denatured state. While $^1{\it J}_{NH}$ measurement holds promise for studying hydrogen bond formation, further optimisation of NMR experiments and utilisation of perdeuterated samples are required to improve the precision of such measurements in large detergent-membrane protein complexes. Naturally occurring split inteins can mediate spontaneous trans-splicing both in vivo and in vitro. Previous studies have demonstrated successful assembly of proteorhodopsin from two separate fragments consisting of helices A-B and helices C-G via a splicing site in the BC loop. To complement the in vitro unfolding/folding studies, pSRII assembly in vivo was attempted by introducing a splicing site in the loop region of the beta-hairpin constituting the BC loop of pSRII. The expression conditions for the N- and C-terminal pSRII-intein segments were optimised, and the two segments co-expressed. However, the native chromophore was not observed. Further optimisation is required for successful in vivo trans-splicing of pSRII and application of this approach towards understanding the roles of helices and loops in the folding of pSRII.

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