| Summary: | Biofilms are one of the most widely distributed modes of life on Earth and have been found to be involved in the geochemical cycles of water, soil, and subsurface environments. The use of biofilm-mediated bioprocesses for various biotechnological applications has been explored due to the easier process control of immobilized biofilms and protection from physicochemical stresses conferred by the biofilm matrix. However, to utilise these beneficial biofilms effectively and efficiently, it is important to engineer a controllable biofilm so that an optimal biofilm thickness can be maintained in these engineered systems. This can be accomplished with optogenetic tools that target the intracellular dynamics of bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP), a ubiquitous nucleotide second messenger that has been found to play a vital role between the switch from the motile and sessile modes of life in bacteria.
However, it may be necessary to further engineer the repertoire of optogenetic tools currently available to improve their performance and achieve improved control of biofilms using light. This can be achieved by mimicking the Darwinian process of evolution in the laboratory in a process called directed evolution which uses a combination of random mutagenesis and targeted screening to find an improved variant with the desired properties. Therefore, in Chapter 3 of this thesis, a three-step directed evolution approach was used to direct the evolution of a near-infrared (NIR) light-responsive diguanylate cyclase (DGC) to obtain a variant, BphS-13, with higher DGC activity and photosensitivity to NIR light. This demonstrates the promising application of directed evolution methods in the field of optogenetics.
In Chapter 4, the intracellular c-di-GMP in Escherichia coli expressing either bphS-13 or bphS-WT was extracted and quantified using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, the ability of BphS-13 to increase biofilm formation was verified by growing the two different strains of E. coli in a continuous flow cell setup and obtaining confocal images of the biofilms after 24 h. In this study, E. coli expressing bphS-13 was able to produce significant amounts of c-di-GMP after just 16 h of exposure to NIR light while E. coli expressing bphS-WT was unable to produce detectable amounts of c-di-GMP even after 24 h of NIR light exposure. Furthermore, in the continuous flow cell set up, the E. coli biofilms expressing bphS-13 were three times thicker and had a greater biovolume than the biofilms expressing bphS-WT after 24 h of growth under NIR light.
In Chapter 5, the SCHEMA-guided design of a chimeric library of Blue Light Utilizing Flavin Adenine Dinucleotide (BLUF) photoreceptors was carried out. The BLUF photoreceptor is responsible for the blue light sensing properties of the phosphodiesterase (PDE), EB1, and its directed evolution was carried out with the goal of improving its recovery kinetics from the lit to dark states. Starting with six BLUF photoreceptors as the parental sequences, the SCHEMA algorithm was used to determine the number of interactions that are disrupted in the creation of a recombinant protein. Next the Recombination as a Shortest Path Problem (RASPP) algorithm was used to identify and select five optimal crossover sites for recombination to occur and a recombinant library of 46,656 BLUF chimeras was created. From this library of BLUF chimeras, 192 recombinants were selected to be expressed and characterized. Subsequently, the empirical information obtained about the recovery kinetics of these hybrid proteins can be used to train Gaussian process models for machine learning-assisted engineering of EB1 in the future. Taken together, this thesis work exemplifies the potential of using directed evolution methods to engineer optogenetic tools for improved control of biofilm dynamics by modulating the intracellular c-di-GMP dynamics of bacteria.
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