Impact of Ammonium Based Ionic Liquids on the Conformation of Bovine serum albumin (BSA)

In this study, impact of different ammonium based ionic liquids on the structure of Bovine serum albumin (BSA) was investigated by different spectroscopic methods like, UV-vis, fluorescence and CD spectroscopy. Fluorescence results reveal that the ILs has no significant impact on the environment of tryptophan residues. From the Far-UV CD results, it was observed that these ILs increase the α-helicity of the protein, BSA. Near-UV CD study confirms the fluorescence results whereas it shows the alteration of tertiary structure of BSA especially around disulphide bonds

Systems and Synthetic Biology in E. coli Cells Quantitative System Characterization, Programming and Engineering Novel Cellular Functions

The emerging field of synthetic biology aims to use artificially designed genetic circuits to program living cells, much as engineers program a computer or control electronic or mechanical systems. This thesis focuses on the design and implementation of synthetic gene circuits in the bacterium Escherichia coli to create new cellular functions, and on the quantitative characterization and modelling of these circuits. Though important in any engineering discipline, quantitative system characterization has been poorly explored in synthetic biology. We have performed a quantitative system characterization by implementing simple gene circuits in Escherichia coli. The work showed that the level and variability of gene expression varied across different cell strains, and we investigated how these effects manifested through the coupled effects of cell division, cellular growth rate, and plasmid copy number regulation. The work suggests that gene circuit modules from a standard library cannot be used universally; the cellular context and the time dependent dynamics must be considered when implementing gene circuits. In order to work precisely as engineering devices, synthetic gene circuits must be appropriately tuned. One standard method of tuning genetic circuits requires altering the DNA sequences by extensive molecular biology work. Part of this thesis focuses on developing easily tunable gene circuits. A set of circuits were developed in E. coli where the shape of the chemically induced signal response curves can be tuned from a band structure to a sigmoidal structure simply by altering the temperature in a single system. Another set of circuits was developed which demonstrate a range of chemically tunable signal response curves along with multiple functions in a single device. One of the ultimate goals of synthetic biology is to program living cell in a human-controlled way. To this end, I developed a set of genetic devices that could work as ‘in cell disease prevention devices,’ preventing an otherwise fatal viral infection in E. coli. The device displays a number of ‘device’ properties: being dormant under normal conditions, detecting the onset of the disease state, turning on automatically to prevent a lethal outcome, and being subject to external deactivation when desired. The combination of design, characterization, and mathematical understanding explored in this work represents a contribution in the direction of developing synthetic biology as a well-founded engineering discipline.Ph

A Logically Reversible Double Feynman Gate with Molecular Engineered Bacteria Arranged in an Artificial Neural Network-Type Architecture

Reversible logic gates are the key components of reversible computing that map inputs and outputs in a certain one-to-one pattern so that the output signals can reveal the pattern of the input signals. One of the main research foci of reversible computing is the implementation of basic reversible gates by various modalities. Though true thermodynamic reversibility cannot be attained within living cells, the high energy efficiency of biological reactions inspires the implementation of reversible computation in living cells. The implementation of synthetic genetic circuits is mostly based on conventional irreversible computing, and the implementation of logical reversibility in living cells is rare. Here, we constructed a 3-input-3-output synthetic genetic reversible double Feynman logic gate with a population of genetically engineered E. coli cells. Instead of following hierarchical electronic design principles, we adapted the concept of artificial neural networks (ANN) and built a single-layer artificial network-type architecture with five different engineered bacteria, named bactoneurons. We used three extracellular chemicals as input signals and the expression of three fluorescence proteins as the output signals. The cellular devices, which combine the input chemical signals linearly and pass them through a nonlinear activation function and represent specific bactoneurons, were built by designing and creating small synthetic genetic networks inside E. coli. The weights of each of the inputs and biases of individual bactoneurons in the bacterial ANN were adjusted by optimizing the synthetic genetic networks. When arranging the five bactoneurons through an ANN-type architecture, the system generated a double Feynman gate function at the population level. To our knowledge, this is the first reversible double Feynman gate realization with living cells. This work may have significance in development of biocomputer technology, reversible computation, ANN wetware, and synthetic biology

A frame-shifted gene, which rescued its function by non-natural start codons and its application in constructing synthetic gene circuits

Abstract Background Frame-shifted genes results in non-functional peptides. Because of this complete loss of function, frame-shifted genes have never been used in constructing synthetic gene circuits. Results Here we report that the function of gene circuits is rescued by a frame-shifted gene, which functions by translating from a non-natural start codon. We report a single nucleotide deletion mutation that developed in the λ-repressor cI within a synthetic genetic NOT gate in Escherichia coli during growth and through this mutation, a non-functional synthetic gene circuit became functional. This mutation resulted in a frame-shifted cI, which showed effective functionality among genetic NOT-gates in Escherichia coli with high regulatory ranges (> 300) and Hill coefficient (> 6.5). The cI worked over a large range of relative copy numbers between the frame-shifted gene and its target promoter. These properties make this frame-shifted gene an excellent candidate for building synthetic gene circuits. We hypothesized a new operating mechanism and showed evidence that frame-shifted cI was translated from non-natural start codon. We have engineered and tested a series of NOT gates made from a library of cI genes, each of which starts from a different codon within the first several amino acids of the frame-shifted cI. It is found that one form with start codon ACA, starting from the 3rd codon had similar repression behavior as the whole frame-shifted gene. We demonstrated synthetic genetic NAND and NOR logic-gates with frame-shifted cI. This is the first report of synthetic-gene-circuits made from a frame-shifted gene. Conclusions This study inspires a new view on frame-shifted gene and may serve as a novel way of building and optimizing synthetic-gene-circuits. This work may also have significance in the understanding of non-directed evolution of synthetic genetic circuits