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Papers by sayak mukhopadhyay

Bioengineering solutions to human space travel must consider microgravity as an important compone... more Bioengineering solutions to human space travel must consider microgravity as an important component. Thus, one of the fundamental challenges of space bioengineering is to create cellular microgravity responsive device, which integrate microgravity as a signal within biochemical and cellular processes. Here, we designed, fabricated and characterized the first biochemical and cellular microgravity responsive device using an engineered genetic circuit in E.coli, which responded to microgravity by changing the expression of a target enhanced green fluorescent gene (EGFP). Our device design was based on the deregulation of HfQ protein in E.coli in microgravity, which was translated through HfQ mediated silencing of EGFP by anti-EGFP synthetic small regulatory RNAs. This resulted a reduced silencing (~28 times) of the EGFP in microgravity. We demonstrated that the basic design of the device is universal in nature for E.coli, by creating multiple successful devices, where target genes (EGF...

Figure S1. Sequencing results for PCRed cI gene. Figure S2 Characterization of wild type (WT) cI ... more Figure S1. Sequencing results for PCRed cI gene. Figure S2 Characterization of wild type (WT) cI and frame-shifted cI. Figure S3 Correlation between the dynamic range and the approximate ratio of plasmid copy number carrying frame-shifted cI and PR-EGFP construct. Figure S4 Open reading frames of wild type cI, frame-shifted cI and truncated cI started from amino acid M41. Figure S5 Generic plasmid maps constructed in this study. Figure S6 Characterization of the NOT gates repression behaviour with postulated truncated variants of cI. Table S1 Curve fitting parameter values. Table S2 Translation initiation rates for EGFP and cI calculated from RBS calculator. Table S3 List of promoters. Table S4 List of plasmids. Table S5 List of primers. (DOCX 4535 kb)

Journal of Biological Engineering

Background: Frame-shifted genes results in non-functional peptides. Because of this complete loss... more 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.

Biological solutions to human space travel must consider microgravity as an important component, ... more Biological solutions to human space travel must consider microgravity as an important component, which is unknown by the biochemical worlds on the Earth. Thus, one of the fundamental challenges of space biotechnology is to create engineered biochemical systems to integrate microgravity as a signal within molecular and cellular processes. Here we created the first molecular or biochemical microgravity sensor by creating a synthetic-small-regulatory-RNA based molecular network in E.coli, which sensed microgravity and responded by altering the expression of a target protein. We demonstrated that the design was universal, could work potentially with any promoter and against any target gene. This device was applied to target cell division process and rescue the deformed cell shape by applying microgravity. The work showed for the first time, a way to integrate microgravity as physical signals within biochemical process of a living cell in a human designed way and thus, opens a new direct...

Biotechnology and Bioengineering

Journal of Biological Engineering

Background: Frame-shifted genes results in non-functional peptides. Because of this complete loss... more 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.

Scientific Reports, 2016

Microgravity is a prominent health hazard for astronauts, yet we understand little about its effe... more Microgravity is a prominent health hazard for astronauts, yet we understand little about its effect at the molecular systems level. In this study, we have integrated a set of systems-biology tools and databases and have analysed more than 8000 molecular pathways on published global gene expression datasets of human cells in microgravity. Hundreds of new pathways have been identified with statistical confidence for each dataset and despite the difference in cell types and experiments, around 100 of the new pathways are appeared common across the datasets. They are related to reduced inflammation, autoimmunity, diabetes and asthma. We have identified downregulation of NfκB pathway via Notch1 signalling as new pathway for reduced immunity in microgravity. Induction of few cancer types including liver cancer and leukaemia and increased drug response to cancer in microgravity are also found. Increase in olfactory signal transduction is also identified. Genes, based on their expression pattern, are clustered and mathematically stable clusters are identified. The network mapping of genes within a cluster indicates the plausible functional connections in microgravity. This pipeline gives a new systems level picture of human cells under microgravity, generates testable hypothesis and may help estimating risk and developing medicine for space missions. The future plan of manned mission to Mars and asteroids 1 requires astronauts to spend years in space. Microgravity is one of the most prominent health hazards for astronauts 2,3. During today's space missions, a short to moderate microgravity exposure (days to months) induces several physiological changes in human body including bone and muscle loss, puffiness in the face, change in cardiovascular physiology, catecholamine cardiomyopathy, insufficient blood flow in the brain, genitourinary issues and disturbance in neurovestibular system 2-7. Further, microgravity induces deregulation of human immune systems 8,9. Multiple gene expression studies showed microgravity-induced signature of early inhibition in T cell activation 10 , impaired endothelial cell function 11 , cellular senescence 12 , alteration of genes related to cell cycle 13,14 , cell adhesion 11 , oxidative phosphorylation 14 and apoptosis 14. It has been showed that the reduced immunity may result from inhibition of NF-κB/Rel pathway, downregulation of early T cell activation genes, IFN-ϒ and EL-2Rα genes 15 and impairment of Jun-N-terminal kinase activity 9. The compromised immune system increases the risk of infection by pathogen like salmonella, virulence of which is increased in microgravity 16. Salmonella infection among astronauts is a well-known health hazard documented starting from Apollo and Skylab missions 16,17. Further, microgravity alters level of micro RNAs (miRNAs), many of which are related with inflammation 18 and multiple cancer types 13,18,19. However, the studies showed controversial inference based on the expression of different microRNAs. For example, expression of hsa-miR-423-5p and hsa-miR-222 in microgravity suggest the induction of breast cancer, whereas expression of hsa-miR-141 suggests the decrease in the same 19. Similar controversial miRNA expression pattern was observed for leukaemia and lung cancer 18,19. Further, as a single miRNA

Bioengineering solutions to human space travel must consider microgravity as an important compone... more Bioengineering solutions to human space travel must consider microgravity as an important component. Thus, one of the fundamental challenges of space bioengineering is to create cellular microgravity responsive device, which integrate microgravity as a signal within biochemical and cellular processes. Here, we designed, fabricated and characterized the first biochemical and cellular microgravity responsive device using an engineered genetic circuit in E.coli, which responded to microgravity by changing the expression of a target enhanced green fluorescent gene (EGFP). Our device design was based on the deregulation of HfQ protein in E.coli in microgravity, which was translated through HfQ mediated silencing of EGFP by anti-EGFP synthetic small regulatory RNAs. This resulted a reduced silencing (~28 times) of the EGFP in microgravity. We demonstrated that the basic design of the device is universal in nature for E.coli, by creating multiple successful devices, where target genes (EGF...

Figure S1. Sequencing results for PCRed cI gene. Figure S2 Characterization of wild type (WT) cI ... more Figure S1. Sequencing results for PCRed cI gene. Figure S2 Characterization of wild type (WT) cI and frame-shifted cI. Figure S3 Correlation between the dynamic range and the approximate ratio of plasmid copy number carrying frame-shifted cI and PR-EGFP construct. Figure S4 Open reading frames of wild type cI, frame-shifted cI and truncated cI started from amino acid M41. Figure S5 Generic plasmid maps constructed in this study. Figure S6 Characterization of the NOT gates repression behaviour with postulated truncated variants of cI. Table S1 Curve fitting parameter values. Table S2 Translation initiation rates for EGFP and cI calculated from RBS calculator. Table S3 List of promoters. Table S4 List of plasmids. Table S5 List of primers. (DOCX 4535 kb)

Journal of Biological Engineering

Background: Frame-shifted genes results in non-functional peptides. Because of this complete loss... more 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.

Biological solutions to human space travel must consider microgravity as an important component, ... more Biological solutions to human space travel must consider microgravity as an important component, which is unknown by the biochemical worlds on the Earth. Thus, one of the fundamental challenges of space biotechnology is to create engineered biochemical systems to integrate microgravity as a signal within molecular and cellular processes. Here we created the first molecular or biochemical microgravity sensor by creating a synthetic-small-regulatory-RNA based molecular network in E.coli, which sensed microgravity and responded by altering the expression of a target protein. We demonstrated that the design was universal, could work potentially with any promoter and against any target gene. This device was applied to target cell division process and rescue the deformed cell shape by applying microgravity. The work showed for the first time, a way to integrate microgravity as physical signals within biochemical process of a living cell in a human designed way and thus, opens a new direct...

Biotechnology and Bioengineering

Journal of Biological Engineering

Background: Frame-shifted genes results in non-functional peptides. Because of this complete loss... more 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.

Scientific Reports, 2016

Microgravity is a prominent health hazard for astronauts, yet we understand little about its effe... more Microgravity is a prominent health hazard for astronauts, yet we understand little about its effect at the molecular systems level. In this study, we have integrated a set of systems-biology tools and databases and have analysed more than 8000 molecular pathways on published global gene expression datasets of human cells in microgravity. Hundreds of new pathways have been identified with statistical confidence for each dataset and despite the difference in cell types and experiments, around 100 of the new pathways are appeared common across the datasets. They are related to reduced inflammation, autoimmunity, diabetes and asthma. We have identified downregulation of NfκB pathway via Notch1 signalling as new pathway for reduced immunity in microgravity. Induction of few cancer types including liver cancer and leukaemia and increased drug response to cancer in microgravity are also found. Increase in olfactory signal transduction is also identified. Genes, based on their expression pattern, are clustered and mathematically stable clusters are identified. The network mapping of genes within a cluster indicates the plausible functional connections in microgravity. This pipeline gives a new systems level picture of human cells under microgravity, generates testable hypothesis and may help estimating risk and developing medicine for space missions. The future plan of manned mission to Mars and asteroids 1 requires astronauts to spend years in space. Microgravity is one of the most prominent health hazards for astronauts 2,3. During today's space missions, a short to moderate microgravity exposure (days to months) induces several physiological changes in human body including bone and muscle loss, puffiness in the face, change in cardiovascular physiology, catecholamine cardiomyopathy, insufficient blood flow in the brain, genitourinary issues and disturbance in neurovestibular system 2-7. Further, microgravity induces deregulation of human immune systems 8,9. Multiple gene expression studies showed microgravity-induced signature of early inhibition in T cell activation 10 , impaired endothelial cell function 11 , cellular senescence 12 , alteration of genes related to cell cycle 13,14 , cell adhesion 11 , oxidative phosphorylation 14 and apoptosis 14. It has been showed that the reduced immunity may result from inhibition of NF-κB/Rel pathway, downregulation of early T cell activation genes, IFN-ϒ and EL-2Rα genes 15 and impairment of Jun-N-terminal kinase activity 9. The compromised immune system increases the risk of infection by pathogen like salmonella, virulence of which is increased in microgravity 16. Salmonella infection among astronauts is a well-known health hazard documented starting from Apollo and Skylab missions 16,17. Further, microgravity alters level of micro RNAs (miRNAs), many of which are related with inflammation 18 and multiple cancer types 13,18,19. However, the studies showed controversial inference based on the expression of different microRNAs. For example, expression of hsa-miR-423-5p and hsa-miR-222 in microgravity suggest the induction of breast cancer, whereas expression of hsa-miR-141 suggests the decrease in the same 19. Similar controversial miRNA expression pattern was observed for leukaemia and lung cancer 18,19. Further, as a single miRNA