Current Issue : January - March Volume : 2013 Issue Number : 1 Articles : 4 Articles
The korAB operon in RK2 plasmids is a beautiful natural example of a negatively and cooperatively self-regulating operon. It\r\nhas been particularly well characterized both experimentally and with mathematical models. We have carried out a detailed\r\ninvestigation of the role of the regulatory mechanism using a biologically grounded mechanistic multi-scale stochastic\r\nmodel that includes plasmid gene regulation and replication in the context of host growth and cell division. We use the\r\nmodel to compare four hypotheses for the action of the regulatory mechanism: increased robustness to extrinsic factors,\r\ndecreased protein fluctuations, faster response-time of the operon and reduced host burden through improved efficiency of\r\nprotein production. We find that the strongest impact of all elements of the regulatory architecture is on improving the\r\nefficiency of protein synthesis by reduction in the number of mRNA molecules needed to be produced, leading to a greater\r\nthan ten-fold reduction in host energy required to express these plasmid proteins. A smaller but still significant role is seen\r\nfor speeding response times, but this is not materially improved by the cooperativity. The self-regulating mechanisms have\r\nthe least impact on protein fluctuations and robustness. While reduction of host burden is evident in a plasmid context,\r\nnegative self-regulation is a widely seen motif for chromosomal genes. We propose that an important evolutionary driver\r\nfor negatively self-regulated genes is to improve the efficiency of protein synthesis....
Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and\r\nfunction. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into\r\nnascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have\r\ndeveloped a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter\r\nproteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate\r\npromoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the\r\nfluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild\r\ntype or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows\r\nmonitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it\r\ncan be readily implemented in other organisms or cell culture systems....
The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily\r\ndetected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh)\r\nwas expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse\r\npromoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in\r\ncontrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for\r\nprotein expression in order to assess promoter strength and inducibility with the different expression systems. Several\r\nmetal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low\r\nlevel of expression. The E. coli promoter Ptrc resulted in constitutive expression in cyanobacteria regardless of whether IPTG\r\nwas added or not. In contrast, a Lac promoter variant, PA1lacO-1, induced EFE-expression in Synechocystis at a level of\r\nexpression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The\r\nregulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing\r\npromoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity\r\nwas observed in Synechocystis, independent of cell density....
Human growth hormone (hGH) is a single-chain polypeptide that participates in a wide range of biological functions such\r\nas metabolism of proteins, carbohydrates and lipids as well as in growth, development and immunity. Growth hormone deficiency in\r\nhuman occurs both in children and adults. The routine treatment for this condition is administration of recombinant human growth\r\nhormone (rhGH) made by prokaryotes. Since nonglycosylated human growth hormone is a biologically active protein, prokaryotic\r\nexpression systems are preferred for its production. Materials and Methods: Different strains of E.coli were transformed by plasmid\r\ncontaining human growth hormone gene and cultured in different conditions. After induction by IPTG, recombinant human growth\r\nhormone production was assessed using ELISA, dot blotting and western blotting techniques. Results: High levels of rhGH were\r\nproduced using E.coli prokaryotic protein production system. Conclusion: This simple and cost effective production process could\r\nbe recruited for large scale production of rhGH....
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