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Smart Healthcare in Smart Cities
Published in Lavanya Sharma, Towards Smart World, 2020
Lac operon is the operon responsible for transport mechanisms in various types of lactose-digested bacteria, such as Escherichia coli. The general opinion is that the multistability in regulatory networks is extensively influenced by the decrease of noise caused by the perturbation. Particularly, at the level, its geometry, great variations in the repressor, and activator protein are probably present with their typical small number of copies in the cell. Although the multistability gene regulatory circuits are not inherent, these fluctuations can induce fast communication (“chat”) between the involved “on” state and excluded “off” condition for the transcription of the genes, inducing less efficiency of the cells and leading to selective deficiencies. Multistability, together with the corresponding hysteresis, allows cells to take part in threshold chemical concentrations (limits) and turn them off at low enough concentrations. This enables the efficiency of penetrating power on/off the decision for gene transcription by the fluctuation of the concentration value of the repressor and activator [88–90].
β-galactosidase Using Gel-Filtration Chromatography
Published in Maik W. Jornitz, Filtration and Purification in the Biopharmaceutical Industry, 2019
In order to determine the extent to which E. coli-PAD overexpresses β-galactosidase, equivalent numbers of permeabilized cells from an overnight culture of E. coli-PAD and a wild-type E. coli strain were assayed for enzyme activity. Because β-galactosidase is an inducible enzyme, its synthesis needed to be induced in the wild-type strain thus, IPTG (isopropyl-l-thio-β-D-galactoside) was added to the culture at a final concentration of 1.0 mM. IPTG, unlike lactose, is non-metabolizable and acts directly by inhibiting the lac repressor. It is the generally preferred inducer for molecular studies involving the regulation and control of the lac operon. By contrast, E. coli-PAD requires no induction because the production of β-galactosidase, driven by plasmid pRS415, is constitutive.
Transcriptional Regulation
Published in Markus W. Covert, Fundamentals of Systems Biology, 2017
The promoter of the lac operon contains a binding site for the CRP-cAMP complex. Thus, the operon is only fully expressed in the presence of CRP-cAMP, which only appears in the absence of glucose. However, what if there is no lactose in the environment? It does not make sense to express the lac genes unless both glucose is absent and lactose is present. E. coli addresses this problem with another transcription factor: LacI (the “I” stands for “inhibitor”). Free LacI inhibits transcription by binding its own operator in the lac operon promoter. However, when lactose is present in the external environment, one of its metabolic products (allolactose) binds LacI, reducing LacI’s binding affinity to the operator and enabling the transcription of the lac operon.
Enhancement of prodigiosin synthetase (PigC) production from recombinant Escherichia coli through optimization of induction strategy and media
Published in Preparative Biochemistry and Biotechnology, 2018
Zhongyu You, Suping Zhang, Xiaoxia Liu, Yujie Wang
Lactose, the natural lac operon inducer, is a low-cost and simple disaccharide. Due to its higher cost and possible toxicity, IPTG is often replaced with lactose for protein induction.[13,27] We investigated the effects of lactose induction on PigC production. As shown in Table 1, lactose induced PigC expression much more efficiently, and the highest specific activities (67.2 ± 1.7 U/mL) were about twofold higher than with IPTG induction. This result contradicts previous studies demonstrating that IPTG is more effective for inducing recombinant protein expression.[11,24] When lactose is used to induce expression from the T7-lacO promoter, it must be converted by 3-galactosidase to allolactose, which binds to the repressor; this is different from IPTG, which binds directly to the repressor protein without modification.[11] The conversion process could reduce the synthesis rate of PigC allowing nascent PigC to fold correctly. Thus, lactose is a more suitable inducer than IPTG for PigC expression.
A synthetic biology approach for the design of genetic algorithms with bacterial agents
Published in International Journal of Parallel, Emergent and Distributed Systems, 2021
A. Gargantilla Becerra, M. Gutiérrez, R. Lahoz-Beltra
Step 4. Fitness evaluation. – Once the bacterial division and mutation take place, the fitness value is calculated (Figure 2). Inspired by synthetic biology BAGA assumes that bacteria include in their plasmid a hypothetical operon (i.e. a group of genes under the control of a region of the DNA called a promoter) as well as a molecule playing a role of operon activator. When activator enters into the interior of the bacterium, it activates the operon being the activator concentration the output of a particular optimisation problem. For example, let us consider a problem consisting in finding the maximum of a function y = f(x), then the activator concentration is given by y. In this example, the value of x results from decoding the plasmid sequence. In terms of synthetic biology, the algorithm assumes as activator the sugar emulator IPTG (isopropyl β-D-1-thiogalactopyranoside), which activates Lac operon (i.e. the operon lactose). For instance, in the above optimisation example, the concentration of IPTG is the value of y, in the 0/1 knapsack problem IPTG concentration is given by where xi represents whether a certain item i has been (1) or not (0) included in the knapsack, being vi its value, etc. In short, in the algorithm IPTG molecules act as an activator of a ‘Z operon’. The Z operon calculates the fitness of a given bacterium from the expression of a Z gene that synthesises a z protein. The concentration of z protein, is the bacterial fitness value, which is given by function z = f(IPTG). The convenience of using a particular function will depend on the optimisation problem. For instance, linear function, Hill function or any other function may be appropriate on a case-by-case basis (Table 1). In the case of the Hill function and this applies to any other chosen function, its parameters (i.e. v, k and n) are set empirically, normalising with z = f(IPTG) function the value of fitness z between 0 and 1.
Optimization of the 503 antigen induction strategy of Leishmania infantum chagasi expressed in Escherichia coli M15
Published in Preparative Biochemistry and Biotechnology, 2018
Luan Tales Costa de Paiva Vasconcelos, Marcos Antônio Oliveira Filho, Vitor Troccoli Ribeiro, Jaciara Silva de Araújo, Francisco Canindé de Sousa Junior, Daniella Regina Arantes Martins, Everaldo Silvino dos Santos
The mechanism of transport of IPTG through the cell membrane is also a considerable factor in the expression of heterologous genes, however, it is still not sufficiently characterized.[34] Despite that, it has been reported for intracellular IPTG control that it can pass through the cell membrane independently of lactose permease[35] and that it is actively transported by lacY.[36] In turn, lactose presents as a competitive inducer and it can be used for both expression and growth. By the dynamics of this compound, the real inducer in this system is the allolactose, which is an intermediate product produced by β-galactosidase (lacY of lac operon) during the conversion of lactose to glucose and galactose. In the case of repression, allolactose is not sufficiently produced and does not bind to the lac operon repressor significantly to provide a good expression.[37] However, since experiments using lactose show differences in biomass levels when compared to assays using IPTG, the efficiency of using lactose as a source of carbon rather than for the production of the antigen is evident. Such behavior suggests the role of cyclic adenosine monophosphate (cAMP) on the regulation of the E. coli’s growth rate and the lac operon expression trade-off. As the transcription of the lac operon is adjusted by the lac repressor and the cAMP receptor protein (CRP).[38] Therefore, in a medium with glucose, this sugar drains phosphate from the IIA component of glucose-specific phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) (EAII[Glc]) that remains desphosphorylated.[39] In this way, the EAII[Glc] is not able to activate membrane-bound enzyme adenylate cyclase that generates the signal metabolite cAMP. It is known that cAMP binds to the CRP forming the cAMP-CRP complex, which in turn binds near the lac promoter and then improves its transcription.[40] Hence, the use of lactose as inducer can keep the EAII[Glc] non-phosphorylated,[41] but while glucose lowers cAMP level inhibiting the lactose permease, lactose lowers the signal molecule concentration without inhibiting its own entry to the cell.[42] Therefore, this control limits the lac operon expression and it can be used for favoring cell growth instead.