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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.
Transport of Nutrients and Carbon Catabolite Repression for the Selective Carbon Sources
Published in Kazuyuki Shimizu, Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production, 2017
The E. coli lac operon is only expressed if allolactose (a lactose isomer formed by ^-galactosidase) binds and inactivates the lac repressor. Lactose cannot be transported into the cell in the presence of glucose, because the lactose permease, LacY is inactive in the presence of glucose (Winler and Wilson 1967). As shown in Fig. 9, phosphorylated EIIAGlc is dominant when glucose is absent and does not interact with LacY, whereas unphosphorylated EIIAGlc can bind and inactivates LacY when glucose is present (Hogema et al. 1999, Nelson et al. 1983). This only occurs if lactose is present (Smirnova et al. 2007). The same mechanism may be seen for the transport of other secondary carbon sources such as maltose, melibiose, raffinose, and galactose (Titgemeyer et al. 1994, Misko et al. 1987).
β-galactosidase Using Gel-Filtration Chromatography
Published in Maik W. Jornitz, Filtration and Purification in the Biopharmaceutical Industry, 2019
The enzyme has three activities that ultimately result in the complete breakdown of the disaccharide lactose into galactose plus glucose. First, β-galactosidase cleaves lactose into galactose plus glucose. Second, the enzyme acts as a transglycosylase, converting lactose into allolactose. Third, it hydrolyzes allolactose into galactose plus glucose. Historically, it has been a puzzle as to why the β-galactosidase protein is so large and why it needs to be a tetramer. The recent elucidation of this enzyme’s multiple and sequential activities may help explain its structural complexity and large mass.6
Cloning and expression of α-amylase in E. coli: genesis of a superior biocatalyst for substrate-specific MFC
Published in International Journal of Green Energy, 2019
Arpita Nandy, Samir Jana, Moumita Khamrai, Vikash Kumar, Shritama Mukherjee, Arindam Bhattacharyya, Patit P. Kundu
pET19b has an IPTG inducible T7 promoter and the gene of interest lies in the multiple cloning site of the vector. After selecting the positive clones, i.e., E. coli BL21(DE3) transformants harboring pETamyA plasmid, individual colony is grown in LB-ampicillin (conc: 50 µg/ml) media at 37°C and 200–300 rpm until Optical density (O.D.) Value reached 0.5–0.6 at 600 nm. Protein expression was induced through the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, a molecular mimic of allolactose, that triggers transcription of the lac operon). After incubation of 3–4 h in shaking condition, cells were pelleted down at 6000 rpm for 10 min at 4°C and stored for observing extracellular enzymatic activity. Intracellular activity of amyA was not measured as the extracellular secretion was confirmed previously by starch agar plates. Control studies were also performed with native E. coli.
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.