Construction of TBSR4-deficient strains of Shewanella

Abstract

This topic is the construction of the missing strain of TBSR4 of Shewanella. In today's global fossil energy shortage and fossil energy exploitation process will cause certain pollution to the environment, microbial fuel cell has become an important research direction. Shewanella is a gram-negative bacterium and a commonly used electrogenic bacterium for microbial fuel cells. Therefore, it has a unique advantage as a microbial fuel cell. Shewanella TBSR4 is dependent on a gene on the TonB iron carrier receptor on the outer membrane. TonB is related to iron homeostasis, which can affect the synthesis of cytochrome c and thus affect microbial electricity production. In this study, the effect of TBSR4 single gene knockout on the synthesis of cytochrome c was verified. After single gene knockout of TBSR4, the content of cytochrome c in the deletion type of TBSR4 was detected to determine whether TBSR4 gene of TBSR4 was related to the synthesis of cytochrome c. In this study, single gene knockout of TBSR4 was performed by using in-frame deletion mutation method. The main steps include: plasmid extraction, overlapping PCR amplification of TBSR4 fusion fragment, construction and transformation of recombinant suicide plasmid, and construction of deletion mutant. Although iron is abundant in nature, access to iron is often a challenge for microorganisms because the element exists primarily in the aerobic environment in the form of extremely insoluble iron Fe3+. To overcome this problem, microorganisms have evolved a variety of iron uptake strategies, one of which is a common strategy by secreting ferriderites, which are endogenously produced iron-chelate metabolites. Ferrifer-mediated iron transport is an alternative route of iron transport under iron-rich conditions and is essential under iron-deficient conditions. In Gram-negative bacteria, the transport of iron-ferriferic complexes from extracellular to periplasmic is primarily dependent on TonB-dependent siderophore receptors on the outer membrane. TBSRs) and TonB-ExbB-ExbD energy transduction system across intima and periplasm. Most TBSRs have similar domains: a barrel-like structure formed by 22 β folds and a globular stopper domain with n-ends. The plug domain binds to the substrate outside the outer membrane. When the iron-ferriderite complex binds to TBSR, the TBSR-TONB interaction domain changes. The TonB complex provides the energy converted by the electrochemical proton dynamic potential and transports the ferriderite complex into the periplasm. Subsequently, the Fe-ferriferal complex crosses the intima via ABC transporter or permease. In the cytoplasm, the iron in the Fe-ferriferal complex is reduced by reductase and released. In addition, under certain conditions, the iron-ferriferic complex can also be degraded in the periplasm, and the subsequently released Fe 3+ is reduced to Fe 2+. Therefore, the study of TBSRs is of great significance for iron transport and cytochrome c synthesis in Shewanella. Through a series of manipulative experiments, the correct missing strains were obtained, and the results of colony PCR and AGAR gel electrophoresis were consistent with expectations.