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The T7 gene is itself under the control of a lac promoter. Normally, both the lac promoter and the T7 promoter are repressed in the E. coli cell by the Lac repressor. In order to initiate transcription, an inducer must bind to the lac repressor and prevent it from inhibiting the gene expression of the T7 gene. Once this happens, the gene can be ...
T7 polymerase is a representative member of the single-subunit DNA-dependent RNAP (ssRNAP) family. Other members include phage T3 and SP6 RNA polymerases, the mitochondrial RNA polymerase (), and the chloroplastic ssRNAP.
T7 DNA polymerase is an enzyme used during the DNA replication of the T7 bacteriophage. During this process, the DNA polymerase “reads” existing DNA strands and creates two new strands that match the existing ones. The T7 DNA polymerase requires a host factor, E. coli thioredoxin, [1] in order to carry out its function
T7 DNA polymerase uses E. coli's endogenous thioredoxin, a REDOX protein, as a sliding DNA clamp during phage DNA replication (though thioredoxin normally has a different function). The sliding clamp functions to hold the polymerase onto the DNA, which increases the rate of synthesis. [20]
T7 DNA helicase (gp4) is a hexameric motor protein encoded by T7 phages that uses energy from dTTP hydrolysis to process unidirectionally along single stranded DNA, separating the two strands as it progresses. It is also a primase, making short stretches of RNA that initiates DNA synthesis. [1] It forms a complex with T7 DNA polymerase.
This function is enabled by the presence of a recombinant λ-prophage (DE3). DE3 carries a T7 RNA polymerase (RNAP) gene under the control of a lacUV5 promoter (lacUV5-T7 gene 1). T7-RNAP is highly specific to the T7 promoter and orthogonal to native E. coli promoters.
The promoters used for these vector are usually based on the promoter of the lac operon or the T7 promoter, [11] and they are normally regulated by the lac operator. These promoters may also be hybrids of different promoters, for example, the Tac-Promoter is a hybrid of trp and lac promoters. [12]
Nonetheless, a basal level of the T7-RNA-polymerase is observable even without induction. T7 lysozyme acts as an inhibitor of the T7-RNA-polymerase. Newly invented strains, containing a helper plasmid (pLysS), constitutively co-express low levels of T7 lysozyme, providing high stringency and consistent expression of the toxic recombinant ...