Difference between revisions of "Part:BBa K1790003"
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The pET expression system is one of the most widely used systems for the cloning and in vivo expression of recombinant proteins in E. coli. This is due to the high selectivity of the pET system's bacteriophage T7 RNA polymerase for its cognate promoter sequences, the high level of activity of the polymerase and the high translation efficiency mediated by the T7 gene 10 translation initiation signals. In the pET system, the protein coding sequence of interest is cloned downstream of the T7 promoter and gene 10 leader sequences, and then transformed into E. coli strains. | The pET expression system is one of the most widely used systems for the cloning and in vivo expression of recombinant proteins in E. coli. This is due to the high selectivity of the pET system's bacteriophage T7 RNA polymerase for its cognate promoter sequences, the high level of activity of the polymerase and the high translation efficiency mediated by the T7 gene 10 translation initiation signals. In the pET system, the protein coding sequence of interest is cloned downstream of the T7 promoter and gene 10 leader sequences, and then transformed into E. coli strains. | ||
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Protein expression is achieved either by IPTG induction of a chromosomally integrated cassette in which the T7 RNA polymerase is expressed from the lacUV5 promoter, or by infection with the polymerase-expressing bacteriophage lambda CE6. Due to the specificity of the T7 promoter, basal expression of cloned target genes is extremely low in strains lacking a source of T7 RNA polymerase. Upon induction the highly active polymerase essentially out-competes transcription by the host RNA polymerase. This phenomenon, together with high-efficiency translation, achieves expression levels in which the target protein may constitute the majority of the cellular protein—after only a few hours. | Protein expression is achieved either by IPTG induction of a chromosomally integrated cassette in which the T7 RNA polymerase is expressed from the lacUV5 promoter, or by infection with the polymerase-expressing bacteriophage lambda CE6. Due to the specificity of the T7 promoter, basal expression of cloned target genes is extremely low in strains lacking a source of T7 RNA polymerase. Upon induction the highly active polymerase essentially out-competes transcription by the host RNA polymerase. This phenomenon, together with high-efficiency translation, achieves expression levels in which the target protein may constitute the majority of the cellular protein—after only a few hours. | ||
Revision as of 11:52, 18 August 2015
GBS
Geobacillus stearothermophilus (formally Bacillus stearothermophilus) is a rod-shaped, Gram-positive bacterium and a member of the division Firmicutes. The bacteria is a thermophile and is widely distributed in soil, hot springs, ocean sediment, and is a cause of spoilage in food products. It will grow within a temperature range of 30-75 degrees Celsius. Some strains are capable of oxidizing carbon monoxide aerobically. It is commonly used as a challenge organism for sterilization validation studies and periodic check of sterilization cycles. Recently, a DNA polymerase derived from these bacteria, Bst polymerase, has become important in molecular biology applications.
HAD
YniC is a sugar phosphatase belonging to the superfamily of haloacid dehalogenase (HAD)-like hydrolases. Its preferred substrate is 2-deoxyglucose-6-phosphate [Kuznetsova06]. The phosphatase activity of YniC was first discovered in a high-throughput screen of purified proteins [Kuznetsova05]. Phosphatase activity of YniC is dependent on the presence of a divalent cation such as Mg2+, which appears to affect substrate binding [Kuznetsova06]. Mutagenesis of the predicted catalytic Asp residues in YniC results in loss of phosphatase activity. A yniC deletion mutant is more sensitive to the presence of 2-deoxyglucose in the growth medium than wild type, while a strain overexpressing yniC tolerates higher concentrations of 2-deoxyglucose [Kuznetsova06]. 2-deoxyglucose is taken up by E. coli and is phosphorylated to 2-deoxyglucose-6P, a toxic analog of glucose-6P [Dietz71].
GlnH
The GlnHPQ high-affinity glutamine transport system is a member of the ATP-Binding Cassette (ABC) Superfamily of transporters [Wu95]. Based on sequence similarity, GlnH is the periplasmic glutamine-binding protein, GlnQ is the ATP-binding component, and GlnP is the membrane component of the ABC transporter. Mutation of glnP results in the impaired ability to transport glutamine as well as the inability to utilized glutamine as a sole source of carbon [Masters81, Nohno86]. Expression of the cloned glnHPQ genes on a plasmid vector restored the glnH, glnP and glnQ mutants' abilities to transport glutamine and utilize glutamine as a sole carbon source [Nohno86].
T7
The pET expression system is one of the most widely used systems for the cloning and in vivo expression of recombinant proteins in E. coli. This is due to the high selectivity of the pET system's bacteriophage T7 RNA polymerase for its cognate promoter sequences, the high level of activity of the polymerase and the high translation efficiency mediated by the T7 gene 10 translation initiation signals. In the pET system, the protein coding sequence of interest is cloned downstream of the T7 promoter and gene 10 leader sequences, and then transformed into E. coli strains.
Protein expression is achieved either by IPTG induction of a chromosomally integrated cassette in which the T7 RNA polymerase is expressed from the lacUV5 promoter, or by infection with the polymerase-expressing bacteriophage lambda CE6. Due to the specificity of the T7 promoter, basal expression of cloned target genes is extremely low in strains lacking a source of T7 RNA polymerase. Upon induction the highly active polymerase essentially out-competes transcription by the host RNA polymerase. This phenomenon, together with high-efficiency translation, achieves expression levels in which the target protein may constitute the majority of the cellular protein—after only a few hours.
Operon lactose
Bacterial operons are polycistronic transcripts that are able to produce multiple proteins from one mRNA transcript. The gene product of lacZ is β-galactosidase which cleaves lactose, a disaccharide, into glucose and galactose. LacY encodes lactose permease, a protein which becomes embedded in the cytoplasmic membrane to enable transport of lactose into the cell. Mechanism: hydrogen from the outside of the cell binds to a carboxyl group on the enzyme that allows it to undergo a conformational change.