Part:BBa_K2872902
DspB Dsp A protein coding fused with T7 promoter at the N-terminus
DspB Dsp A protein coding fused with T7 promoter at the N-terminus
Usage and Biology
The composite improvement part T7DsbADspB allows for the regulation of DsbADspB expression via the T7 promoter. This part is meant for use in a common host strain BL-21 (DE3) E. coli. This strain possesses a chromosomal copy of the T7 RNA polymerase gene, a bacteriophage RNA polymerase. The T7 RNA polymerase recognizes the T7 promoter upstream of DsbADspB and initiates its expression. Besides high level of transcription, to reduce basal transcription the host strain carries another plasmid called pLysS. This plasmid contains genes coding for lysozyme, and chloramphenicol resistance to maintain selection on the plasmid in culture. The low level of lysozyme expressed from pLysS prevents basal transcription of T7 RNA polymerase from the T7 promoter hence it has as an addition layer of regulation over the expression of the DsbADspB. In situations where constitutive expression of DsbADspB is required, site directed mutagenesis of the T7 promoter sequence to result in a constitutive promoter. Sequence and Features
Characterization
Dsp plasmid transformed into BL21DE3 plysS:
- Transformed cells
- Inoculated single 3 separate colonies into 3ml Lb+chl, shaking at 37 degrees Celcius
- Grew until OD 40-60 using a Klett colorimeter
- Added 1mM IPTG. 1ml aliquot taken at t=0, t=3h and t=24h
- Extracellular separated from intracellular using centrifugation at 13,000 rpm for 10min
- UV Vis Spectrophotometric reading of total protein taken to note if any increase in extracellular protein was present
Agarose gel electrophoresis of Dsp plasmid transformed into BL21DE3 plysS and digested with EcoRI and PstI to check size
Transformants grew successfully on LB- Chl plates
The black arrows show bands of interest
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 259
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 415
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