Part:BBa_K1601016
T7 promoteur,RBS - Cytochrome C S.oneidensis - HisTag
T7 promoter with RBS and Cytochrome C Shewanella oneidensis. A tag 10xHis is located to the end of the protein (Cterm) to enable its purification
We tested this part in the pSB1C3 backbone and the chassis BL21 (DE3). Cells were grown to an OD of between 0.2 and 0.4, the culture was the left for 2 hours without shaking to cause it to become anaerobic and then induced by adding 0.5mM IPTG and left for 3 hours at 37°C to allow protein production.
To test for protein production harvested cells were broken by sonication and analyzed by SDS-PAGE gel 15% (Figure 1). To determine the localisation of the cytochrome C the cell lysate was divided into cytoplasmic and membrane fractions. As we can see, the cytochrome c is located in both the cytoplasmic and membrane fractions. In the cytoplasmic fraction the protein is mainly present as dimers and partly as monomers, of the expected size for the full length protein containing the C terminal His tag. In the membrane fraction there is more monomeric protein present. This shows that the part is functional producing a protein of the expected size. The fractionation suggests that the cytochrome C is associated with the membrane.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |