Part:BBa_K2602012
Vitreoscilla hemoglobin under medium strong Anderson promoter
Vitreoscilla hemoglobin under a medium strength Anderson promoter
Introduction
This composite part contains the Vitreoscilla hemoglobin found in the aerobic bacteria Vitreoscilla. It is expressed under an Anderson promoter of relative strength 0.47 and a medium strong ribosome binding site. A double terminator sequence is included to allow for a tight expression.
The entire sequence is included in the set of composite parts created by Team Lund 2018 to allow for a modulated expression of the Vitreoscilla hemoglobin. This allows the user to screen various expression levels of the protein so that a suitable effect on the cell productivity is obtained when expressed under low oxygen environments. This could be used by co-expressing the hemoglobin with a target protein of interest to increase yields in e.g. reactor settings or to increase cell survivability under stressful environments [1].
Experience
Successful insertion of this biobrick into pSB1C3 and transformation into Escherichia coli was confirmed through agarose gel electrophoresis.
After expression in E. coli BL21, we also performed SDS-PAGE (fig. 1). VHb has a molecular weight of 15.8 kDa, which is expected to be just above the second band of the ladder. The gel shows that all our transformations have a band with the expected molecular weight. However, we cannot confirm that it corresponds to VHb as the negative control also has a band with the same molecular weight. It is possible that there are other proteins expressed in E. coli of the same molecular weight, making the VHb difficult to discern.
Figure 1: SDS-PAGE results. From left to right: ladder, negative control
(biobrick BBa_R0010), K2602010-K2602016.
Source
The coding sequence is based on the Vitreoscilla hemoglobin basic part, K2602000. The Anderson promoter J23106 and ribosome binding site B0034 is used to express the protein and double terminators, B0012 and B0011, are used to avoid leakage.
References
[1] Geckil H, Gencer S, Kahraman H, Erenler SO (2003) Genetic engineering of Enterobacter aerogenes with the Vitreoscilla hemoglobin gene: cell growth, survival, and antioxidant enzyme status under oxidative stress. Res Microbiol 154:425–431
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |