Part:BBa_K2602013
VHb under weak Anderson promotor
Vitreoscilla hemoglobin under a weak 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 0.16 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 VVitreoscilla 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 sequencing and 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.
When expressing VHb, we found that addition of Aminolevulinic acid (ALA) increased the red color which is characteristic of hemoglobin (fig. 2). ALA is a precursor to heme, a ligand necessary for the function of hemoglobin. Bubbling the cultivations with carbon monoxide (CO), which is commonly done when expressing human hemoglobins, also increased the red color.
Figure 2: BBa_K2602010 and BBa_K2602013 expressed in E. coli BL21, as well as BBa_R0010 as a negative control.
Pellets in the back to the right have a stronger red color, characteristic of hemoglobin.
The part was expressed in E. coli BL21 at 37ºC and 150 rpm. The growth curve (fig. 3) was analyzed to see if there were any differences between different parts in the collection with respect to the promoter strength. The results of the analysis show that the part has a positive effect on the growth rate compared to the negative control, significant at a confidence level of 90%. For more details visit our modelling results.
Figure 3: OD600 as a function of time for the biobrick expressed in E. coli.
The blue line is the estimated sigmoidal function.
Source
The coding sequence is based on the Vitreoscilla hemoglobin basic part, K2602000. The Anderson promoter J23100 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 |