Part:BBa_K1585312
B0034.GlgC
This is the translational unit of GlgC, the ADP-glucose pyrophophorylase. It creates ADP-glucose from glucose-1-phosphate for glycogen synthesis.
Usage and Biology
The BioBrick is based on BBa_K118016 by Team Edinburg 2008, but was fused with the well-characterized RBS B0034. For expression tests, BBa_K1585312 was cloned into a pSB1A30 expression vector and transformed in BL21 Gold (DE3).
|
| SDS-PAGE of GlgC in comparison to mRFP glgC was expressed in pSB1A30 and compared to mRFP in pSB1A30 (T7 promoter) expression. After induction with IPTG at OD=0.6, glgC was expressed, indicated by the small arrows pointing to the GlgC bands of 48 kDa. No difference between induced and uninduced was observed. After overnight expression the strong bands were no longer present. |
On top of that, the function of the construct was proven by an iodine staining with BL21 Gold (DE3) and BL21 Gold (DE3) expressing glgC in pSB1K30. The iodine staining is performed with Lugol's iodine which dyes glycogen resulting in a brown color. If more glycogen is present, the color of stainend cultures is darker. In the picture below, the brown color of BL21 Gold (DE3) expressing glgC indicates that the enzyme has the expected activity.
|
| Iodine staining of BL21 Gold (DE3) with glgC in pSB1K30 compared to BL21 Gold (DE3) wild type Iodine staining was performed with LB and LB + 40 mM glucose overnight cultures which were adjusted to the same OD. The cultures grown in medium with extra glucose were darker than those grown on just LB medium. The darker color of the strain expressing glgC in both samples shows that more glycogen was present. Therefore, the functionality of the ADP-glucose pyrophosphorylase is proved. |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 212
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |