Part:BBa_K1585310
B0034.GlgA
This is the translational unit of GlgA (Glycogen synthase), codon-optimized for E.coli. The enzyme elongates linear glycogen chains by using ADP-glucose units and forming α-1,4-glycosidic bonds. Through autophosphorylation, GlgA is also the starting point of glycogen synthesis in bacteria.
Usage and Biology
The construct was confirmed by sequencing. Moreover, the expression of glgA was tested by an SDS-PAGE. In the picture below, you can see that glgA is expressed in strains containing BBa_K1585310 in a pSB1K30 expression vector.
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| SDS-PAGE of GlgA in comparison to mRFP After induction with IPTG at OD=0.6, glgA was expressed. Samples were taken 6 and 18.5 hours after induction. The small arrows indicates the expected bands for GlgA. mRFP in pSB1K30 (T7 promoter) was used as the negative control. |
On top of that, the function of the construct was proven by an iodine staining with BL21 Gold (DE3) and BL21 Gold (DE3) expressing glgA 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 glgA indicates that the enzyme has the expected activity.
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| Iodine staining of BL21 Gold (DE3) with glgA in pSB1K30 compared to BL21 Gold (DE3) wild type Iodine staining was performed with overnight cultures which were adjusted to the same OD. The darker color of the strain expressing glgA shows that more glycogen is present. Therefore, the functionality of the glycogen synthase is shown. |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 934
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