Part:BBa_K1585321

glgCAB for polycistronic expression of glycogen synthesis genes in E.coli

This composite part combines all 3 enzymes involved in glycogen synthesis. The ADP-glucose pyrophophorylase (GlgC) forms ADP-glucose from ATP and glucose-1-phosphate, the glycogen synthase (GlgA) elongates alpha-1,4-linked chains and the branching enzyme (GlgB) catalyzes the formation of alpha-1,6-linked branches. This construct is an extension and improvement of Part:BBa_K118016.

Usage and Biology

In order to upregulate the whole glycogen synthesis pathway, the polycistronic plasmid was built. The glgC coding sequence is based on the part BBa_K118016 from Team Edinburgh 2008, but the RBS B0034 was added to the existing Biobrick. The construct was confirmed by sequencing. The expression of all three enzymes GlgC, GlgA and GlgB was tested in BL21 Gold (DE3) strains containing BBa_K1585321 in a pSB1A30 expression vector.

SDS-PAGE of glgCAB in pSB1A30 glgCAB in pSB1A30 was expressed in BL21 Gold (DE3) strains and IPTG induced. The small arrows indicates the expected bands for all three enzymes . The BL21 Gold (DE3) wild type was used as negative control.

The combined functionality was characterized by iodine staining (see picture below). It was performed with Lugol's iodine which dyes glycogen in a brownish color. If more glycogen is present, the color of stainend cultures is darker. The darker staining of BL21 Gold (DE3) transformants of BBa_K1585321 indicates considerably more glycogen accumulations compared to the wild type.

Iodine stained samples of Bl21 Gold (DE3) with glgCAB compared to the wild type The samples were taken from overnight cultures in LB + 20 mM glucose and were adjusted to the same OD before staining with 200 µl Lugol's iodine solution.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 212
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2259
  Illegal BsaI site found at 3253