Difference between revisions of "Team:Aachen/Composite Part"

Latest revision as of 02:45, 19 September 2015

Our BioBrick [http://parts.igem.org/Part:BBa_K1585321 BBa_K1585321] for polycistronic expression of glycogen synthesis genes is a composite part that combines all 3 glycogen formation enzymes. The ADP-glucose pyrophophorylase (GlgC) forms ADP-glucose from ATP and glucose-1-phosphate, the glycogen synthase (GlgA) elongates α-1,4-linked chains and the branching enzyme (GlgB) catalyzes the formation of α-1,6-linked branches. This construct is an extension and improvement of the Part [http://parts.igem.org/Part:BBa_K118016 BBa_K118016]

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 the 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 stained 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 staining BL21 Gold (DE3) + glgCAB vs. wild type
Cultivated in LB + 20 mM glucose, BL21 Gold (DE3) + glgCAB stained distinctly darker than the BL21 Gold (DE3) wild type.

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 {{Team:Aachen/Header}}
-A composite part is a functional unit of DNA consisting of two or more basic parts assembled together. BBa_I13507 is an example of a composite part, consisting of an RBS, a protein coding region for a red fluorescent protein, and a terminator.
+{{Team:Aachen/StartTour}}
-New composite BioBrick devices can be made by combining existing BioBrick Parts (like Inverters, Amplifiers, Smell Generators, Protein Balloon Generators, Senders, Receivers, Actuators, and so on).
-=GlgCAB for polycistronic expression of glycogen synthesis genes in E.coli=
+Our BioBrick [http://parts.igem.org/Part:BBa_K1585321 BBa_K1585321] for polycistronic expression of glycogen synthesis genes is a composite part that combines all 3 glycogen formation enzymes. The ADP-glucose pyrophophorylase (GlgC) forms ADP-glucose from ATP and glucose-1-phosphate, the glycogen synthase (GlgA) elongates α-1,4-linked chains and the branching enzyme (GlgB) catalyzes the formation of α-1,6-linked branches. This construct is an extension and improvement of the Part [http://parts.igem.org/Part:BBa_K118016 BBa_K118016]
-[http://parts.igem.org/Part:BBa_K1585321 BBa_K1585321]
+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.
-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:[http://parts.igem.org/Part:BBa_K118016 BBa_K118016]
+{{Team:Aachen/Figure|Aachen_glgCAB for registry.png|title=SDS-PAGE of ''glgCAB'' in pSB1A30|subtitle=''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 the negative control.|size=large}}
-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 he 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.
+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 stained cultures is darker. The darker staining of BL21 Gold (DE3) transformants of BBa_K1585321 indicates considerably more glycogen accumulations compared to the wild type.
-{{Team:Aachen/Figure|Aachen_glgCAB for registry.png|title=SDS-PAGE of ''glgCAB'' in pSB1A30|subtitle=''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 the negative control.|size=large}}
-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 picture indicates considerably more glycogen accumulations compared to the wild type.
 {{Team:Aachen/Figure|Aachen_glgCAB , WT_v2.png|title=Iodine staining BL21 Gold (DE3) + ''glgCAB'' vs. wild type |subtitle=Cultivated in LB + 20 mM glucose, BL21 Gold (DE3) + ''glgCAB''  stained distinctly darker than the BL21 Gold (DE3) wild type.|size=medium}}