Part:BBa_K1618026
GlgX with IPTG-inducible promoter
GlgX is a genetic sequence which encodes for a glycogen debranching enzyme which will cleave alpha-1,6 linkages in glycogen, therefore reduce branching in glycogen to create a more linear molecule. GlgX is preceded by the LacI IPTG-inducible promoter an an RBS.
iGEM15_NRP-UEA used this part in E. coli. Cells were transformed and grown10 mL LB media overnight at 37 °C with shaking. Each culture was used to inoculate 2 x 10 mL of fresh media, grown to an OD of approximately 0.6 and then IPTG was added to one of each duplicate culture and the cultures continued to grow, with samples taken after 1 hour, 3 hours and overnight.
Glycogen was extracted from the cell pellet according to the following glycogen extraction protocol:
1. Harvest E. coli cells from liquid cultures by centrifugation at 5000 rpm for 10 minutes and resuspended in 10 mL water in a 50 mL Falcon tube. 2. Pellet resuspended cells by spinning at 5,000 × g for 10 minutes. Discard supernatant and resuspend pellet in 10 mL fresh water. 3. Sonicate at room temperature at 10 micron amplitude for 3 minutes, 1 second on and 2 seconds off. 4. Transfer to 50 mL centrifuge tubes and centrifuge at 30,000 × g for 15 minutes. 5. Transfer supernatant to a 50 mL Falcon tube. Add 5 mL of 0.2 M glycine, pH 10.5 and 5 mL chloroform. Shake vigorously and spin at 2000 rpm for 3 minutes to separate into aqueous and organic layers. 6. Transfer top, aqueous layer to a new 50 mL Falcon tube with a pipette and repeat step 5. 7. Transfer top, aqueous layer to a round-bottomed flask and remove any remaining chloroform using rotary evaporation. 8. Transfer to a 30 kDa spin filter and concentrate to ~8 mL by spinning at 5000 × g for approximately 55 minutes. Check after 30 minutes of centrifugation. 9. Transfer to 8 × 1 mL ultracentrifuge tubes and balance all to within 1 mg of each other. 10. Spin in Ultracentrifuge at 108,000 × g (55,000 rpm) at 4ºC for 2-3 hours. 11. Discard supernatant and resuspend pellets in 2 mL total volume of water and add to 50 mL Falcon tube.v 12. Precipitate glycogen with 8 mL cold ethanol. 13. Spin at 4,000 × g for 10 minutes and discard supernatant. 14. Dissolve pellet in 2 mL of water and freeze-dry overnight to yield the glycogen as an amorphous white powder.
The powder can be confirmed as glycogen by transmission electron microscopy (TEM):
Glycogen yield obtained were 50 % less in induced cultures. Non-induced:glycogen extract mass = 31.8 mg Induced with IPTG: glycogen extract mass = 14.6 mg
This confirms functional because GlgX enzyme will de-branch glycogen resulting in linear glucan strands, which are lost from the glycogen molecule.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1124
- 1000COMPATIBLE WITH RFC[1000]
| None |