Difference between revisions of "Part:BBa K1618026"

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Glycogen was extracted from the cell pellet according to the following glycogen extraction protocol:
 
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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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.
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(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
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(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.
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(12) Precipitate glycogen with 8 mL cold ethanol.
13. Spin at 4,000 × g for 10 minutes and discard supernatant.
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(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.
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(14) Dissolve pellet in 2 mL of water and freeze-dry overnight to yield the glycogen as an amorphous white powder. The powder was confirmed as glycogen  by transmission electron microscopy (TEM):
  
 
https://static.igem.org/mediawiki/2015/0/0a/NRP-Mark-Results4Image2.png[https://static.igem.org/mediawiki/2015/0/0a/NRP-Mark-Results4Image2.png]
 
https://static.igem.org/mediawiki/2015/0/0a/NRP-Mark-Results4Image2.png[https://static.igem.org/mediawiki/2015/0/0a/NRP-Mark-Results4Image2.png]
  
The powder can be confirmed as glycogen  by transmission electron microscopy (TEM):
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Glycogen yield obtained were 50 % less in induced cultures.   
 
Glycogen yield obtained were 50 % less in induced cultures.   
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Induced with IPTG: glycogen extract mass = 14.6 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.
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This confirms functionality because GlgX enzyme will de-branch glycogen resulting in linear glucan strands, which are lost from the glycogen molecule.
 +
 
 +
Additionally,  glycogen  was dissolved in 100 uL 100 mM sodium acetate buffer, pH 4 and then treated with <i>Pseudomonas</i> Isoamylase (1 Unit) at 37 °C for 3 hours. Isoamylase removes α-1,6 linkages and  de-branches the glycogen sample. A control sample of commercial glycogen was also debranched by this method. An aliquot of the debranched glycogen was then diluted 100 fold in matrix solution (1mg/mL Dihydroxybenzoic acid in 30% aq. Acetonitrile) and analysed by MALDI mass spectrometry.
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When GlgX is overexpressed the glycogen is less branched, hence there is a shift toward longer chain lengths:
 +
 
 +
[[File:Marc-Maldi_glgX.jpg]]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 11:57, 21 September 2015

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 was confirmed as glycogen by transmission electron microscopy (TEM):

NRP-Mark-Results4Image2.png[1]


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 functionality because GlgX enzyme will de-branch glycogen resulting in linear glucan strands, which are lost from the glycogen molecule.

Additionally, glycogen was dissolved in 100 uL 100 mM sodium acetate buffer, pH 4 and then treated with Pseudomonas Isoamylase (1 Unit) at 37 °C for 3 hours. Isoamylase removes α-1,6 linkages and de-branches the glycogen sample. A control sample of commercial glycogen was also debranched by this method. An aliquot of the debranched glycogen was then diluted 100 fold in matrix solution (1mg/mL Dihydroxybenzoic acid in 30% aq. Acetonitrile) and analysed by MALDI mass spectrometry. When GlgX is overexpressed the glycogen is less branched, hence there is a shift toward longer chain lengths:

Marc-Maldi glgX.jpg

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1124
  • 1000
    COMPATIBLE WITH RFC[1000]