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Team:Groningen/14 June 2010
From 2010.igem.org
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| - | '''Week 24 | + | '''Week 24''' |
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| + | '''Arend Jan''' | ||
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'''David & Peter''' | '''David & Peter''' | ||
| - | '''Extraction of Streptomyces cellwalls''' | + | '''Extraction of Streptomyces cellwalls & purification of chaplin proteins''' |
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| - | + | Harvesting of chaplin coated mycelium of Streptomyces with a razorblade. | |
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| - | [[ExtractionCellwallsGR]] | + | The mycelium was disrupted to extract the cell walls ([[ExtractionCellwallsGR]]) of Streptomyces. |
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| - | + | After extraction the freeze dried cellwalls consist for approx 5% of their dry weight of assembled chaplin proteins. Further purification and monomerisation of the chaplin proteins is achieved by TFA treatment ([[TFA treatmentGR]]) of extracted cellwalls. | |
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| - | + | TFA treated chaplins are monomeric and can be rediluted in demiwater or trisbuffer. Note that after redulution the monomeric chaplins will immediately start to reassemble in amyloid fibres. This proces is accelerated by shaking of the liquid. | |
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{{Team:Groningen/Footer}} | {{Team:Groningen/Footer}} | ||
Latest revision as of 19:27, 24 October 2010
Hydrophobofilm
pushing coatings into a greener future
Week 24
Arend Jan
Control digestion of 4 clones of pNZ8901-bbs (bbs = biobrick sites). The original plasmid does have a XbaI, SpeI, and PstI site (which were replaced in the PCR with these sites in the correct orientation), but no EcoRI site. Therefore the control digestion was done with EcoRI. Also, HindIII was removed in the PCR but not replaced so clones should not be cut with HindIII.
- 10ul plasmid - 1.5ul buffer EcoRI/R - 0.5ul EcoRI/HindIII - 3ul MQ
EcoRI cuts in all clones except only partially in 4. HindIII does not cut in all clones (positive control would have been nice, new enzyme however). Clones 1, 2, and 3 are good for sure.
David & Peter
Extraction of Streptomyces cellwalls & purification of chaplin proteins
Harvesting of chaplin coated mycelium of Streptomyces with a razorblade.
The mycelium was disrupted to extract the cell walls (ExtractionCellwallsGR) of Streptomyces.
After extraction the freeze dried cellwalls consist for approx 5% of their dry weight of assembled chaplin proteins. Further purification and monomerisation of the chaplin proteins is achieved by TFA treatment (TFA treatmentGR) of extracted cellwalls.
TFA treated chaplins are monomeric and can be rediluted in demiwater or trisbuffer. Note that after redulution the monomeric chaplins will immediately start to reassemble in amyloid fibres. This proces is accelerated by shaking of the liquid.
