Difference between revisions of "Part:BBa K1783000:Experience"
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In order to express tagged miraculin, we expressed K1783000 using the BL21 strain of E. coli. Four 5 mL cultures of transformed cells in LB media were grown to an OD of 0.6, then induced with 10% arabinose to a final %arabinose of 0.00%, 0.05%, 0.10%, and 0.20%. Cells were induced at 37oC for 18 hours. Cells were then spun down for 5 minutes at 18000xg, then lysed with 10% SDS. Supernatant was separated from insoluble pellet by spinning at 18000xg for 15 minutes. Presence of protein was confirmed using SDS PAGE. | In order to express tagged miraculin, we expressed K1783000 using the BL21 strain of E. coli. Four 5 mL cultures of transformed cells in LB media were grown to an OD of 0.6, then induced with 10% arabinose to a final %arabinose of 0.00%, 0.05%, 0.10%, and 0.20%. Cells were induced at 37oC for 18 hours. Cells were then spun down for 5 minutes at 18000xg, then lysed with 10% SDS. Supernatant was separated from insoluble pellet by spinning at 18000xg for 15 minutes. Presence of protein was confirmed using SDS PAGE. | ||
| − | + | https://static.igem.org/mediawiki/2015/7/7c/UMDmiracgel.jpg | |
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| + | Ladder is NEB P7708 Prestained Ladder (7-175 kDa) | ||
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| + | Lanes 1-4: Growth media (no cells) for 0%, 0.05%, 0.1%, and 0.2% arabinose induction, respectively | ||
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| + | Lanes 5-8: Resuspended cell pellet for 0%, 0.05%, 0.1%, and 0.2% arabinose induction, respectively | ||
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| + | Lanes 9-12: Supernatant of lysed cells for 0%, 0.05%, 0.1%, and 0.2% arabinose induction, respectively | ||
For future expression and purification, we advise using LB media supplemented with 1% glucose. An IMAC resin column charged with Nickel or Cobalt should be used to selectively purify the his-tagged miraculin. | For future expression and purification, we advise using LB media supplemented with 1% glucose. An IMAC resin column charged with Nickel or Cobalt should be used to selectively purify the his-tagged miraculin. | ||
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Latest revision as of 20:57, 17 September 2015
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Applications of BBa_K1783000
In order to express tagged miraculin, we expressed K1783000 using the BL21 strain of E. coli. Four 5 mL cultures of transformed cells in LB media were grown to an OD of 0.6, then induced with 10% arabinose to a final %arabinose of 0.00%, 0.05%, 0.10%, and 0.20%. Cells were induced at 37oC for 18 hours. Cells were then spun down for 5 minutes at 18000xg, then lysed with 10% SDS. Supernatant was separated from insoluble pellet by spinning at 18000xg for 15 minutes. Presence of protein was confirmed using SDS PAGE.
Ladder is NEB P7708 Prestained Ladder (7-175 kDa)
Lanes 1-4: Growth media (no cells) for 0%, 0.05%, 0.1%, and 0.2% arabinose induction, respectively
Lanes 5-8: Resuspended cell pellet for 0%, 0.05%, 0.1%, and 0.2% arabinose induction, respectively
Lanes 9-12: Supernatant of lysed cells for 0%, 0.05%, 0.1%, and 0.2% arabinose induction, respectively
For future expression and purification, we advise using LB media supplemented with 1% glucose. An IMAC resin column charged with Nickel or Cobalt should be used to selectively purify the his-tagged miraculin.
- Warning: Expression of K1783000 in DH5α using identical induction conditions as above was found to lead to significantly reduced product concentrations. This is likely due to the presence of proteases in DH5α that are not found in BL21.
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