Difference between revisions of "Part:BBa K1783000:Experience"
 
(12 intermediate revisions by the same user not shown)
Line 5: Line 5:
 
===Applications of BBa_K1783000===
 
===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.  
 
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
 +
 +
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.
 
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.
Line 23: Line 33:
 
<!-- End of the user review template -->
 
<!-- End of the user review template -->
 
<!-- DON'T DELETE --><partinfo>BBa_K1783000 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K1783000 EndReviews</partinfo>
 +
[[File:Example.jpg]]

Latest revision as of 20:57, 17 September 2015

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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.

UMDmiracgel.jpg

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.

User Reviews

UNIQ71a45b597d81a637-partinfo-00000000-QINU UNIQ71a45b597d81a637-partinfo-00000001-QINU Example.jpg