Difference between revisions of "Part:BBa K1111007:Experience"
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After the plasmid was complete, bacteria were transformed with it and plated. A colony was then picked and the plasmid was sent for sequencing while a glycerol stock of the colony was made. After the sequence was verified, part of the stock was inoculated overnight and then induced by adding 50ul of 20% arabinose to the 5ml liquid culture medium, the expected result being secretion (or at least expression) of the MMP2 protein. A Western blot was performed with anti-His tag antibodies on the super stand and lysate of the bacteria. The result was unfortunately negative. A His-tag purification was then performed and followed by an SDS-PAGE.
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After the plasmid was complete, bacteria were transformed with it and plated. A colony was then picked and the plasmid was sent for sequencing while a glycerol stock of the colony was made. This indicated that a STOP codon had appeared just before the GFP sequence, after the linker. Isolation should still be possible since the His tag was added to the beginning of the MMP2 protein CDS. After the sequence was verified, part of the stock was inoculated overnight and then induced by adding 50ul of 20% arabinose to the 5ml liquid culture medium, the expected result being secretion (or at least expression) of the MMP2 protein. A Western blot was performed with anti-His tag antibodies on the super stand and lysate of the bacteria. The result was unfortunately negative. A His-tag purification was then performed and followed by an SDS-PAGE.
  
  

Revision as of 18:58, 1 October 2013

After the plasmid was complete, bacteria were transformed with it and plated. A colony was then picked and the plasmid was sent for sequencing while a glycerol stock of the colony was made. This indicated that a STOP codon had appeared just before the GFP sequence, after the linker. Isolation should still be possible since the His tag was added to the beginning of the MMP2 protein CDS. After the sequence was verified, part of the stock was inoculated overnight and then induced by adding 50ul of 20% arabinose to the 5ml liquid culture medium, the expected result being secretion (or at least expression) of the MMP2 protein. A Western blot was performed with anti-His tag antibodies on the super stand and lysate of the bacteria. The result was unfortunately negative. A His-tag purification was then performed and followed by an SDS-PAGE.


Applications of BBa_K1111007

This plasmid can be used for targeted secretion of MMP2 protein. The promoter driving the expression can be replaced by one whose induction conditions suit the user.

User Reviews

UNIQd461fe6c4955441c-partinfo-00000000-QINU UNIQd461fe6c4955441c-partinfo-00000001-QINU