Difference between revisions of "Part:BBa K1111007:Experience"

Latest revision as of 14:40, 4 October 2013

After the plasmid was complete, bacteria were transformed with it and plated. A colony was then picked and the plasmid miniprep was sent for sequencing while a glycerol stock of the colony was made. The sequencing indicated that a STOP codon had appeared just before the GFP sequence, after the linker, thus preventing us to see a green glow in the tubes when the plasmid was induced since the GFP was not expressed.
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 culture medium and lysate of the bacteria. The result was unfortunately negative. A His-tag purification under native conditions was then performed and followed by a NanoDrop quantification and SDS-PAGE. The NanoDrop indicated the presence of protein in the wash but not in the final eluate. This could be due to the protein folding over the His-tag and preventing it from binding to the purification column.
Comparison of the SDS-PAGE results between the induced colonies and negative control should be suffiscient to prove the expression of our protein upon arabinose induction if there is an additional band on the induced colonies' results. Unfortunately, the SDS-PAGE did not work due to a Coomassie stain issue and we could not verify the functioning of our plasmid.

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

UNIQ5cc3cc7905e42e1c-partinfo-00000000-QINU UNIQ5cc3cc7905e42e1c-partinfo-00000001-QINU

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+After the plasmid was complete, bacteria were transformed with it and plated. A colony was then picked and the plasmid miniprep was sent for sequencing while a glycerol stock of the colony was made. The sequencing indicated that a STOP codon had appeared just before the GFP sequence, after the linker, thus preventing us to see a green glow in the tubes when the plasmid was induced since the GFP was not expressed. <BR>
+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.<BR>
+A Western blot was performed with anti-His tag antibodies on the culture medium and lysate of the bacteria. The result was unfortunately negative. A His-tag purification under native conditions was then performed and followed by a NanoDrop quantification and SDS-PAGE. The NanoDrop indicated the presence of protein in the wash but not in the final eluate. This could be due to the protein folding over the His-tag and preventing it from binding to the purification column.<BR>
+Comparison of the SDS-PAGE results between the induced colonies and negative control should be suffiscient to prove the expression of our protein upon arabinose induction if there is an additional band on the induced colonies' results. Unfortunately, the SDS-PAGE did not work due to a Coomassie stain issue and we could not verify the functioning of our plasmid.
 ===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===