Difference between revisions of "Part:BBa K1111009:Experience"
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Sequencing of the part revealed the presence of an unexpected STOP codon before the superfolder GFP sequence. This could be due to a mistake in the design of the primers or in their fabrication. Isolation of the expressed protein should still be possible since a His tag was added to its CDS together with the Gibson assembly primers.
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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>
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Bacteria were transformed, inoculated and grown overnight so that an assay could be performed. 50ml of 20% arabinose was then added to the 5ml of their LB+Chl growth medium, to activate the pBAD/araC promoter and induce gelE expression. Western blots with anti-His antibodies were then performed on the culture medium and cell lysates but revealed themselves negative. As a last resort, Ni-NTA spin kit purification (for His tags) of bacteria prepared in the same way was done.
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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>
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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>
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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.<BR>
  
 
===Applications of BBa_K1111009===
 
===Applications of BBa_K1111009===

Revision as of 14:39, 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_K1111009

This part can be used to induce the expression of a gelatin-degrading protein upon a desired signal. Another promoter, responding to the specific requirements if the user, can be incorporated into the plasmid instead of the pBAD/araC promoter by Gibson assembly.

User Reviews

UNIQ591a0c8fa7902230-partinfo-00000000-QINU UNIQ591a0c8fa7902230-partinfo-00000001-QINU