Difference between revisions of "Part:BBa K1111009:Experience"
| Line 1: | Line 1: | ||
| − | |||
__NOTOC__ | __NOTOC__ | ||
| − | This | + | 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. |
| − | + | 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. | |
===Applications of BBa_K1111009=== | ===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=== | ===User Reviews=== | ||
Revision as of 18:52, 1 October 2013
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. 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.
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
UNIQf8bac9786a02ab11-partinfo-00000000-QINU UNIQf8bac9786a02ab11-partinfo-00000001-QINU