Team:Ruia-Mumbai/Improve
IMPROVE
pBAD_ompT_xylE:BBa_K2749003
This part consists of an arabinose induced promoter pBAD which in the presence of arabinose allows transcription of ompT-an
aspartic type endopeptidase allowing extracellular production of enzyme catechol 2,3-dioxygenase, causing cleavage of the oxidative ring of catechol.
aspartic type endopeptidase allowing extracellular production of enzyme catechol 2,3-dioxygenase, causing cleavage of the oxidative ring of catechol.
Fig 1: Gel-Red stained agarose gel. The expected size of ompT_xylE is 3.2kb and its two bands lie between 3 & 3.5kb of the ladder suggesting successful cloning of the improved part.
Characterisation:
2-HMS Assay-
The supernatant of the overnight grown culture in minimal medium (M9) with arabinose is collected after spinning down at maximum speed and analysed for the concentration of 2-Hydroxymuconate semialdehyde.
As ompT would cause the enzyme to be released extracellularly, the concentration of enzyme can be easily detected in the supernatant and hence determine the characterisation of the ompT_xylE.
Observation:
No yellow colouration was observed and hence can be concluded that the enzyme is not synthesized in the construct itself.
SDS-PAGE Coomassie Brilliant Blue staining-
From the SDS-PAGE gel it can be said that pBAD_ompT_xylE construct is not synthesizing the expected enzyme catechol-2,3-dioxygenase.
Comments:
The failure of this construct can be predicted to be due to-
- The protein being degraded in the the supernatant ie; media. For future prospects, we can use a Protease inhibitor after 16 hours incubation of the culture and then test for the enzyme production.
- As the protein secretion efficiency varies based on the complexity of protein due to disulfide bonds, proteases in the
periplasm, so the total quantity of protein excreted might be very low wherein for future project we can use a different
technique for protein staining which is more sensitive to low quantities of proteins like the silver nitrate staining.