Part:BBa_K2749003
pBAD_ompT_xylE
This part consists of an arabinose induced promoter pBAD which in presence of arabinose allows transcription of ompT-an aspartic type endopeptidase allowing extracellular production of enzyme catechol 2,3-dioxygenase causing cleavage of oxidative ring of catechol.
Characterisation:
2-HMS Assay-
The supernatant of the overnight grown culture in minimal medium with arabinose (M9) is collected after spinning down at maximum speed and analysed for the concentration of 2-HMS. 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 itself in the construct.
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- 1) 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. 2) 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 a different technique for protein staining which is more sensitive to low quantities of proteins like the silver nitrate staining can be used. 3) pelB can be used as an alternative to ompT for extracellular nature of the enzyme desired in DH5a cells.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 891
Illegal BamHI site found at 65 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |