Difference between revisions of "Part:BBa K2749005"

Latest revision as of 18:57, 5 December 2018

pBAD_xylE

The part consists of promoter pBAD which in presence of arabinose initiates transcription and hence synthesis of the enzyme catechol-2,3-dioxygenase, which degrade catechol to 2-hydroxymuconate semialdehyde.

Characterization of the part pBAD_xylE:

2-HMS Assay-

In this assay, 2-Hydroxymuconate Semialdehyde (2-HMS) is detected which is degradation product of catechol, catalysed by enzyme catechol-2,3-dioxygenase. This product has a characteristic Amax at 380 nm. Here in this assay, we used 415 nm and a range of concentration of catechol prepared (0.1 - 0.4 mM) was used for 2-HMS detection at intervals of 5 mins where total volume of test was 100 uL with the prepared cell suspension culture(18-24 hrs old) of 0.5 OD.

Link to the protocol followed: https://docs.google.com/document/d/1nQblRhNW9mQdp4-v2tOf3AuiAgS5yaU2f5CmxueqWlc/edit

Observation:

pBAD_xylE with Glucose as substrate- Absorbance readings at 415 nm at different time intervals for range of catechol from 0.1 mM to 0.4 mM

pBAD_xylE with Arabinose as substrate Absorbance readings at 415 nm at different time intervals for range of catechol from 0.1 mM to 0.4 mM

From the above graph of Absorbance v/s Time of the reaction system at 0.35 mM catechol concentration, it can be interpreted that- With advancement in time, the absorbance of the system increases when arabinose is used in the substrate but remains constant with glucose. The experiment performed involved a range of catechol concentration from 0.1 to 0.4 mM out of which the concentration of 0.35 mM was prominent where maximum increase of the absorbance value was observed for arabinose provided cells. With increasing catechol concentration, the absorbance value increased till 0.35 mM catechol concentration. After the concentration of 0.35 mM of catechol, the absorbance readings of the construct for both of the substrates containing glucose as well as arabinose is found to decrease, which can be suggested from the fact that catechol at high concentrations is toxic for the cell growth. The pBAD_xylE construct is specifically activated in presence of arabinose and hence concludes the non leaky expression of the promoter, which is evident from the graph having sharp increase in 2-HMS concentration where cells are provided with the substrate containing arabinose but not not in glucose. Hence, the enzyme is specifically being produced in presence of only arabinose.

SDS-PAGE Coomassie Brilliant Blue stained-

From the above SDS-PAGE Coomassie Brilliant Blue stained gel it is interpreted that- The dark protein bands are observed slightly above the 33 kDa ladder which is equal to the expected size of the enzyme catechol-2,3-dioxygenase of 35 kDa. {ref.https://www.ncbi.nlm.nih.gov/pubmed/8713131}

DH5a pBAD_xylE transformants grown in Glucose do not show any protein bands at this size and so can be determined to be negative control consistent with the the fact that pBAD promoter is specifically induced by arabinose only to synthesise the enzyme catechol-2,3-dioxygenase.

DH5a pBAD_xylE transformants grown in Arabinose show the bands at the expected size. Hence it can be concluded that in presence of Glucose in the substrate, the enzyme is not synthesised, while in presence of Arabinose, the enzyme is synthesised, and therefore can be used in the project design where kill switch and enzyme module can be made functional at specific time points wrt to presence of a single molecule arabinose which acts as both inducer for enzyme module as well as repressor for kill switch module.

Sequence and Features