Part:BBa_M36363:Experience

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Stanford Location

Plasmid name: pJ814

DNA 2.0 Gene ID: 133500

Organism: E coli

Device type: Actuator

Barcode Numbers of Glycerol Stocks: 0133009700 (SEW-S), 0133009649 (SEW-E), 0133011827 (SEW-W)

Box Label: BIOE 44 F13

Preliminary Characterization of pJ814 DbjA Activity

Preliminary testing of this part has been insufficient to determine whether it is functional.

Preliminary Assay

Description

Cells transformed with the pJ814 plasmid were grown overnight in a media of LB with either 100 μM rhamnose (LB+R), 0.2% glucose (LB+G), or no additional solutes (LB). 4 replicates of cells were grown for each medium. The LB+R medium was used to induce DbjA expression, while the LB+G and LB+R media were expected to produce cells with minimal DbjA expression. Cells were subsequently lysed with a chloroform & SDS procedure. 60 μL of each lysate was split into two tubes, one of which contained 1.375 mL of assay buffer, and the other of which contained 1.375 mL of assay buffer with 10 mM 1,2-dichloropropane substrate. Our assay buffer, in accordance with the protocol in [http://www.sciencedirect.com/science/article/pii/S0167701298000086 Holloway, Trevors, and Lee], consisted of 1 mM HEPES (titrated to pH 8.2), 20 mM sodium sulfate, 1 mM EDTA, and 0.375 mL phenol red pH indicator (concentration information was not provided with the package). We allowed the lysate to react in the assay buffer and substrate (if present) for 10 minutes, and then measured the OD540 of each sample in a spectrophotometer calibrated against water. Enzymatic activity was expected to lower the pH of the reaction solution, and thus to decrease its OD540 as the indicator shifted away from red towards yellow.

Data

Our preliminary data indicates that our assay procedure is not sufficiently optimized for us to characterize the behavior of this part. We measured roughly identical OD540 values for all reaction solutions with added substrate, and this value was equal to the average OD540 of the solutions derived from cells grown in LB without added substrate. Among the average OD540 values of reaction solutions without added substrate, the solutions derived from both LB+R and LB+G cell cultures both had lower OD540 values than the solutions derived from LB cultures.

Control Assay

Description

The same procedure in the preliminary assay was repeated, but instead using cells transformed with a pComet plasmid. Thus, these cells were not expected to demonstrate enzymatic activity on 1,2-dichloropropane.

Data

We measured roughly identical OD540 values for all reactions without added substrate, and this value was equal to the average OD540 of the solutions derived from cells grown in LB+R with added substrate. Among the average OD540 values of reaction solutions with added substrate, the solutions derived from both LB and LB+G cell cultures both had higher OD540 values than the solutions derived from LB+R cultures. The pH of one replicate solution representative of each sample type was measured, and the pHs of all solutions were within the range of 7.37-7.52. Although we did not have enough time to do more thorough pH measurements, this result suggests that the pHs of all solutions were roughly equal.

Interpretation of Data

Data from the control assay suggests that the presence of rhamnose or glucose in cell cultures does not affect the OD540 of reaction solutions without substrate. It also indicates that the presence of substrate in the reaction solution increases the OD540 of solutions derived from LB and LB+G medium, and it decreases the OD540 of solutions from LB+R medium. We do not know how to explain this difference, though it may be possible that 1,2-dichloropropane is involved in unexpected interactions with rhamnose, the reaction solution buffer, and/or phenol red.

Data from the preliminary assay indicates that the presence of rhamnose or glucose in cell cultures does not affect the OD540 of reaction solutions with substrate. It also shows that the presence of rhamnose or glucose in cell culture leads to a reaction solution, when not mixed with substrate, with lower OD540 values. We do not know how to interpret these results, in light of data from the control assay.

We can conclude that our current assay protocol is not optimized enough to produce meaningful data. Further work should include additional controls known to work with the published assay for haloalkane dehalogenase activity.

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