Team:NTU-Singapore/Project/Wetlab

Conclusive Results

Our working constructs!

• The first construct, J23119-B0034-NorR-B0015, is a system construct, in that it was intended for our system to feature this as a functional part.
• The second construct, J23119-B0034-GFP-B0015, is a characterization construct to characterize the modular function of the J23119 promoter in the transcription of NorR.
• The third construct, Lac(R0010)-B0034-CHE-B0015, is also a test construct to only test and characterize the enzymatic activity of CHE.
• The fourth construct, J23119-B0034-IFP-B0015-J23119-B0034-HO1(I15008)-B0015, is a test construct to only test the reporting ability of IFP.

J23119-B0034-NorR-B0015 (K256007)

NorR is an important NO sensor that triggers the transcription at pNO. Thus, we want an NorR producing construct to improve on the sensitivity of pNO.

Double Digest verification

Successful construct verification of J23119-B0034-NorR-B0015 by Double Digest!

Since the sole purpose of J23119-B0034-NorR-B0015 is to produce NorR, we shall dwell into the factors that might affect the production of the protein. The transformed cells were incubated at 3 different environmental temperatures (30 / 37 / 42 deg C).

SDS PAGE characterization was carried out (top right).

It has been observed that at elevated temperature, i.e. 42 deg C, production of NorR had decreased as shown by the diminished intensity (see 2nd lane). The production of NorR at 30 deg C and 37 deg C carried no significant difference.

Characterization of J23119 (K256003)

Since J23119 was used as the constitutive promoter for NorR producing construct, it is necessary for us to understand its modular function and how it would affect NorR production.

R0010-B0034-CHE-B0015 (K256028)

To examine the enzymatic activity of cholesteryl esterase, a test construct was synthesized. CHE production was induced by l0mM lactose. The cells were sonicated, and the cell lysate containing CHE was characterized using Amplex® Red Cholesterol Assay Kit. The characterization results is shown below.

Cholesterol Assay with CHE Construct

From the graph, the cell lysate containing CHE had achieved higher fluorescence readings than the negative control. The increase in fluorescence reading is due to enzyme-coupled reaction that detects free cholesterol concentration. Hence, we have proven that CHE had indeed been produced from this construct and had been working to our expectation.