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Team:NTU-Singapore/Project/Wetlab
From 2009.igem.org
Conclusive Results
Successful Constructs
The following are the constructs that we have shown to be working.
- J23119-B0034-NorR-B0015 (system construct)
- J23119-B0034-GFP-B0015 (characterization construct)
- pLac(R0010)-B0034-CHE-B0015 (test construct)
- J23119-B0034-IFP-B0015-J23119-B0034-HO1(I15008)-B0015 (test construct)
The first construct is a system construct, in that it was intended for our system to feature this as a functional part.
The second construct is a characterization construct to characterize the modular function of the J23119 promoter in the transcription of NorR.
The third construct is also a test construct to only test and characterize the enzymatic activity of CHE.
The fourth construct 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'
Lanes 2 and 3 are the subjects of interest. Double digestion of J23119-B0034-NorR-B0015 with Xba1 & PstI indicated that the ligation in lane 2 had failed while lane 3 contained the successful construct. Length of J23119-B0034-NorR-B0015 corresponds to the expected length of ~1750 bp.
SDS PAGE verification of NorR
Successful expression of NorR
55 kDa NorR has been successfully produced by the construct. NorR band is highlighted with white arrow.
SDS PAGE characterization of NorR
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 results for NorR expressed at different inoculation temperatures
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.
