Team:NTU-Singapore/Wetlab

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NTU@iGEM Project Introduction Methodology Team Introduction Team background Notebook Dry Lab Introduction ODEs used Modeling Constructs Parameters used Deterministic Modeling Sensitivity Analysis Stochastic Modeling Wetlab Introduction Materials and Equipment Protocols Verification of Detection & Lysis System Our New Parts Characterization Introduction Standard Promoters Characterization of pLacI-GFP pLacI-GFP images and clips Characterization of pLsrA-YFP New characterization Proposal Parameter Estimation & Correlations Acknowledgements

Introduction

Wet laboratory experiments are held in our faculty laboratories and include the following experiments:

1) Creation of Standard Biobrick parts
2) Digestion and Ligation of NTU@iGEM System
3) Characterization of standard Biobrick promoters via GFP activity
4) System verification using OD measurement

• An overview of how our experiments are conducted is represented in the above figure.
  

Our major biobrick parts: Lysis protein generator, E7-Immunity protein generator, LsrA promoter were created using standard biobrick construction protocol. The genes for these parts were extracted from their wild bacteria strains (Escherichia Coli MG1655, BW and W3110) and modified to become standard biobrick modules. The parts were then verified by Gel Electrophoresis for its base pair length.
After the parts were created, our “system assembly factory” starts to fuse the individual biobrick parts to from devices, and eventually to our overall system. Again, Gel electrophoresis confirmed that our system assembly process was successful as the end products had base pair lengths that match our prediction. Our Detection & Lysis system was tested with Ai2 induction and results have shown that our system works wonderfully! On addition of Ai2 in LB growth culture, optical density measurement starts to show a constant value over time as compared to our control experiment which shows a trend of ever increasing OD values. This illustrates that when AI2 is added, cell transformed with our lysis systems starts producing lysis gene which cause cell lysis and death, and hence a constant OD values. Without induction of Ai2, cells continue to multiply, leading to higher OD values.
While our system is being tested, we also performed characterization experiments for 2 promoters:

• Existing LacI promoter which is regulated by lactose concentration
• NTU@iGEM new LsrA promoter which is regulated by Ai2 concentration.

For more information characterization results, please see section: Characterization.