Project Overview

We studied the efficiency of rho-independent transcription terminators which are essential parts of synthetic genetic circuits using both theoretical calculation and experimental methods. We first developed a theoretical model. This model calculates the free energy of RNA folding and predicts the secondary structure of terminators. Based on the secondary structure, we developed an algorithm that predicts the efficiency of the terminator. The software (TTEC) based on the algorithm is available at (Figure.1). We also collected existing data from various publications and built up a database for comprehensive study on terminator efficiency (Figure.2).

Figure 1, TTEC website

Figure 2, Terminator Efficiency database

To validate our theoretical prediction, we performed biological experiments. We constructed a vector expressing RFP and GFP and designed 100 terminators to be inserted between RFP and GFP sequences (Figure 3). We measured the fluorescence intensity of RFP and GRP, respectively, which were further used to calculate the efficiency of the inserted terminator. We found that the experimental results are in a good agreement with theoretical prediction, and our model is useful for studying transcriptional regulation. Our terminator efficiency measurement protocol has been submitted to Biobrick foundation as a technical standard(BBF RFC 90).

Figure 3: Plasmid design

TTEC is the first published software to predict the efficiency of terminators. Our work will help to characterize the transcription regulation and predict gene expression.

South University of Science and Technology of China