Transcription Terminator Efficiency Calculator (TTEC) - PowerPoint PPT Presentation

Transcription Terminator Efficiency Calculator (TTEC) SUSTC-Shenzhen-B

Outline 1 Background & Motivation 2 Terminator Efficiency Calculator Algorithm Database Experiment 3 Human Practice

Background & Motivation RBS CDS Promoter Terminator A rho-independent terminator

Terminator efficiency Case 1: Completely terminate transcription. Double terminators, 100% efficiency ! Case 2 : Imperfectly terminate transcription. Control gene expression. 2 : 1 � Promoter RBS CDS1 CDS2 � Ter ermina minator or ef efficienc iciency : : 50% 50% �

Terminator is not well characterized Partsregistry Database Predicting promoter efficiency PNAS ( 2010) vol. 107 no. 72854 Number of working parts Predicting RBS efficiency Promoter 67 Nature Biotechnology (2009) 27, 946 RBS 55 Predicting terminator efficiency: terminator 16 ? √ √ Efficiency: ？ � Promoter RBS CDS Terminator

Outline 2 Terminator Efficiency Calculator Algorithm

Algorithm • Step 1 – Input the sequence of terminator 1. 50bp on average � 2. poly A, GC r i ch part,poly T � AAAAAAACCCCGCGGTTTTCCGCGGGGTTTTTTTTTTTTTTT

Algorithm • Step 2 – Predict the secondary structure of terminator – Recognize A tail, stem loop, T tail RNA folding algorith m: Minimal free energy model Stemloop � A Tail � T Tail �

Algorithm • Step 3 – Define a d score based on Gibbs free energy � n=1 the n nucleotide is T the nucleotide is not T

Algorithm • Step 4 From d score to terminator efficiency 1. International Open Facility Advancing Biotechnology (BIOFAB) from Stanford and UC Berkely � 2. Terminator efficiency experimental data available Linear fitting

Algorithm • Step 4 From d score to terminator efficiency Class Efficiency High efficiency Low efficiency significant difference in d score of high and low efficiency terminators �

Outline 2 Terminator Efficiency Calculator Database

Database Data Sources � Database Information � Efficiency � Partsregist 16 � Sequence � ry � Direction � Literature � 25 � Enviroment Biofab � 34 �

Validate our model by existing database �  Correlation coefficient between model prediction and data: 0.84 Data used in the figure � Partsregistry: 4 � Literatures: 18 �

Database � Current problem in existing terminator data: Inconsistent methods of measuring terminator efficiency 1. Gel 2. Fluorescence protein + Flow cytometry

Outline 2 Terminator Efficiency Calculator Experiment

Experiment Design RFP terminat o r GFP The Experimental Group Efficiency GFP level RFP GFP � 0% As much as control group The Control Group 100% none

Experiment Procedure Terminator Insertion Plasmid Fluorescence Construction Measurement Sequence Flow cytometry Efficiency Selection Analysis 3 4 2 1 5

Experiment Validation Flow cytometry measurement Fluorescent Microscope Results of GFP intensity the expression of GFP

Experiment Validation Agreement between prediction and experiment Correlation coefficient: 0.93

Experimental Standard Technical Standard Technical standards framework is formed to reduce the complexity and cost of producing synthetic living organisms by leveraging time-honored engineering principles of abstraction and standardization. BBF RFC 90 ： � The Measurement of rho-independent Transcription Terminator Efficiency. We create the standard for future terminator efficiency measurement

Demo

Online version www.terminatorefficiency.com

Terminator Efficiency Calculator In the future To Integrate promoter, RBS and terminator prediction method; To create a tool to predict the efficiency of whole transcription . √ √ √ Promoter RBS CDS Terminator

Outline 3 Human Practice

Human Practice (Open Classes) Lecture 1 Introduction to Synthetic Biology Lecture 2 Interesting Stories Lecture 3 Central Dogma Lecture 4 BioBrick and Basic Idea Lecture 5 Lab Work

Human Practice (Open Classes) Number of audience “This lecture is very 2000 Audiences interesting and very easy to understand. “ Rapidly Increase 1800 1600 1400 Comments 1200 1000 800 “I’m not in this field and know nothing abou 600 t synthetic biology, but 400 now I’m quite intereste d in it!” 200 9.24 9.26 9.28 9.30 10.2 10.4 Date

Summary • We developed a theoretical model to predict the efficiency of terminator • We performed experiment to validate our model • We created a large database of efficiency of terminators • We developed a technical standard on terminator efficiency measurement

Thanks to: Prof. Huang Wei (Hong Kong University) Dr.Ying-Ja Chen ( Synthetic Biology Center, MIT ) 2012 iGEM team: Shenzhen 2012 iGEM team: Shenzhen-SUSTC-A 2012 iGEM team: OUC