Transcription Terminator Efficiency Calculator (TTEC)

SUSTC-Shenzhen-B

Outline Background & Motivation 1

Terminator Efficiency Calculator 2

Human Practice 3

Algorithm

Database

Experiment

Promoter RBS CDS

Terminator

Background & Motivation

A rho-independent terminator

Terminator efficiency

Case 1:

Completely terminate transcription. Double terminators, 100% efficiency !

Case 2:

2 : 1

Terminator efficiency : 50%

Promoter RBS CDS1 CDS2

Imperfectly terminate transcription. Control gene expression.

Terminator is not well characterized

Efficiency:

Promoter RBS CDS Terminator

√ √ ？

Number of working parts

Promoter 67 RBS 55 terminator 16

Predicting promoter efficiency PNAS (2010) vol. 107 no. 72854

Predicting RBS efficiency Nature Biotechnology (2009) 27, 946 Predicting terminator efficiency: ?

Partsregistry Database

Outline

Terminator Efficiency Calculator 2 Algorithm

•  Step 1 –  Input the sequence of terminator

1.  50bp on average2.  poly A,GC rich part,poly T

AAAAAAACCCCGCGGTTTTCCGCGGGGTTTTTTTTTTTTTTT

Algorithm

•  Step 2 – Predict the secondary structure of terminator – Recognize A tail, stem loop, T tail

RNA folding algorithm: Minimal free energy model

A Tail T Tail

Stemloop

Algorithm

•  Step 3 – Define a d score based on Gibbs free energy

Algorithm

n=1

the n nucleotide is T

the nucleotide is not T

1.  International Open Facility Advancing Biotechnology (BIOFAB) from Stanford and UC Berkely

2.  Terminator efficiency experimental data available

• Step 4 From d score to terminator efficiency

Algorithm

Linear fitting

• Step 4 From d score to terminator efficiency

Algorithm

Class Efficiency

High efficiency Low efficiency

significant difference in d score of high and low efficiency terminators

Outline

Terminator Efficiency Calculator 2

Database

Database

Data Sources

Partsregistry

16

Literature 25

Biofab 34

Database Information Efficiency Sequence Direction Enviroment

�Correlation coefficient between model prediction and data: 0.84

Data used in the figurePartsregistry: 4Literatures: 18

Validate our model by existing database

Current problem in existing terminator data: Inconsistent methods of measuring terminator efficiency

Database

1.  Gel 2.  Fluorescence protein + Flow cytometry

Outline

Terminator Efficiency Calculator 2

Experiment

Experiment Design

The Experimental Group

The Control Group

RFP GFP

RFP terminator GFP

Efficiency GFP level

0% As much as control group

100% none

Experiment Procedure

Sequence Selection

1

Plasmid Construction

2

Terminator Insertion

3

Fluorescence Measurement

Flow cytometry

4

Efficiency Analysis

5

Experiment Validation

Fluorescent Microscope Results

the expression of GFP

Flow cytometry measurement of GFP intensity

Experiment Validation

Agreement between prediction and experiment

Correlation coefficient: 0.93

Experimental Standard

Technical Standard

BBF RFC 90：

The Measurement of rho-independent Transcription Terminator Efficiency.

We create the standard for future terminator efficiency measurement

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.

Demo

Online version

www.terminatorefficiency.com

To Integrate promoter, RBS and terminator prediction method; To create a tool to predict the efficiency of whole transcription.

Terminator Efficiency Calculator

In the future

Promoter RBS CDS Terminator

√ √ √

Outline

Human Practice 3

Lecture 5 Lab Work

Lecture 4 BioBrick and Basic Idea

Lecture 3 Central Dogma

Lecture 2 Interesting Stories

Lecture 1 Introduction to Synthetic Biology

Human Practice (Open Classes)

Human Practice (Open Classes)

9.24 9.26 9.28 9.30 10.2 10.4

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“I’m not in this field and know nothing about synthetic biology, but now I’m quite interested in it!”

“This lecture is very interesting and very easy to understand. “

Comments

Date

Number of audience

•  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

Summary

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