Transcription Terminator Efficiency Calculator (TTEC) - - PowerPoint PPT Presentation

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

Ter ermina minator

• r ef

efficienc iciency : : 50% 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 average
• 2. 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 Partsregist ry 16 Literature 25 Biofab 34 Database Information

Efficiency

Sequence Direction Enviroment

Correlation coefficient between model prediction and data: 0.84

Data used in the figure Partsregistry: 4 Literatures: 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

• f 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

• rganisms 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 10.4 200 400 600 800 1000 1200 1400 1600 1800 2000 Audiences

Rapidly Increase

“I’m not in this field and know nothing abou t synthetic biology, but now I’m quite intereste d 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
• f 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