transcription terminator efficiency calculator (ttec)
TRANSCRIPT
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