Introduction to Synthetic Biology. Standard for Physical DNA Composition. Vincent Rouilly Bioengineering Department Imperial College London
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1 Introduction to Synthetic Biology Topic 1 Topic 2 Topic 3 Topic 4 Topic 5 Standard for Physical DNA Composition Vincent Rouilly Bioengineering Department Imperial College London
2 A Programming Language ACAGGGTGACCTAAGACCTTATTGTCTAATTGAGAAGGCTTCGGAATTTGACCTAAG ACCTTATTGTCTAATTGAGAAGGGCCGGTTGAACAGGGCTTCGGAATTTGACCTAAG ACCTTATTGTCTAATTGAGATGACCTAAGACCTTATTGTCTAATTGAGAAGGAGGGCC GGTTGAACAGGTGACCTAAGACCTTATTGTCTAATTGAGAAGGGCTTCGGAATTTGAC CTAAGACCTTATTGTCTAATTGAGAAGGGCCGGTTGAACAGGGCTTCGGAATTTGACC TAAGACCTTATTGTCTTGACCTAAGACCTTATTGTCTAATTGAGAAGGAATTGAGAAGG GCCGGTTGAACAGGTGACCTAAGACCTTATTGTCTAATTGAGAAGGGCTTCGGAATTT GACCTAAGACCTTATTGTCTAATTGAGAAGGGCCGGTTGAACAGGGCTTCGGAATTTG ACTGACCTAAGACCTTATTGTCTAATTGAGAAGGCTAAGACCTTATTGTCTAATTGAGA AGGGCCGGTTGAACAGGGCTTCGGAATTTGACCTAAGACCTTATTGTCTAATTGAGAAG GGCCGGTTGAACAGGTGACCTAAGACCTTATTGTCTAATTGAGAAGGGCTTCGGAATTT GACCTAAGACCTTATTGTCTAATTGAGAAGGGCCGGTTGAACAGGGTGACCTAAGAC ACCTTATTGTCTAATTGAGAAGGGCCGGTTGAACAGGGCTTCGGAATTTGACCTAAG ACCTTATTGTCTAATTGAGATGACCTAAGACCTTATTGTCTAATTGAGAAGGAGGGCC GGTTGAACAGGTGACCTAAGACCTTATTGTCTAATTGAGAAGGGCTTCGGAATTTGAC CTAAGACCTTATTGTCTAATTGAGAAGGGCCGGTTGAACAGGGCTTCGGAATTTGACC TAAGACCTTATTGTCTTGACCTAAGACCTTATTGTCTAATTGAGAAGGAATTGAGAAGG GCCGGTTGAACAGGTGACCTAAGACCTTATTGTCTAATTGAGAAGGGCTTCGGAATTT GACCTAAGACCTTATTGTCTAATTGAGAAGGGCCGGTTG
3 Sizes E.Coli Genome Size Reg. Binding Site Promoter Gene 4.6 M bp 4500 genes Yeast (S. cerevisae) 130 M bp 6600 genes Mammalian (Human Fibroblast) 3000 M bp ~25000 genes ~10bp ~10bp ~10bp ~100 bp ~1000 bp ~10000 to bp ~1000 bp ~1000 bp ~10000 to bp from An Introduction to Systems Biology, Uri Alon, 2004
4 Imaginary Project Bird Flu Drug Production in E.Coli 5 Genes 4 Promoters 4 RBS 1 Plasmid ~6000 bp 4 Terminators
5 DNA Toolbox (cut, copy, paste, read & write) DNA Synthesis Restriction Enzymes PCR DNA Sequencing DNA Ligase
6 DNA Synthesis current price : from 0.80 euro/bp production time: from 2 to 4 weeks
7 DNA Synthesis is the solution... from Carlson, 2003
8 DNA Synthesis
9 Back to our Project Bird Flu Drug Production in E.Coli 5 Genes 4 Promoters 4 RBS 4 Terminators 1 Plasmid ~6000 bp Maybe, we want to tune the level of expression for each gene Maybe, we have different mutants for each gene How many plasmids to synthesise?
10 Traditional cloning techniques
11 Traditional cloning techniques
12 Traditional cloning techniques
13 Lack of Standardisation The lack of standardization in assembly techniques for DNA sequences forces each DNA assembly reaction to be both an experimental tool for addressing the current research topic, and an experiment in and of itself. One of our goals is to replace this ad hoc experimental design with a set of standard and reliable engineering mechanisms to remove much of the tedium and surprise during assembly of genetic components into larger systems Tom Knight, MIT. from Idempotent Vector Design for Standard Assembly of Biobricks, Tom Knight (MIT) 2003.
14 Wish List for DNA composition Library of DNA Parts that can easily be plugged together Efficient and Reliable process Support an Abstraction Hierarchy
15 Towards a BioBrick Standard
16 Towards a BioBrick Standard Standard Sequence Standard Sequence
17 Towards a BioBrick Standard Prefix Sequence Suffix Sequence
18 Towards a BioBrick Standard RS1 Prefix Sequence RS2 RS3 Suffix Sequence RS4
19 BioBrick Standard (Tom Knight, MIT) GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C T ACTAGT A GCGGCCGC CTGCAG A TGATCA T CGCCGGCG GACGTC from Idempotent Vector Design for Standard Assembly of Biobricks, Tom Knight (MIT) 2003.
20 BioBrick: 4 Operations Create Front-Insert GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C *AATTC GCGGCCGC A TCTAGA G G CGCCGGCG T ACATCT C T ACTAGT A GCGGCCGC CTGCAG A TGATCA T CGCCGGCG GACGTC TA A TGATC*
21 BioBrick: 4 Operations Create Back-Insert GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C *CTAGAG TC T ACTAGT A GCGGCCGC CTGCAG A TGATCA T CGCCGGCG GACGTC T ACTAGT A GCGGCCGC CTGCA* A TGATCA T CGCCGGCG G
22 BioBrick: 4 Operations Create Front-Vector GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C G CTTAA* *CTAGA G TC T ACTAGT A GCGGCCGC CTGCAG A TGATCA T CGCCGGCG GACGTC T ACTAGT A GCGGCCGC CTGCAG A TGATCA T CGCCGGCG GACGTC
23 BioBrick: 4 Operations Create Back-Vector GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C T ACTAGT A GCGGCCGC CTGCAG A TGATCA T CGCCGGCG GACGTC TA A TGATC* *G ACGTC
24 BioBricks: Composition Techniques Prefixing Composition = Front Vector + Front Insert GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C T ACTAGT A GCGGCCGC CTGCAG 1 A TGATCA T CGCCGGCG GACGTC T ACTAGT A GCGGCCGC CTGCAG 2 A TGATCA T CGCCGGCG GACGTC
25 BioBricks: Composition Techniques Prefixing Composition = Front Vector + Front Insert GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C T ACTAGT A GCGGCCGC CTGCAG 1 A TGATCA T CGCCGGCG GACGTC T ACTAGT A GCGGCCGC CTGCAG 2 A TGATCA T CGCCGGCG GACGTC
26 BioBricks: Composition Techniques Prefixing Composition = Front Vector + Front Insert *CTAGA G TC G CTTAA* *AATTC GCGGCCGC A TCTAGA G G CGCCGGCG T ACATCT C T ACTAGT A GCGGCCGC CTGCAG 1 A TGATCA T CGCCGGCG GACGTC TA 2 A TGATC*
27 BioBricks: Composition Techniques Prefixing Composition = Front Vector + Front Insert G*AATTC GCGGCCGC A TCTAGA G CTTAA* G CGCCGGCG T ACATCT C 2 T A*CTAGA G A TGATC*T C 1 T ACTAGT A GCGGCCGC A TGATCA T CGCCGGCG CTGCAG GACGTC
28 BioBricks: Composition Techniques Postfixing Composition = Back Vector + Back Insert GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C T ACTAGT A GCGGCCGC CTGCAG 1 A TGATCA T CGCCGGCG GACGTC T ACTAGT A GCGGCCGC CTGCAG 2 A TGATCA T CGCCGGCG GACGTC
29 BioBricks: Composition Techniques Postfixing Composition = Back Vector + Back Insert GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C T ACTAGT A GCGGCCGC CTGCAG 1 A TGATCA T CGCCGGCG GACGTC GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C T ACTAGT A GCGGCCGC CTGCAG 2 A TGATCA T CGCCGGCG GACGTC
30 BioBricks: Composition Techniques Postfixing Composition = Back Vector + Back Insert GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C *CTAGAG TC *G ACGTC TA 1 A TGATC* T ACTAGT A GCGGCCGC CTGCA* 2 A TGATCA T CGCCGGCG G
31 BioBricks: Composition Techniques Postfixing Composition = Back Vector + Back Insert GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C 1 T A*CTAGA G A TGATC*T C 2 T ACTAGT A GCGGCCGC CTGCA* *G A TGATCA T CGCCGGCG G ACGTC
32 Idempotence and Abstraction Hierarchy Standard Assembly
33 Idempotence and Abstraction Hierarchy Standard Assembly Rolling Assembly
34 BioBricks Standard Summary Upstream Sequence GAATTC GCGGCCGC A TCTAGA G CTTAAG CGCCGGCG T ACATCT C Downstream Sequence T ACTAGTA GCGGCCGC CTGCAG A TGATCAT CGCCGGCG GACGTC Restriction sites clean-up Verification primers Verification forward 5 ttg tct cat gag cgg ata ca 3 Verification reverse 5 att acc gcc ttt gag tga gc
35 Behind the Standard: Technical considerations Enzyme choice: - Easy to use/reliable, - Compatible buffers/temperature, - Heat killed, - Few required bases outside of their recognition sites, - Low star activity, - Four base overhangs to enhance ligation efficiency - Avoiding accidental creation of ATG sequence, - Avoid methylation sensitive sites at the end... as always... concessions
36 How to make a BioBrick: using PCR Natural DNA Sequence
37 How to make a BioBrick: using PCR Forward Primer with Std Prefix Natural DNA Sequence Reverse Primer with Std Suffix + Restriction sites clean-up
38 How to make a BioBrick: Direct Synthesis DNA Sequence
39 How to make a BioBrick: Direct Synthesis Prefix DNA Sequence + Restriction sites clean-up Suffix
40 Registry of standard biological parts
41 Competing / Alternative standards BioBrick++, Austin Che (MIT) Pam Silver's Lab Biobrick Version JC Anderson's Lab Biobrick Version
42 References > Drew Endy Talks > Austin Che Presentations > OpenWetWare Folks > igem Competition > BioBricks Foundation
43 to be added 3 Antibiotics Assembly Automated assembly
44 to be added Study of the scare (frame shift) detail of the protein fusion (Silver's version) Specific BB version for Coding Sequence parts
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