j5: DNA assembly design automation

Transcription

1 j5: DNA assembly design automation Nathan J. Hillson BIOEN Workshop on Synthetic Biology October 26, 2010

2 The DNA assembly goal Biofuel metabolic pathway: OrfA OrfB OrfC OrfD OrfE Metabolic pathway expression vector: orfe dbl laci ampr origin 2

3 The DNA assembly challenge Metabolic pathway expression vector: orfe dbl laci ampr origin Composite biological parts: orfe dbl laci ampr origin 3

4 The traditional approach T7 inator *+,0)))%*+/1- /"0)%*+$2- /"%)%*+,+- $%&.)%*+,"- '(&)%*+!,- $"))%*+#/- -&.)%*+#/- f1 origin!"#()%*+,,- Multiple Cloning Site (MCS) KanR2-12)%*3"$- -(2)%*3"+- -%&)%*3,#-!"#$%&'()*!"#$%&' T7 promoter laci pbr322 origin Assembly strategy not standardized (EMD/Novagen) 4 Difficult to automate

5 The BioBrick approach EcoRI BglII BamHI XhoI EcoRI BglII partb BamHI XhoI EcoRI BglII BamHI/XhoI BamHI XhoI BglII partb BglII/XhoI BamHI XhoI (Shetty et al. 2008, Anderson et al. 2010) EcoRI BglII Ligation BamHI/BglII scar G! GATCT! CCTAG! A! partb BamHI XhoI BamHI GGATCC! CCTAGG! BglII AGATCT! TCTAGA! Standardized assembly strategy Automatable (Anderson Lab, UC Berkeley) 5

6 BioBrick parallel assembly orfe EcoRI BglII orfe BamHI XhoI 6

7 BioBrick assembly considerations Introduction of scar sites After assembly: difficult to modify central parts Potential combinatorial diversity reduction orfe EcoRI BglII BamHI XhoI orfe 7

8 The SLIC approach Sequence and Ligation-Independent Cloning (Li et al. 2007) Linearized destination vector PCR product T4 DNA polymerase 3 Exo activity (no dntps) T4 DNA polymerase (+ dctp) Anneal (~25 bp overlaps) ssdna gap Transformation 8

9 The Gibson approach (Gibson et al. 2009) Linearized destination vector PCR product T5 exonuclease 5 Exo activity Anneal (~25 bp overlaps) Simultaneous/ one-pot 9 Phusion polymerase/taq ligase Transformation

10 The CPEC approach Circular Polymerase Extension Cloning (Quan et al. 2009) Linearized destination vector PCR product Melt dsdna/ anneal (~25 bp overlaps) nick Phusion polymerase extension nick Transformation 10

11 SLIC/Gibson/CPEC similarities Identical starting materials and final assembled products Linearized destination vector PCR product Automatable assembly strategies Scar-less Destination vector independent (BioBrick compatible) 11

12 SLIC/Gibson/CPEC similarities E Bg orfe Ba X dbl laci ampr origin E Bg orfe Ba X dbl laci ampr origin One-pot concurrent multi-part assembly All parts accessible; combinatorial diversity preserved Must design oligos specifically for each assembly junction 12

13 j5: DNA assembly design Given a list of biological parts to assemble, designs the most cost-effective strategy Non-degenerate Part IDs and Sources Part Location ID Number Name Source Plasmid Reverse Complement Start (bp) End (bp) Size (bp) Sequence 0 pes119_sbfi_asci_vector_fragment pes119 FALSE CGCGCCTT 1 atfa_3prime pes119 FALSE GGACTCAA 2 BBa_B1002_ BBa_B1002 FALSE CGCAAAAA 3 lacuv5_laco pbbe5a-rfp FALSE CCTGTGGC 4 EcoRI_mutation pes119 FALSE A 5 pdc_without_3prime_end pes119 FALSE AATTCAAA 6 pdc_3prime_and_adhb pes119 FALSE TGATGGTC 7 BBa_B1006_ BBa_B1006 FALSE AAAAAAAA 8 ITesA_5prime pes119 FALSE AATTCAAA Target Part Ordering/Selection/Strategy Part Order ID Number Name Orientation Strategy 0 0 pes119_sbfi_asci_vector_fragment forward Linearized Destination Vector 1 1 atfa_3prime forward PCR 2 2 BBa_B1002_ forward Embed_in_primer_reverse 3 3 lacuv5_laco forward SOE 4 4 EcoRI_mutation forward Embed_in_primer_reverse 5 5 pdc_without_3prime_end forward PCR 6 6 pdc_3prime_and_adhb forward PCR 7 7 BBa_B1006_ forward Embed_in_primer_reverse 8 3 lacuv5_laco forward SOE 9 4 EcoRI_mutation forward Embed_in_primer_reverse 10 8 ITesA_5prime forward SOE j5! 13

14 j5: DNA assembly design Outputs oligo synthesis, PCR reaction, and assembly piece information Oligo Synthesis ID Number Name Length Tm Cost ($) Sequence 16 NJH00005_(lacUV5_lacO)_pure_forward CCTGTGGC 17 NJH00017_(lacUV5_lacO)_forward CGGGGTTT 18 NJH00007_(lacUV5_lacO)_pure_reverse TGAAATTG 19 NJH00008_(lacUV5_lacO)_(EcoRI_mutation)_reverse CTTCCTTTG 20 NJH00018_(ITesA_5prime)_pure_forward AATTCAAAG 21 NJH00019_(ITesA_5prime)_forward TGTGAGCG 22 NJH00020_(ITesA_5prime)_pure_reverse CCAGTCCT 23 NJH00021_(ITesA_5prime)_reverse GATGCTGT PCR/SOE Reactions Forward Reverse Oligo ID Number Template Name Name Note Mean Oligo Tm!Oligo Tm Length Amplified Sequence 0 pes119 NJH00001_NJH00003_ PCR GGACTCAACATAATTT 1 pbbe5a-rfp NJH00005_NJH00007_ SOE CCTGTGGCGCCGGTG 2 pes119 NJH00009_NJH00011_ PCR AATTCAAAGGAAGGA 3 pes119 NJH00013_NJH00015_ PCR TGATGGTCCGTACAA 4 pbbe5a-rfp NJH00005_NJH00007_ SOE CCTGTGGCGCCGGTG 5 pes119 NJH00018_NJH00020_ SOE AATTCAAAGGAAGGA Assembly Pieces (including the Linearized Destination Vector) ID Number Type Type ID CPEC Tm Prev CPEC Tm Next Overlap Prev Overlap Next Length Sequence 0 Linearized Destination Vector CGCGCCTT 1 PCR ATAGTGCT 2 SOE GGGGTTTT 3 PCR TGTGAGCG 4 PCR AAGTTATG 5 SOE CGGGGTTT 6 SOE TGTGAGCG 14

15 j5: DNA assembly design Identifies assembly piece incompatibilities and suggests hierarchical assembly E Bg 0 Ba X orfe dbl laci ampr origin 15

16 j5: DNA assembly design Generates an annotated genbank sequence file for the resulting assembly AmpR origin dbl laci Plac )*&*&+,$/$-!"#)*&+,$##- $%&.*&+!$,!-!'(**&+!$%"- orfe Plac!"#$"%&'()*$!+,-.+/!"#$%&'( Plac 16

17 The Golden Gate approach A variation of (Engler et al. 2008, Engler et al. 2009) BsaI GGTCTCNNNNNN! CCAGAGNNNNNN! Automatable assembly strategy Scar-less Largely vector independent PCR-linearized destination vector PCR product Simultaneous/ one-pot BsaI/ Ligase 17

18 Combinatorial Golden Gate assembly PCR-linearized destination vector PCR products X X BsaI/ Ligase One-pot concurrent multi-part assembly All parts accessible; combinatorial diversity preserved Only 4 bp junction identity required for full oligo re-use Must find a compatible set of overhang sequences Must design the resulting oligos for each assembly junction 18

19 j5: DNA assembly design Outputs oligo synthesis, PCR reaction, and assembly piece information Oligo Synthesis ID Number Name Length Tm Cost ($) Sequence 4 NJH00146_(atfA_3prime)_pure_forward GGACTCAACATAATTTCTG 5 NJH00166_(atfA_3prime)_forward TTTTTTGGTCTCACAGGAC 6 NJH00148_(atfA_3prime)_pure_reverse ATCCTTAATTGGCTGTTTT 7 NJH00167_(atfA_3prime)_(BBa_B1002_)_reverse TTTTTTGGTCTCAGGGCG 8 NJH00025_(lacUV5_lacO)_pure_forward CCTGTGGCGCCGGTGATG 9 NJH00168_(lacUV5_lacO)_forward TTTTTTGGTCTCAGCCCTG PCR Reactions Forward Reverse ID Number Template Oligo Oligo Mean Oligo Tm! Oligo Tm Length Amplified Sequence 0 pes119 NJH00162_ NJH00164_ CGCGCCTTCCGGCTCTG 1 pes119 NJH00146_ NJH00148_ GGACTCAACATAATTTCT 2 pbbe5a-rfp NJH00025_ NJH00094_ CCTGTGGCGCCGGTGAT 7 PCR Reaction 0 NJH00163_ NJH00165_ TTTTTTGGTCTCAGACTG 8 PCR Reaction 1 NJH00166_ NJH00167_ TTTTTTGGTCTCACAGGA 9 PCR Reaction 2 NJH00168_ NJH00169_ TTTTTTGGTCTCAGCCCT Assembly Pieces (including the Linearized Destination Vector) ID Number Type Type ID 5' Overhang 3' Overhang Relative Overhang Length Sequence 0 PCR Linearized Destination Vector 7 GACT CAGG TTTTTTGGTCTCAGACTG 1 PCR 8 CAGG GCCC TTTTTTGGTCTCACAGGA 2 PCR 9 GCCC AAAA TTTTTTGGTCTCAGCCCT 3 PCR 10 AAAA CCAT TTTTTTGGTCTCAAAAAT 4 PCR 11 CCAT TTCC TTTTTTGGTCTCACCATG 5 PCR 12 TTCC TCAA TTTTTTGGTCTCATTCCTG 6 PCR 13 TCAA GACT TTTTTTGGTCTCATCAAA 19

20 Device Editor: a BioCAD canvas Visually represents and captures DNA assembly design schematic details localization tag or BMC X linker GFPuv ssra degradation tag long standard or X gfp uv X or Douglas Densmore, Joanna Chen ccmn short enhanced 20

21 j5 and DeviceEditor online j5 web interface j5 user s manual Available at no cost to academic, non-profit, and government labs Commercial licensing and 14-day trial periods available Contact TTD@lbl.gov for more information 21

22 Synthetic Biology an integrated process BioCAD tools JBEI Parts Registry Deposit in registry New part Selected component parts Automated assembly j5! 22

23 Experimental efforts underway Execution and validation of j5 DNA assembly designs arac camr arac camr t0 P BAD E ori SC101 pbbs8c-rfp SLIC/Gibson/CPEC dbl EcoRI/XhoI arac camr X P BAD E Bg Ba X rfp t0 dbl ori SC101 gfp uv _sig.pep t0 dbl ori SC101 ps8c-gfp uv _sig.pep PCR/SOE, then SLIC/ Gibson/ CPEC assembly (silent mutations) X Ba gfp uv _sig.pep pgfpuv_sig.pep localization tag or BMC ccmn X Combinatorial Golden-gate linker GFPuv ssra degradation tag long standard or short X gfp uv X or enhanced Integration of high-throughput devices into DNA assembly workflows Liquid-handling robotics Droplet-based microfluidic chips (Gerdts et al., 2008) 23

24 Acknowledgements DeviceEditor Douglas Densmore Joanna Chen Timothy Ham Zinovii Dmytriv j5-designed assembly validation John W. Thorne Jay Kinney, Anna Chen Rafael Rosengarten Robotics discussions Masood Hadi Microfluidics discussions Anup Singh Rajiv Bharadwaj Feedback and discussions Jay Keasling Keasling Lab JBEI Scientific Divisions Operations support JBEI staff Funding DOE Genomics/GTL/BRC (JBEI) Berkeley Lab LDRD 24

25 j5: DNA assembly design Exhaustively searches for the optimal set of compatible overhang sequences Compatible sets of overhang sequences X 5 -TGCCTACC! ATGG! AGTT! TCAAACGG-5! Desired overhang annealing X 5 -TGCCTACC! ATGG! TAGC! ATCGGCAA-5! No overhang annealing 5 -TGCCTACC! ATGG! TATT! ATAAAGGG-5! Undesired overhang annealing 25

26 j5: DNA assembly design Exhaustively searches for the optimal set of compatible overhang sequences Golden Gate overhang sequence selection options Preceding AGTTTG-3! Part TCAAAC! 5 -CCTACC Subsequent! GGATGG Part! (Neutral)! 5 -TTTGCCTACC! GGATGG! AG! TCAAAC-5! 5 -TGCCTACC! ATGG! AGTT! TCAAACGG-5! 5 -CCTACC! GG! AGTTTG! TCAAACGGAT-5! Assembled AGTTTGCCTACC! Sequence TCAAACGGATGG! 26

27 Device Editor: a BioCAD canvas Facilitates the creation of Eugene design specification rules Eliminates undesired assembly combinations 27

28 Device Editor: a BioCAD canvas Generates j5 input files and controls j5 parameters 28

29 JBEI Registry of Biological Parts Facilitates access to parts distributed across multiple labs Part information Advanced search features Tracks intellectual property and funding sources Automates sequence validation Open source and freely available Vector Editor Timothy Ham, Zinovii Dmytriv 29