Synthetic Biology: Complexity and Challenges

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1 Synthetic Biology: Complexity and Challenges James Johnson Manager, GeneMill Facility University of Liverpool

2 DNA SEQUENCING TRANSCRIPTOME PROFILING AND GENE REGULATION SEQUENCE CAPTURE MICROARRAYS DESIGN AND SYNTHESIS EXPRESSION AND SCREENING METABOLOMICS FACILITIES ACCESS METAGENOMICS AND METAGENETICS ADVANCED LASER MICROSCOPY FLOW CYTOMETRY BIOINFORMATICS

3 Centre For Genomic Research Our Technologies All major Next Gen and 3 rd generation sequencers including Illumina HiSeqs, MiSeqs and PacBio Affymetrix and Agilent microarray platforms NanoString ncounter

include: Fluidigm C1 single-cell auto-prep system Sony

4 Centre For Genomic Research Single Cell Genomics Laboratory Precise single cell isolation platforms include: Fluidigm C1 single-cell auto-prep system Sony SH800Z cell sorter Zeiss laser micro-dissection microscope with PALM microbeam

5 GeneMill Gene Foundry Led by Mark Caddick and Christiane Hertz-Fowler ~ 4M Initial investment DNA Foundry, Next Generation DNA Synthesis (Liverpool, Oxford, Bristol, Birmingham, Skalene) and Phenotyping Platform (Liverpool, EGF) Knowledge exchange JGI Remove the complexity of manufacture for the user GeneMill allows researchers to focus on the design and testing of the parts, devices, pathways and networks

6 What Is SynBio? Engineering/Re-engineering of Genes/Pathways/Networks An approach to Biology Not a particular technique Makes use of many Designing Biology

7 SynBio Overview

8 A New Industrial Revolution Standardised production Automation Productivity Removed Art of manufacture

9 Level of Interaction C++ Binary Functional Part Gene Structure DNA Sequence

10 21 st Century Platform For Innovation One of the 8 Great Technologies Bioengineering $150Bn Industrial Biotech market

11 Location

12 Workflow

13 Modular Cloning High throughput construct assembly ~ 3 days once library in place

14 Cloning Larger Fragments E. coli plasmids limited ~40kb Large fragments brought together by Yeast Homologous Recombination >100kb per reaction.

15 CRISPR/Cas9 Guide RNA (grna) 20nt complimentary to target Seq on +/- strand. Protospacer Adjacent Motif (PAM) site critical for binding and cleavage Canonical NGG Sequence Different Sp. & Mutants ID ed Cas9 Nuclease complexes with grna and cuts both DNA strands

16 CRISPR/Cas9 Options

17 Scalable Expression Testing High throughput small scale protein expression testing - 5 days from Parts library to expression testing

Example Robolector Data Biomass 800 700 600 500 400 300 200

Intensity 800 700 600 500 400 300 200 100 0-100 0 5 10 15 20

18 Example Robolector Data Biomass o C Yield IPTG Time (h) GFP Intensity Time (h) Yield mM 0.1mM 0.5mM

19 Metabolomics Compound identification Ion-Mobility Q-ToF Services Untargeted Discovery Metabolomics High mass accuracy Ion-Mobility Time-of-Flight provides exceptional resolution and sensitivity for the most demanding analytical challenges for compound discovery. Biomarker Discovery - Multivariate Data Modelling Target Compound Quantification Triple Quadrupole LC-MS provides high sensitivity. LC-Triple Quad. GC-MS Compound Identification Ion-Mobility, MS/MS, and High mass accuracy combined for structural elucidations of unknowns Advanced Data Modelling Exploratory data analysis for significance testing and class structure; predictive quantification calibrations; correlations between Multi-Omic data sets. Pathway Level Analysis & Mapping Organism specific metabolic pathway building; metabolite mapping, and Multi-Omic data mapping Heavy label isotope distribution analysis. Pathways Modelling & Flux Analysis

20 Metabolite Analysis Mechanically disrupt samples Add solvent 96-well solid-phase extraction Or online extraction 5min run per sample -> 8hr run time IMS data adds another dimension Data analysis and modelling

21 Services Include DNA Synthesis and cloning Plasmid libraries Modular cloning Large constructs DNA Synthesis Pooled and Cloned Final Library Genome Editing Tools CRISPR Designer Proteins Enzyme Prototyping Culture Optimisation Metabolic Phenotyping Training/ Consultancy Grant Applications & Vouchers

22 Workflow

Practicalities Parts & reagents stored in

23 Practicalities Parts & reagents stored in automated freezer. Picklist generated of required barcodes Tubes retrieved from Arktic De-capped Loaded onto TTP Labtech Mosquito. nl volumes dispensed Tubes re-capped and returned to freezer Cloning reactions performed PCR Machine or CyBio Platform

tubes arrayed in pre-determined order/position in SBS rack

24 Compact Workflow Arktic 140,000 tubes (0.5ml) Small footprint Fast retrieval Secure storage 2D Barcode tubes arrayed in pre-determined order/position in SBS rack Downstream-optimised 96-well plate Mosquito X1 and HTS. Cherry-picking or HT No 2 plates/racks.

25 Cherry Pick Many Reformatting and amalgamation of samples from one or more plates into a more useful format for the system or other devices such as the Labcyte Echo. Reduces the number of plates required Helps to protect any sensitive and valuable sample material which may degrade or evaporate during a long procedure. The method requires a picklist to indicate the required material and will generate an output file which lists the new material in the output plate.

26 Method workflow Destination plate barcode scanned and picklist loaded Source plate barcode scanned System checks source barcode and loads transfers for that plate Are there more source plates? System writes Output tracking/logging files The method follows a simple workflow: Yes 1. The destination plate is scanned and the picklist loaded 2. Each source plate is scanned as it is presented to the system. If it is listed in the picklist it will be used, if not a new plate is requested. 3. Information is loaded from the picklist and the plate processed 4. If more source plates are specified in the picklist, a new source plate is requested 5. Upon completion an output file is generated which details the contents of the destination plate.

27 Output file Destination plate contents file generated. For each used well the following information is supplied: The well co-ordinate Sample ID Volume

28 Requirement for LIMS

29 Leaf LIMS In-house development LIMS built from scratch Built for SynBio but flexible. Tasks and Workflows can be built as needed CRM Integration eaflims.github.io

30 Inventory Basic Substance types Measures Stock levels Tracked Alerts Lot # Order # Projects used in External Integration ICE J5 SBOL igem Mosaic DICOM-SB Etc.

Leaf Cover(s) Submitted Projects Submitted Samples Through all projects/final designs used Tube barcodes, information & metadata Reagents and consumables Levels used

31 Leaf Cover(s) Submitted Projects Submitted Samples Through all projects/final designs used Tube barcodes, information & metadata Reagents and consumables Levels used through projects Levels remaining for stock Workflow requirements Tasks built into workflows Modular Outputs sample picklists/worklists for specific equipment Flexibility

32 Workflows......

Advances with Acoustics

Advances with Acoustics Evolution of Acoustic Dispensing and Next Generation Developments SLAS Europe Compound Management March 14, 2017 2016 LABCYTE INC. All rights reserved. Labcyte, Echo, the Labcyte