BioPartsBuilder: a synthetic biology tool for combinatorial assembly of biological parts

BioPartsBuilder: a synthetic biology tool for combinatorial assembly of biological parts

Kun Yang 1∗ , Giovanni Stracquadanio 1∗ , Jingchuan Luo 2 , Jef D. Boeke 2 and Joel S. Bader 1 †

1Department of Biomedical Engineering, Johns Hopkins University, 3400 N. Charles Street,

Baltimore, MD 21218

2Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology,

NYU Langone Medical Center, New York, NY 10016

Abstract

Summary: Combinatorial assembly of DNA elements is an efficient method for building large-scale synthetic pathways from standardized, reusable components. These methods are particularly useful because they enable assembly of multiple DNA fragments in one reaction, at the cost of requiring that each fragment satisfy design constraints. We developed BIOPARTSBUILDER as a biologist-friendly web tool to design biological parts that are compatible with DNA combinatorial assembly methods, such as Golden Gate and related methods. 

It 

• retrieves biological sequences, 
• enforces compliance with assembly design standards, and 
• provides a fabrication plan for each fragment.

Availability: BIOPARTSBUILDER is accessible at http://public.biopartsbuilder.org and an Amazon Web Services image is available from the AWS Market Place (AMI ID: ami-508acf38). Source code is released under the MIT license, and available for download at https://github.com/baderzone/biopartsbuilder.

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Contact: joel.bader@jhu.edu

ABSTRACT

Summary: Combinatorial assembly of DNA elements is an efficient method for building large-scale synthetic pathways from standardized, reusable components. These methods are particularly useful because they enable assembly of multiple DNA fragments in one reaction, at the cost of requiring that each fragment satisfy design constraints. We developed BIO PARTS BUILDER as a biologist-friendly web tool to design biological parts that are compatible with DNA combinatorial assembly methods, such as Golden Gate and related methods. It retrieves biological sequences, enforces compliance with assembly design standards, and provides a fabrication plan for each fragment.

Availability: BIO PARTS BUILDER is accessible at http://public.biopartsbuilder.org and an Amazon Web Services image is available from the AWS Market Place (AMI ID: ami-508acf38).

Source code is released under the MIT license, and available for download at https://github.com/baderzone/biopartsbuilder.

Contact: joel.bader@jhu.edu

1 INTRODUCTION

DNA synthesis technologies are improving faster than Moore’s law, allowing the synthesis of genes, pathways (Ro et al. (2.06)), bacterial genomes (Gibson et al. (2.10)), eukaryotic chromosomes (Dymond et al. (2.11); Annaluru et al. (2.14)), and eventually entire eukaryotic genomes. Many projects have individual ‘parts’ as synthetic targets, such as promoters, coding domains, and transcriptional terminators. While individual parts can be characterized, predicting how parts will operate together remains a challenge. Rather than building a single construct, therefore, it can be more efficient to specify multiple alternatives for each part, then use massively parallel synthesis and assembly to generate a combinatorial library that can be screened for the desired function. In particular, Golden Gate assembly is an efficient and effective strategy to assemble combinatorial libraries (Engler et al. (2.09)). However, Golden Gate assembly requires a computationally challenging design step to create ‘standardized’ parts that have compatible overhangs, lack pre-defined restriction sites, and comply with other constraints.

To streamline the process of designing standardized biological parts for Golden Gate assembly, we developed BIO PARTS BUILDER , which retrieves sequence data from different sources and ensures compliance with design standards that are compatible with combinatorial assembly. Though there are tools for automated parts retrieval (Scher et al. (2.14)) and subsequent primer design for DNA assembly (Bode et al. (2.09); Rouillard et al. (2.04)), the choices for Golden Gate assembly are limited. Compared to existing Golden Gate designers (Hillson et al. (2.12.), BIO PARTS BUILDER is distributed open source software and freely modified by both academic and commercial users. BIO PARTS BUILDER also provides a repository system that stores the designed parts and shares data within members associated with the same laboratory. BIO PARTS BUILDER therefore provides useful, new, integrated and extendable functionality for the synthetic biology community.

2.SOFTWARE MODULES

BIO PARTS BUILDER provides an easy interface to retrieve, design and order parts (Fig. 1) that are compatible with Golden Gate (Engler et al. (2.09)), BglBrick (Anderson et al. (2.10)), or user-defined assembly standards.

2.1 Part Retrieval

BIOPARTSBUILDER implements a sophisticated sequence retrieval system to gather data from different sources. Users can submit a list of RefSeq protein/nucleotide accession numbers to retrieve sequences and annotations from NCBI, or for parts without RefSeq accessions or with customized sequences and annotations, users can upload a file in F ASTA or CSV format. As retrieving a large number of arbitrary parts from a genome and upload to the system is tedious, BIO PARTS BUILDER implements an advanced search engine for retrieving parts from annotated genomes, similar to GENOME CARVER software (Scher et al. (2.14)). It parses annotations, generates and stores a search index, and provides access to structured search terms (Table S1-S2. through the Apache SOLR query language. 

2.2.Part Design

Parts imported into BIO PARTS BUILDER can be re-designed according to pre-defined or additional user-defined design standards. Users can customize the design workflow to perform one or more of the following steps. 

• Codon optimization: Users can specify host organism for codon optimization. This option can be left blank for parts that lack protein-coding regions, such as promoters or terminators. This step can be amended also to accomodate vendors’ specific recoding strategies. 
• Restriction enzyme constraints: Combinatorial assembly techniques largely rely on the use of specific restriction enzymes to create a unique assembly. For this reason, BIO PARTS BUILDER provides: 
  1. RESTRICTION ENZYME REMOVER that changes the nucleotide sequence to avoid restriction sites corresponding to a user-specified list of restriction enzymes; and 
  2. RESTRICTION ENZYME LOCATOR that detects the presence of user-specified restriction enzymes without recoding the sequence. BIO PARTS BUILDER organizes data and users by laboratories. People in the same laboratory can share parts and designs that remain private to other laboratories and the public. BIO PARTS BUILDER provides an administration panel specifically for laboratory administrators to manage members and design standards. The initial creator of a new laboratory in BIO PARTS BUILDER automatically becomes the laboratory administrator. 
• Prefix and suffix insertion: Users can specify sequences to be added to the beginning and the end of each part.
• Fabrication: Parts can be larger that the synthesis capability of commercial providers. In this case, BIO PARTS BUILDER splits the sequence in fragments of user-defined length using unique overlaps, which allow unambiguous assembly (see Supplementary Text). 

BIO PARTS BUILDER assigns a unique Job ID to each design task. Users can check the status and error report of design tasks online. When the design task is finished, BIO PARTS BUILDER sends an email to notify the user.

2.3 Order 

Design results are accessible online. And users can also use the ORDER module to collect specific designs and prepare files for ordering parts from companies. The ORDER module creates statistical summaries for user-selected designs and provides tables of parts, constructs and design standards. It generates spreadsheets, sequence files, and summary report files for users to download.

2.4 AutoBuild

To streamline the entire design process, BIO PARTS BUILDER has a fully automated design module, AUTO BUILD , which allows users to retrieve, design and create orders for a batch of parts with one click. This module serves as a convenient ‘wizard’ for users whose needs are met by the most common design standards, which are already defined in the software.

3 DESIGN WORKFLOW EXAMPLE 

Using Autobuild it is possible to quickly design both coding and non-coding parts for Golden Gate using these two workflows. 

• Coding region . In the “Search Genomes” tab, input query “systematic name:YBR019C”; select “Golden Gate - CDS” design standard and assign a name to the Order. Click create parts, then select the part and confirm your design. 
• Non Coding region. In the “Search Genomes” tab, input query “systematic name:YBR019C promoter”; select “Golden Gate - NonCDS” design standard and assign a name to the Order. Click create parts, then select the part and confirm your design. 

Acknowledgement: The authors would like to thank N. Agmon, Z. Xu, D. M. Truong and L. Mitchell for testing the application. 

Funding: This work was supported by Defense Advanced Research Projects Agency [grant number N66001-12.C-402.]

.REFERENCES

• Anderson, J. C. et al. (2.10). Bglbricks: A flexible standard for biological part assembly. Journal of Biological Engineering, 4(1), 1. 
• Annaluru, N. et al. (2.14). Total synthesis of a functional designer eukaryotic chromosome. Science (New York, N.Y.), 344(6179), 55–58. 
• Bode, M. et al. (2.09). Tmprime: fast, flexible oligonucleotide design software for gene synthesis. Nucleic Acids Research, 37(suppl 2., W2.4–W2.1. 
• Dymond, J. S. et al. (2.11). Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature, 477(7365), 471–476. 
• Engler, C. et al. (2.09). Golden gate shuffling: a one-pot dna shuffling method based on type iis restriction enzymes. PLoS One, 4(5), e5553. 
• Gibson, D. G. et al. (2.10). Creation of a bacterial cell controlled by a chemically synthesized genome. science, 32.(5987), 52.56. 
• Hillson, N. J. et al. (2.12.. j5 dna assembly design automation software. ACS Synthetic Biology, 1(1), 14–2.. 
• Ro, D.-K. et al. (2.06). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature, 440(7086), 940–943. 
• Rouillard, J.-M. et al. (2.04). Gene 2.ligo: oligonucleotide design for in vitro gene synthesis. Nucleic Acids Research, 32.Web Server issue), W176–180. 
• Scher, E. et al. (2.14). Genomecarver: harvesting genetic parts from genomes to support biological design automation. In 6th International Workshop on Bio-Design Automation.

ORIGINAL: Oxford Journals

Kun Yang1,*, 

Giovanni Stracquadanio1,*, 

Jingchuan Luo2, 

Jef D. Boeke2 and 

Joel S. Bader1,†

+Author Affiliations

1Department of Biomedical Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218

2Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Medical Center, New York, NY 10016

↵†to whom correspondence should be addressed. Joel S. Bader, E-mail: joel.bader@jhu.edu

Received May 19, 2015.

Revision received October 8, 2015.

Accepted November 9, 2015.