rpbasicdesign

A command-line tool to convert rpSBML files into SBOL and CSV files ready-to-be used with DNA-Bot.

rpbasicdesign extracts enzyme IDs from rpSBML files -- produced by the RP suite available in the SynBioCAD Galaxy platform -- to generate genetic constructs compliant with the BASIC assembly approach. CSV files produced are ready to be used with DNA-Bot to generate instructions for automated build of the genetic constructs using OpenTrons liquid handling robots.

Installation

sh conda install -c brsynth -c conda-forge rpbasicdesign

Usage

Simple call: sh conda activate <myenv> python -m rpbasicdesign.cli --rpsbml_file tests/input/muconate_example.xml

Output folders for dnabot-ready files and SBOL export can be set using o_dnabot_dir and o_sbol_dir options: sh python -m rpbasicdesign.cli \ --rpsbml_file tests/input/muconate_example.xml \ --o_dnabot_dir out/dnabot_input \ --o_sbol_dir out/sbol_export

The number of constructs to design is tuned using sample_size: sh python -m rpbasicdesign.cli \ --rpsbml_file tests/input/muconate_example.xml \ --sample_size 5

The complete list options is provided the embedded help, which can be printed using the --help or -h keywords: ``` python -m rpbasicdesign.cli -h

usage: python -m rpbasicdesign.cli [-h] --rpsbmlfile RPSBMLFILE [--partsfiles PARTSFILES [PARTS_FILES ...]] [--lmsid LMSID] [--lmpid LMPID] [--backboneid BACKBONEID] [--samplesize SAMPLESIZE] [--cdspermutation CDSPERMUTATION] [--maxenzperrxn MAXENZPERRXN] [--odnabotdir ODNABOTDIR] [--osboldir OSBOLDIR]

Convert rpSBML enzyme info in to BASIC construct. UniProt IDs corresponding enzyme variants are extracted rpSBMl files. Promoters and RBSs are randomly chosen from a default list. CDSs, in other words gene variants, of enzymes are randomly chosen from amongst the UniProt IDs extracted. Constructs generated can be stored as (i) a CSV file ready to be used by DNA-Bot, (ii) as SBOL files.

optional arguments: -h, --help show this help message and exit --rpsbmlfile RPSBMLFILE rpSBML file from which enzymes UniProt IDs will be collected. --partsfiles PARTSFILES [PARTSFILES ...] List of files providing available linkers and user parts (backbone, promoters, ...) for constructs. Default: [data/biolegioparts.csv, userparts.csv] --lmsid LMSID part ID to be used as the LMS methylated linker. Default: LMS --lmpid LMPID part ID to be used as the LMP methylated linker. Default: LMP --backboneid BACKBONEID part ID to be used as the backbone. Default: BASICSEVA37CmR-p15A.1 --samplesize SAMPLESIZE Number of construct to generate.Default: 88 --cdspermutation CDSPERMUTATION Whether all combinations of CDS permutation should be built Default: true --maxenzperrxn MAXENZPERRXN Maximum number of enyzme to consider per reaction. If more enzymes are available for a given reaction, then only the last one listed in the MIRIAM annotation section will be kept. --maxgeneperconstruct MAXGENEPERCONSTRUCT Maximum number of genes per construct. If more genes are required, i.e. more reactions are described in the inputet SBML file, then the execution will failed. --odnabotdir ODNABOTDIR Output folder to write construct and plate files. It will be created if it does not exist yet. Existing files will be overwritten. Default: out/dnabotin --osboldir OSBOL_DIR Output folder to write SBOL depictions of constructs. Existing files will be overwritten. Default: not output. ```

Lycopene example

If one wishes to only use a subset of BASIC parts, the way to go is to provide a restricted list of parts with the --parts_file option.

The command below generates up to 88 constructs for the lycopene producing pathway (CrtEBI pathway) defined in examples/lycopene_CrtEBI_from_selenzy.xml.xml, using the parts described in examples/parts_for_lycopene.csv. Output files will be written in examples/lycopene_sbol folder for SBOL files and examples/lycopene_dnabot for DNA-Bot. At the end 88 constructs should be outputted.

bash python -m rpbasicdesign.cli --rpsbml_file examples/lycopene_CrtEBI_from_selenzy.xml --sample_size 88 --parts_files examples/parts_for_lycopene.csv --o_sbol_dir examples/lycopene_sbol_crtEBI --o_dnabot_dir examples/lycopene_dnabot_crtEBI --max_enz_per_rxn 1

Inputs

This section documents input files required / optional, their purpose, and how information should be structured.

rpSBML file [required]

SBML with retropath-like annotations. UnitProt IDs of enzyme are expected to be listed here. More information of rpSBML file at https://github.com/brsynth/rptools. Some examples or rpSBML files are provided in tests/input.

Parts files [optional]

These are CSV files listing the linker IDs available for the constructs (BASIC linkers), as well as the user parts (backbone, promoters, ...). The format should be comma separated on 4 columns with header. Example below: id,type,sequence,comment L1,neutral linker,, L2,neutral linker,, L3,neutral linker,,

By default, the rpbasicdesign/data/biolegio_parts.csv file is used which corresponds to the BioLegio commercial plate (link). A second predefined file corresponding to older version of the BioLegio plate is also described in rpbasicdesign/data/legacy_parts.csv.

For linkers, the type annotation should be one of neutral linker, methylated linker, peptide fusion linker or RBS linker. For user parts, type should be one of backbone or constitutive promoter. Other type will raise a warning and will be omited. By default, biolegio_parts.csv and user_parts.csv are used.

Use the parts_files arguments to override.

Important: - IDs should match the linker naming conventions (see below). - IDs should match the IDs used in the plate file inputed to dnabot. As example -- but also ready to be used -- the biolegio_plate.csv is a valid input files for dnabot, with consistent IDs between biolegio_parts.csv and biolegio_plate.csv.

For developers

Installation

sh git clone https://github.com/brsynth/rpbasicdesign.git cd rpbasicdesign conda env create -f environment.yaml -n <myenv> conda develop -n <myenv> .

Tests

sh conda activate <myenv> python -m pytest -v --cov=rpbasicdesign --cov-report html

Constraints and limitations

The BASIC linker set is a major piece of the BASIC assembly method. For a detailed explanation of the BASIS approach, see Storch et. al., ACS Synth. Biol., 2015 (doi: 10.1021/sb500356d).

Polycistronic constructs

Only polycistronic constructs are enabled at the moment.

Predefined set of linkers

By default, the set of linkers used is the one presented available in from the commercial plate from BioLegio. If one wants to use its own set of linkers, the user is advised to do it carefully and to look for more information.

Linker naming conventions

Due to DNA-Bot implementation: - RBS linkers should start with the Un suffix, where n could be any alphanumeric character. - Any linkers should have its two half linkers ending with the -P and -S suffixes listed in the "plate" file, ie in the file that provides the well locations containing the DNA fragment. See the BASIC approach paper, and especially the supplementary files for more information.

Controlled vocabulary for parts file

Parts and linkers provided in the *_parts.csv files have to match on the following type:

• neutral linker
• methylated linker
• RBS linker
• peptide fusion linker
• backbone
• constitutive promoter

Providing CDSs

As of today, CDS are obtained only by parsing rpSBML files.

Custom linkers

For advanced users wishing to play with custom linkers: - Linkers and parts can be provided using a custom file with the --parts_files argument. - Linkers described user_parts.csv are not considered. - RBS linker IDs have to be in the form AAA-BBB with AAA being the linker suffix ID. - Linker prefixes and suffixes coordinates on the plate have to be listed in [biolegio|legacy]_plate.csv.

Maximum number of CDSs per construct

The maximum number of genes in a construct limited to 3 with the default biolegio_plate.csv RBS library, because there is only 3 different RBS suffix in the commercial BioLegio library. Anyway, if needed, this max number of genes can be relaxed and increased using the --max_gene_per_construct parameter.

TODO

• Better handle logs and add verbose option

References

• Galaxy-SynBioCAD: https://doi.org/10.1101/2020.06.14.145730
• DNA-Bot: https://doi.org/10.1093/synbio/ysaa010
• BASIC assembly method: https://doi.org/10.1021/sb500356d