An implementation of the Yeast ToolKit for the Python MoClo library.
This module is tested against the officials parts available in the Yeast ToolKit (YTK), and also against the Pichia ToolKit (PTK) parts since they were designed to be compatible with each other.
The documentation of this module is mostly adapted from the Lee et al. supplementary data. Each item also has specific sections that are organized as follow:
moclo-ytk
library.References
moclo.kits.ytk.
YTKProduct
(Product)[source]¶A MoClo Yeast ToolKit product.
As the YTK entry vector does not contain the required BsaI restriction site, the site must be contained in the product sequence.
Caution
The standard construction describe in the Lee et al. paper directly inserts the beginning of the BsaI recognition site inside of the two BsmBI overhangs at both ends of the product. Other valid constructs that do not proceed like so won’t be considered a valid product, although they contain the required BsaI site.
References
Lee et al., Supplementary Figure S19.
__init__
(record)¶Initialize self. See help(type(self)) for accurate signature.
cutter
¶alias of Bio.Restriction.Restriction.BsmBI
is_valid
()¶Check if the wrapped record follows the required class structure.
Returns: | True if the record is valid, False otherwise. |
---|---|
Return type: | bool |
overhang_end
()¶Get the downstream overhang of the target sequence.
Returns: | the downstream overhang. |
---|---|
Return type: | Seq |
overhang_start
()¶Get the upstream overhang of the target sequence.
Returns: | the downstream overhang. |
---|---|
Return type: | Seq |
structure
()[source]¶Get the module structure, as a DNA regex pattern.
Warning
If overloading this method, the returned pattern must include 3 capture groups to capture the following features:
target_sequence
()¶Get the target sequence of the module.
Modules are often stored in a standardized way, and contain more than the sequence of interest: for instance they can contain an antibiotic marker, that will not be part of the assembly when that module is assembled into a vector; only the target sequence is inserted.
Returns: | the target sequence with annotations. |
---|---|
Return type: | SeqRecord |
Note
Depending on the cutting direction of the restriction enzyme used during assembly, the overhang will be left at the beginning or at the end, so the obtained record is exactly the sequence the enzyme created during restriction.
moclo.kits.ytk.
YTKEntryVector
(EntryVector)[source]¶A MoClo Yeast ToolKit entry vector.
Any plasmid with two BsmBI restriction sites can be used to create a YTK entry, although the toolkit-provided entry vector (pYTK001) is probably the most appropriate plasmid to use.
Caution
To the contrary of the usual MoClo entry vectors described in the Weber et al. paper, the YTK entry vectors do not provide another BsaI restriction site enclosing the placeholder sequence. As such, YTK Level -1 modules must embed the BsaI binding site.
__init__
(record)¶Initialize self. See help(type(self)) for accurate signature.
assemble
(module, *modules, **kwargs)¶Assemble the provided modules into the vector.
Parameters: |
|
---|---|
Returns: | the assembled sequence with sequence annotations inherited from the vector and the modules. |
Return type: |
|
Raises: |
|
cutter
¶alias of Bio.Restriction.Restriction.BsmBI
is_valid
()¶Check if the wrapped record follows the required class structure.
Returns: | True if the record is valid, False otherwise. |
---|---|
Return type: | bool |
overhang_end
()¶Get the downstream overhang of the vector sequence.
overhang_start
()¶Get the upstream overhang of the vector sequence.
placeholder_sequence
()¶Get the placeholder sequence in the vector.
The placeholder sequence is replaced by the concatenation of modules during the assembly. It often contains a dropout sequence, such as a GFP expression cassette that can be used to measure the progress of the assembly.
structure
()¶Get the vector structure, as a DNA regex pattern.
Warning
If overloading this method, the returned pattern must include 3 capture groups to capture the following features:
target_sequence
()¶Get the target sequence in the vector.
The target sequence if the part of the plasmid that is not discarded during the assembly (everything except the placeholder sequence).
moclo.kits.ytk.
YTKEntry
(Entry)[source]¶A MoClo Yeast ToolKit entry.
YTK entries are stored and shared as plasmids flanked by BsaI binding sites at both ends of the target sequence.
Danger
Although the BsaI binding sites is not located within the target
sequence for almost all the standard toolkit parts, special Type 234r
parts have these sites reversed, because these parts are used to
assemble cassette vectors and require the final construct to contain
a BsaI site to allow assembly with other parts. Those parts will
not match the default YTKEntry
, and must be
used as YTKPart234r
instances for the assembly
logic to work as expected.
__init__
(record)¶Initialize self. See help(type(self)) for accurate signature.
cutter
¶alias of Bio.Restriction.Restriction.BsaI
is_valid
()¶Check if the wrapped record follows the required class structure.
Returns: | True if the record is valid, False otherwise. |
---|---|
Return type: | bool |
overhang_end
()¶Get the downstream overhang of the target sequence.
Returns: | the downstream overhang. |
---|---|
Return type: | Seq |
overhang_start
()¶Get the upstream overhang of the target sequence.
Returns: | the downstream overhang. |
---|---|
Return type: | Seq |
structure
()¶Get the module structure, as a DNA regex pattern.
Warning
If overloading this method, the returned pattern must include 3 capture groups to capture the following features:
target_sequence
()¶Get the target sequence of the module.
Modules are often stored in a standardized way, and contain more than the sequence of interest: for instance they can contain an antibiotic marker, that will not be part of the assembly when that module is assembled into a vector; only the target sequence is inserted.
Returns: | the target sequence with annotations. |
---|---|
Return type: | SeqRecord |
Note
Depending on the cutting direction of the restriction enzyme used during assembly, the overhang will be left at the beginning or at the end, so the obtained record is exactly the sequence the enzyme created during restriction.
moclo.kits.ytk.
YTKCassetteVector
(CassetteVector)[source]¶A MoClo Yeast ToolKit cassette vector.
The YTK provides a canonical integration plasmid, preassembled from several other parts, that can be used as a cassette vector for an assembly of Type 2, 3 and 4 parts. Type 8, 8a and 678 parts are also considered as cassette vectors.
References
Lee et al., Figure 2.
__init__
(record)¶Initialize self. See help(type(self)) for accurate signature.
assemble
(module, *modules, **kwargs)¶Assemble the provided modules into the vector.
Parameters: |
|
---|---|
Returns: | the assembled sequence with sequence annotations inherited from the vector and the modules. |
Return type: |
|
Raises: |
|
cutter
¶alias of Bio.Restriction.Restriction.BsaI
is_valid
()¶Check if the wrapped record follows the required class structure.
Returns: | True if the record is valid, False otherwise. |
---|---|
Return type: | bool |
overhang_end
()¶Get the downstream overhang of the vector sequence.
overhang_start
()¶Get the upstream overhang of the vector sequence.
placeholder_sequence
()¶Get the placeholder sequence in the vector.
The placeholder sequence is replaced by the concatenation of modules during the assembly. It often contains a dropout sequence, such as a GFP expression cassette that can be used to measure the progress of the assembly.
structure
()¶Get the vector structure, as a DNA regex pattern.
Warning
If overloading this method, the returned pattern must include 3 capture groups to capture the following features:
target_sequence
()¶Get the target sequence in the vector.
The target sequence if the part of the plasmid that is not discarded during the assembly (everything except the placeholder sequence).
moclo.kits.ytk.
YTKPart1
(YTKPart, YTKEntry)[source]¶A YTK Type 1 part (Upstream assembly connector).
Parts of this type contain non-coding and non-regulatory sequences that are used to direct assembly of multigene plasmids, such as ligation sites for other Type IIS endonucleases (e.g. BsmBI).
Note
Official toolkit Type 1 parts also include a EcoRI and XbaI site just after the upstream overhang for BioBrick compatibility of the assembled cassettes and multi-gene plasmids.
moclo.kits.ytk.
YTKPart2
(YTKPart, YTKEntry)[source]¶A YTK Type 2 part (Promoter).
Parts of this type contain a promoter. The downstream overhang doubles as the start codon for the subsequent Type 3 or Type 3a coding sequence.
Note
Official toolkit Type 2 parts also include a BglII site immediately preceding the start codon (overlapping the downstream overhang) for BglBrick compatibility.
moclo.kits.ytk.
YTKPart3
(YTKPart, YTKEntry)[source]¶A YTK Type 3 part (Coding sequence).
Parts of this type contain a coding sequence, with the start codon located on the upstream overhang. If a stop codon is omitted from the part, and two bases are added before the downstream overhang, the resulting site can be used as a two amino acid linker to a Type 4 or 4a C-terminal fusion.
Note
Official toolkit Type 3 parts also include a BamHI recognition site at the end of the included CDS (overlapping the downstream overhang) for BglBrick compatibility.
moclo.kits.ytk.
YTKPart3a
(YTKPart, YTKEntry)[source]¶A YTK Type 3a part (N-terminal coding sequence).
moclo.kits.ytk.
YTKPart3b
(YTKPart, YTKEntry)[source]¶A YTK Type 3b part (C-terminal coding sequence).
Note
As with Type 3 parts, official toolkits Type 3b parts also include a BamHI recognition site at the end of the included CDS (overlapping the downstream overhang) for BglBrick compatibility.
moclo.kits.ytk.
YTKPart4
(YTKPart, YTKEntry)[source]¶A YTK Type 4 part (Transcriptional terminator).
As Type 3 parts do not include a stop codon, parts of this type should
encode an in-frame stop codon before the transcriptional terminator.
Commonly used C-terminal fusions, such as purification or epitope tags,
but it is recommended to use YTKPart4a
and
YTKPart4b
subtypes instead.
Note
Official toolkit Type 4 parts all start by a stop codon directly after the upstream overhang, followed by a XhoI recognition site which enables BglBrick compatibility, then followed by the terminator sequence itself.
moclo.kits.ytk.
YTKPart4a
(YTKPart, YTKEntry)[source]¶A YTK Type 4a part (C-terminal tag sequence).
Type 4a parts contain additional coding sequences that will be fused to the C-terminal extremity of the protein. These parts include, but are not limited to: localisation tags, purification tags, fluorescent proteins.
Caution
In contrast to the Type 3 and 3b parts, the convention for 4a parts is to include the stop codon rather than enable read-through of the downstream overhang, although that convention it is not enforced.
Note
Official toolkit Type 4a parts contain a stop codon after the CDS, itself immediately followed by a XhoI recognition site just before the downstream overhang, for BglBrick compatibility.
moclo.kits.ytk.
YTKPart4b
(YTKPart, YTKEntry)[source]¶A YTK Type 4b part (Terminator sequence).
Type 4b contain transcriptional terminators, but are not required to encode an in-frame start codon, as it should be located in the Type 4a part that precedes it.
moclo.kits.ytk.
YTKPart5
(YTKPart, YTKEntry)[source]¶A YTK Type 5 part (Downstream assembly connector).
As with Type 1 parts, parts of this type provide sequences such as restriction enzymes recognition sites, for instance in order to direct multigene expression plasmids.
Note
Official toolkit parts also include a SpeI and PstI site at the end of the part sequence for BioBrick compatibility of the assembled cassettes and multi-gene plasmids.
moclo.kits.ytk.
YTKPart6
(YTKPart, YTKEntry)[source]¶A YTK Type 6 part (Yeast marker).
Parts of this type contain a selectable marker for S. cerevisiae, as a full expression cassette (promoter, ORF, and terminal) for conferring the selectable phenotype (such as drug-resistance or bioluminescence).
moclo.kits.ytk.
YTKPart7
(YTKPart, YTKEntry)[source]¶A YTK Part Type 7 part (Yeast origin / 3’ homology).
Depending on the expression organism (E.coli or S. ceverisiae), this sequence will either hold a yeast origin of replication, or a 3’ homology sequence for integration in the bacterial genome.
moclo.kits.ytk.
YTKPart8
(YTKPart, YTKCassetteVector)[source]¶A YTK Type 8 part (Bacterial origin & marker).
Parts of this type contain a bacterial origin of replication, as well as an antibiotic resistance marker. They act as the Golden Gate Assembly vector when assembling a cassette, and as such should also embbed a dropout sequence, such as a fluorescent protein expression cassette.
Note
Official toolkit parts use an mRFP coding sequence as the dropout, and also include NotI restriction site at each end of the part to allow the verification of new assemblies.
moclo.kits.ytk.
YTKPart8a
(YTKPart, YTKCassetteVector)[source]¶A YTK Part 8a part (Bacterial origin & marker).
Parts of this type, like Type 8 parts, include a bacterial origin of replication and an antibiotic resistance marker, and act as Assembly vectors.
Note
Official toolkit parts use an mRFP coding sequence as the dropout, and also include NotI restriction site at each end of the part so the integration plasmid can be linearized prior to transformation into yeast.
moclo.kits.ytk.
YTKPart234
(YTKPart, YTKEntry)[source]¶A YTK Type 234 part (Composite 2, 3, 4).
Type 234 parts are composed of a complete expression cassette (promoter, coding sequence, and terminator) fused into a single part, instead of separate Type 2, 3 and 4 parts.
moclo.kits.ytk.
YTKPart234r
(YTKPart, YTKEntry)[source]¶A YTK Type 234 part (Composite 2, 3, 4) with reversed BsaI sites.
Type 234r parts are designed so that the BsaI sites are kept within the final cassette. They are used to assemble canonical integration vectors, where the Type 234 part acts as a placeholder until replaced by actual Type 2, 3 and 4 parts in the final construct.
moclo.kits.ytk.
YTKPart678
(YTKPart, YTKCassetteVector)[source]¶A YTK Type 678 part (Composite 6, 7, 8).
Type 678 parts are used when there is no requirement for yeast markers and origins to be included in the final assembly, for instance when assembling an intermediary plasmid acting as a vector for a multi-gene construct.
moclo.kits.ytk.
YTKDeviceVector
(DeviceVector)[source]¶A MoClo Yeast ToolKit multigene vector.
Parts of Type 1 and 5 are used to order the cassette plasmids within
the multigene assembly. The vector always contains a ConLS
and ConRE
parts.
References
Lee et al., Supplementary Figure S21.
cutter
¶alias of Bio.Restriction.Restriction.BsmBI