# coding: utf-8
"""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:
**Note**:
this section describes a behaviour that is not part of the YTK
standard, but that is implemnted in all YTK official parts, and encouraged
to follow by the YTK authors.
**Caution**
this section describes a behaviour that goes against the MoClo standard,
but which you are entitled to follow for your parts to be valid YTK parts.
**Danger**
this section describes a quirk specific to the ``moclo-ytk`` library.
References:
1. `Lee, M. E., DeLoache, W. C., Cervantes, B., Dueber, J. E. (2015).
A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly.
ACS Synthetic Biology, 4(9), 975–986.
<https://doi.org/10.1021/sb500366v>`__
2. `Obst, U., Lu, T. K., Sieber, V. (2017).
A Modular Toolkit for Generating Pichia pastoris Secretion Libraries.
ACS Synthetic Biology, 6(6), 1016–1025
<https://doi.org/10.1021/acssynbio.6b00337>`__
3. `Weber, E., Engler, C., Gruetzner, R., Werner, S., Marillonnet, S. (2011).
A Modular Cloning System for Standardized Assembly of Multigene Constructs.
PLOS ONE, 6(2), e16765.
<https://doi.org/10.1371/journal.pone.0016765>`__
"""
import abc
import six
from Bio.Restriction import BsaI, BsmBI
from ..core import modules, vectors, parts
__author__ = "Martin Larralde <martin.larralde@ens-paris-saclay.fr>"
__version__ = (
__import__("pkg_resources")
.resource_string(__name__, "ytk.version")
.strip()
.decode("ascii")
)
### VECTORS
[docs]class YTKEntryVector(vectors.EntryVector):
"""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.
"""
cutter = BsmBI
[docs]class YTKCassetteVector(vectors.CassetteVector):
"""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.
"""
cutter = BsaI
[docs]class YTKDeviceVector(vectors.DeviceVector):
"""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 = BsmBI
### MODULES
[docs]class YTKProduct(modules.Product): # FIXME ?
"""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.
"""
cutter = BsmBI
[docs] @staticmethod
def structure(): # noqa: D105
return (
"CGTCTC" # BsmBI
"N"
"(NNGG)" # Product overhangs (start)
"(TCTC" # BsaI (first 2 nucleotides in the overhang)
"N"
"NNNN" # Type specific overhang (start)
"N*?" # Template
"NNNN" # Type specific overhang (end)
"N"
"GA)" # BsaI (last 4 nucleotides in the overhang)
"(GACC)" # Entry overhangs (end)
"N"
"GAGACG" # BsmBI
)
[docs]class YTKEntry(modules.Entry):
"""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 `~moclo.kits.ytk.YTKEntry`, and must be
used as `~moclo.kits.ytk.YTKPart234r` instances for the assembly
logic to work as expected.
"""
cutter = BsaI
[docs]class YTKCassette(modules.Cassette):
"""A MoClo Yeast ToolKit cassette.
"""
cutter = BsmBI
### PARTS
@six.add_metaclass(abc.ABCMeta)
class YTKPart(parts.AbstractPart):
"""A Yeast MoClo ToolKit standard part.
A part is a plasmid with standardized flanking overhangs sequences
that allows immediate type recognition.
Note:
The base MoClo framework defines entries and vectors for each level
of assembly, but the Yeast ToolKit authors instead identify their
sequences as "parts" of different, strongly-defined types. In order
to follow the base MoClo frameworks, parts of Type 8, 8a, and 678 are
handled as cassette vectors, while other parts are handled as entries.
"""
cutter = BsaI
signature = NotImplemented
[docs]class YTKPart1(YTKPart, YTKEntry):
"""A YTK Type 1 part (**Upstream assembly connector**).
.. image:: type1.svg
:align: center
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.
"""
signature = ("CCCT", "AACG")
[docs]class YTKPart2(YTKPart, YTKEntry):
"""A YTK Type 2 part (**Promoter**).
.. image:: type2.svg
:align: center
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.
"""
signature = ("AACG", "TATG")
[docs]class YTKPart3(YTKPart, YTKEntry):
"""A YTK Type 3 part (**Coding sequence**).
.. image:: type3.svg
:align: center
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.
"""
signature = ("TATG", "ATCC")
[docs]class YTKPart3a(YTKPart, YTKEntry):
"""A YTK Type 3a part (**N-terminal coding sequence**).
.. image:: type3a.svg
:align: center
"""
signature = ("TATG", "TTCT")
[docs]class YTKPart3b(YTKPart, YTKEntry):
"""A YTK Type 3b part (**C-terminal coding sequence**).
.. image:: type3b.svg
:align: center
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.
"""
signature = ("TTCT", "ATCC")
[docs]class YTKPart4(YTKPart, YTKEntry):
"""A YTK Type 4 part (**Transcriptional terminator**).
.. image:: type4.svg
:align: center
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 `~moclo.kits.ytk.YTKPart4a` and
`~moclo.kits.ytk.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.
"""
signature = ("ATCC", "GCTG")
[docs]class YTKPart4a(YTKPart, YTKEntry):
"""A YTK Type 4a part (**C-terminal tag sequence**).
.. image:: type4a.svg
:align: center
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.
"""
signature = ("ATCC", "TGGC")
[docs]class YTKPart4b(YTKPart, YTKEntry):
"""A YTK Type 4b part (**Terminator sequence**).
.. image:: type4b.svg
:align: center
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.
"""
signature = ("TGGC", "GCTG")
[docs]class YTKPart234(YTKPart, YTKEntry):
"""A YTK Type 234 part (**Composite 2, 3, 4**).
.. image:: type234.svg
:align: center
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.
"""
signature = ("AACG", "GCTG")
[docs]class YTKPart234r(YTKPart, YTKEntry):
"""A YTK Type 234 part (**Composite 2, 3, 4**) with reversed BsaI sites.
.. image:: type234r.svg
:align: center
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.
"""
signature = ("AACG", "GCTG")
@staticmethod
def structure(): # noqa: D105
return "(AACG)(NGAGACCN*?GGTCTCN)(GCTG)"
[docs]class YTKPart5(YTKPart, YTKEntry):
"""A YTK Type 5 part (**Downstream assembly connector**).
.. image:: type5.svg
:align: center
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.
"""
signature = ("GCTG", "TACA")
[docs]class YTKPart6(YTKPart, YTKEntry):
"""A YTK Type 6 part (**Yeast marker**).
.. image:: type6.svg
:align: center
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).
"""
signature = ("TACA", "GAGT")
[docs]class YTKPart7(YTKPart, YTKEntry):
"""A YTK Part Type 7 part (**Yeast origin** / **3' homology**).
.. image:: type7.svg
:align: center
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.
"""
signature = ("GAGT", "CCGA")
[docs]class YTKPart8(YTKPart, YTKCassetteVector):
"""A YTK Type 8 part (**Bacterial origin & marker**).
.. image:: type8.svg
:align: center
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.
"""
signature = ("CCGA", "CCCT")
[docs]class YTKPart8a(YTKPart, YTKCassetteVector):
"""A YTK Part 8a part (**Bacterial origin & marker**).
.. image:: type8a.svg
:align: center
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.
"""
signature = ("CCGA", "CAAT")
[docs]class YTKPart8b(YTKPart, YTKEntry):
"""A YTK Type 8b part (**5' homology**).
.. image:: type8b.svg
:align: center
As with certain Type 7 parts, parts of this type contain long sequences
of homology to the genome that is upstream of the target locus.
"""
signature = ("CAAT", "CCCT")
[docs]class YTKPart678(YTKPart, YTKCassetteVector):
"""A YTK Type 678 part (**Composite 6, 7, 8**).
.. image:: type678.svg
:align: center
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.
"""
signature = ("TACA", "CCCT")