Failed to create any GoldenGate assemblies

[GabrielleJohnston]

I have used the example goldengate_plasmid.py without any modifications and have received the following error:

    154             filename = self._filename() + ".fasta"
    155 
--> 156         self._check_output()
    157         return SeqIO.write(self.output, filename, "fasta")
    158 

...\synbio\protocol.py in _check_output(self)
    346         """
    347         if not self.containers:
--> 348             self.run()
    349 
    350             if not self.output:

...\synbio\protocols\goldengate.py in run(self)
     75         # get all the unique contents and set them up in their own wells
     76         mixed_wells = self._create_mixed_wells()
---> 77 
     78         for step in [
     79             Setup(

...\synbio\protocols\goldengate.py in _create_mixed_wells(self)
    125         if not mixed_wells:
    126             raise RuntimeError(f"Failed to create any GoldenGate assemblies")
--> 127 
    128         return sorted(mixed_wells)

RuntimeError: Failed to create any GoldenGate assemblies

Gibson assembly examples work perfectly for me, but none of the GoldenGate examples do.

Thank you!

Edit: The examples work if linear = False is specified in the design.

[jjti]

Hey I tried this and could replicate. I thought it might be a dependency issue (where a bumped dependency broke the package) but I can't resolve the issue even when pinning the depends.

When you ran with a linear = False in design, how long did it take for it to assemble? it was taking >2 minutes on my laptop so I just stopped it

[GabrielleJohnston]

Thank you for your quick response!

I believe it is because the genbank files are for circular DNA, but unless it is specified it is interpreted as linear. On my laptop it runs in less than 5 seconds.

In examples/goldengate_plasmid.py I replaced the design with this:

    (read(r)
    for r in [
        "J23100_AB.gb",
        "B0032m_BC.gb",
        "C0012m_CD.gb",
        "B0015_DE.gb",
        "DVK_AE.gb",
    ]), 
    linear = False
)

For examples/goldengate_combinatorial.py I did find that assembly took too long to run, but I suspect that it is because the data directory is so long and multiple plasmids are read in. If the file read in method from tests/goldengate_test.py is used, assembly is also very fast.

I've copied and pasted my entire goldengate_combinatorial.py below as there are several changes:

"""Example of a Combinatorial Golden Gate assembly with steps and output."""

import os

from Bio.SeqIO import parse

from synbio.designs import Combinatorial
from synbio.protocols import GoldenGate

DIR_NAME = os.path.abspath(os.path.dirname(__file__))
DATA_DIR = os.path.join(DIR_NAME, "..", "..", "data", "goldengate")

def read(filename):
    """Read in a single Genbank file from the test directory."""

    return next(parse(os.path.join(DATA_DIR, filename), "genbank"))

records = []
for (_, _, filenames) in os.walk(DATA_DIR):
    for file in filenames:
        gb = os.path.join(DATA_DIR, file)
        if not gb.endswith(".gb"):
            continue
        for record in parse(gb, "genbank"):
            records.append(record)

record_sets = []
for f_type in ["promoter", "RBS", "CDS", "terminator"]:

    def test(r):
        return any(
            f.type == f_type and f.location.start < 50 for f in r.features
        )

    new_bin = [r for r in records if test(r)][:5]
    record_sets.append(new_bin)  # add a new bin

records = [r for record_set in record_sets for r in record_set] + [
    read("DVK_AE.gb")
]

# create a combinatorial library design from multiple "bins"
design = Combinatorial(records, linear = False)

# create a protocol using Golden Gate as the sole composite step and run
protocol = GoldenGate(
    name="Combinatorial Golden Gate", design=design, include=["KanR"], min_count=5
)

# export all the output plasmids to a multi-FASTA
protocol.to_fasta("composite_parts.fasta")

# export plate layouts
protocol.to_csv("plate_layouts.csv")

# export human protocol
protocol.to_txt("protocol.txt")

# export a hamilton picklist
protocol.to_picklists("robotic_picklist.gwl", platform="tecan")