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kunkel_mutagenesis.py
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kunkel_mutagenesis.py
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import math
import json
import csv
import datetime
from autoprotocol.util import make_dottable_dict
from autoprotocol.pipette_tools import *
from autoprotocol.container import Container, WellGroup
from autoprotocol.protocol import Ref
from autoprotocol.unit import Unit
import transcriptic
config = transcriptic.Config.from_file('~/.transcriptic')
def return_media(mtype):
'''
Dicts of all plates available that can be purchased.
'''
if mtype == 'solid':
media = {"50_ugml_Kanamycin": "ki17rs7j799zc2",
"100_ugml_Ampicillin": "ki17sbb845ssx9",
"100_ugmL_Spectinomycin": "ki17sbb9r7jf98",
"noAB": "ki17reefwqq3sq"}
elif mtype == 'liquid':
media = {"50_ugml_Kanamycin": "lb-broth-50ug-ml-kan",
"100_ugml_Ampicillin": "lb-broth-100ug-ml-amp",
"100_ugmL_Spectinomycin": "lb-broth-100ug-ml-specto"}
else:
raise ValueError("mtype has to be solid or liquid")
return(media)
def det_new_group(i, base=0):
'''
Helper to determine if new_group should be added. Returns true when i matches the base, which defaults to 0.
'''
assert isinstance(i, int), "Needs an integer."
assert isinstance(base, int), "Base has to be an integer"
if i == base:
new_group = True
else:
new_group = False
return new_group
def printdatetime(time=True):
printdate = datetime.datetime.now().strftime('%Y-%m-%d_%H:%M:%S')
if not time:
printdate = datetime.datetime.now().strftime('%Y-%m-%d')
return printdate
def provision_to_tube(protocol, name, tube, resource_id, volume, discard=True, storage=None):
'''
provision_to_tube allows us to provision into a tube/well that can then be used
in transfers on the workcell.
'''
assert isinstance(volume, (Unit, int, float)), "Volume must be of type int, float or Unit."
if isinstance(volume, Unit):
volume = volume.value
if storage:
dest = protocol.ref(name, None, tube, storage=storage).well(0)
else:
dest = protocol.ref(name, None, tube, discard=discard).well(0)
protocol.provision(resource_id, dest, "%s:microliter" % volume)
return(dest)
def transfer_kwargs(pre_buffer, one_tip=False, one_source=False):
kwargs = {"one_tip": one_tip,
"one_source": one_source,
"pre_buffer": "%s:microliter" % pre_buffer,
"blowout_buffer": True}
return(kwargs)
def thermocycle_ramp(start_temp, end_temp, total_duration, step_duration):
'''
Create a ramp instruction for the thermocyler. Used in annealing protocols.
'''
assert Unit.fromstring(total_duration).unit == Unit.fromstring(step_duration).unit, ("Thermocycle_ramp durations"
" must be specified using the"
" same unit of time.")
thermocycle_steps = []
start_temp = Unit.fromstring(start_temp).value
num_steps = int(Unit.fromstring(total_duration).value // Unit.fromstring(step_duration).value)
step_size = (Unit.fromstring(end_temp).value - start_temp) // num_steps
for i in xrange(0, num_steps):
thermocycle_steps.append({
"temperature": "%d:celsius" % (start_temp + i * step_size), "duration": step_duration
})
return thermocycle_steps
def ref_kit_container(protocol, name, container, kit_id, discard=True, store=None):
'''
Still in use to allow booking of agar plates on the fly
'''
kit_item = Container(None, protocol.container_type(container))
if store:
protocol.refs[name] = Ref(name, {"reserve": kit_id, "store": {"where": store}}, kit_item)
else:
protocol.refs[name] = Ref(name, {"reserve": kit_id, "discard": discard}, kit_item)
return(kit_item)
def find_aliquot_by_name(name):
# looks up code for the sample
resp = config.get('samples?q=%s&show_containers=false' % name)
for aq in resp.json()['results']:
if aq['name'] == name:
return aq
def kunkel_mutagenesis(protocol, params):
def find_part(part_name, check_primer=False):
if check_primer and (params.t7pro or params.t7term):
return 'primer'
else:
aq = find_aliquot_by_name(part_name)
if not aq:
raise ValueError(
"Couldn't find aliquot with name '%s'" % part_name)
container = None
for r in protocol.refs.itervalues():
if r.opts.get('id') == aq['container']['id']:
container = r.container
break
if container is None:
container = protocol.ref(
aq['container']['label'] or aq['container']['id'],
aq['container']['id'],
aq['container']['container_type']['shortname'],
discard=False,
storage="cold_20"
)
return container.well(aq['well_idx']).set_volume("%s:microliter" % aq['volume_ul'])
def make_10_atp(vol):
# account for tube dead_vol
vol = vol + 15
atp = protocol.ref("atp_10mM", cont_type='micro-1.5', discard=True).well(0)
protocol.provision('rs16pccshb6cb4', atp, "%s:microliter" % (vol/10))
protocol.provision('rs17gmh5wafm5p', atp, "%s:microliter" % (vol - (vol/10)))
return atp
def provision_reagents(reagents, dest):
for key, reagent in reagents.iteritems():
protocol.provision(reagent[0],
dest,
"%s:microliter" % ((len(params.mutants) + 1.0) * reagent[1] * add_mm))
def isLast(itr):
old = itr.next()
for new in itr:
yield False, old
old = new
yield True, old
params = make_dottable_dict(params)
params.ssDNA = find_part(params.ssDNA)
params.mutants = []
# read in oligos and mutants
with open('kunkel_mutants.csv', 'rU') as rxtmap:
reader = csv.reader(rxtmap)
current_mutant_label = ''
current_sequencing_primer = ''
for idx, (is_last, row) in enumerate(isLast(reader)):
if idx == 0 and row[0] == 'mutant_label':
continue
if row[0] != current_mutant_label:
if current_mutant_label != '':
params.mutants.append({
'sequencing_primer': find_part(current_sequencing_primer, True),
'mutant_label': current_mutant_label,
'oligos': my_oligos
})
current_mutant_label = row[0]
current_sequencing_primer = row[1]
my_oligos = []
my_oligos.append({"oligo_label": row[2],
"sequence": row[3],
"scale": row[4],
"purification": row[5]})
if is_last:
params.mutants.append({
'sequencing_primer': find_part(current_sequencing_primer, True),
'mutant_label': current_mutant_label,
'oligos': my_oligos
})
# mastermix vol to make - needs to be adjusted based on testing
add_mm = 1.3
# Get unique set of oligos based on sequence data
# Oligosynthesize
oligos_to_synthesize = []
for i, mutant in enumerate(params.mutants):
mutant["mutant_label"] = mutant["mutant_label"] or "mutant_%s" % (i + 1)
oligos_to_synthesize.append(mutant["oligos"])
flattened = [val for sublist in oligos_to_synthesize for val in sublist]
oligos_to_synthesize = list({v['sequence']: v for v in flattened}.values())
# re-factor to remove add_properites
oligo_containers = []
oligos = []
for i, oligo in enumerate(oligos_to_synthesize):
label = oligo["oligo_label"] or "seq_%s" % i
oligo_containers.append(protocol.ref(label, None, "micro-2.0",
storage="cold_4").well(0))
oligo_containers[i].add_properties({"sequence": oligo["sequence"]})
oligos.append({"sequence": oligo["sequence"],
"destination": oligo_containers[i],
"scale": oligo["scale"],
"purification": oligo["purification"]})
protocol.oligosynthesize(oligos)
# Kinase
kinase_oligo_plate = protocol.ref("kinase_oligo_plate", None, "96-pcr",
storage="cold_20")
wells_to_kinase = kinase_oligo_plate.wells_from("A1", len(oligos))
# provision atp for entire protocol
atp_needed = ((len(oligos) * add_mm) + (len(params.mutants) + 1) * 0.4 * add_mm)
atp = make_10_atp(atp_needed)
kinase_mix = []
for i in range(int(math.ceil(len(oligos)/60.0))):
kinase_mix.append(protocol.ref("kinase_mix-%s" % (i + 1), None, "micro-1.5", discard=True).well(0))
reagents = {'pnkbuffer': ['rs16pc9rd5sg5d', 3],
'water': ['rs17gmh5wafm5p', 18],
'pnk': ['rs16pc9rd5hsf6', 1]}
provision_reagents(reagents, kinase_mix)
protocol.transfer(atp, kinase_mix, "%s:microliter" % ((len(params.mutants) + 1) * 1 * add_mm), new_group=True)
protocol.transfer(kinase_mix,
wells_to_kinase,
"23:microliter",
**transfer_kwargs(15, True, True))
for i, oligo in enumerate(oligo_containers):
protocol.transfer(oligo,
wells_to_kinase[i],
"7:microliter",
mix_after=False,
new_group=det_new_group(i),
aspirate_source=aspirate_source(depth=depth("ll_following",
lld="pressure",
distance="0.0:meter")),
**transfer_kwargs(10))
protocol.seal(kinase_oligo_plate)
protocol.thermocycle(kinase_oligo_plate,
[{"cycles": 1, "steps": [
{"temperature": "37:celsius",
"duration": "60:minute"},
]}
], volume="30:microliter")
# make ssDNA_mastermix
mix_plate = protocol.ref("mix_plate", None, "96-pcr", discard=True)
ssDNA_mix = mix_plate.well(0)
protocol.transfer(params.ssDNA,
ssDNA_mix,
"%s:microliter" % ((len(params.mutants) + 1) * 2.0 * add_mm),
**transfer_kwargs((len(params.mutants) + 1) * 1))
protocol.provision('rs17sh5rzz79ct', ssDNA_mix, "%s:microliter" % ((len(params.mutants) + 1) * 0.2 * add_mm))
# Dilute
protocol.unseal(kinase_oligo_plate)
diluted_oligo_plate = protocol.ref("dilute_oligo_plate", None, "96-flat", discard=True)
diluted_oligo_wells = diluted_oligo_plate.wells_from(0, len(params.mutants))
water = [provision_to_tube(protocol, "water%s" % (i + 1), "micro-2.0", "rs17gmh5wafm5p", 1900)
for i in range(int(math.ceil(len(params.mutants)/float(9.5))))
]
protocol.transfer(water,
diluted_oligo_wells,
"200:microliter",
disposal_vol="0:microliter",
**transfer_kwargs(40, True, True))
mutants = [m for m in params.mutants if m]
mutants = sorted(mutants, key=lambda mutant: mutant["mutant_label"])
for i, m in enumerate(mutants):
for j, kin_oligo in enumerate(m["oligos"]):
if i == 0 and j == 0:
new_group = True
else:
new_group = False
index = next((i for i, olig in enumerate(oligo_containers) if olig.properties["sequence"] == kin_oligo["sequence"]), -1)
protocol.transfer(kinase_oligo_plate.well(index),
diluted_oligo_plate.well(i),
"2:microliter",
mix_after=True,
mix_vol="2:microliter",
new_group=new_group,
**transfer_kwargs(10))
protocol.cover(diluted_oligo_plate)
protocol.spin(diluted_oligo_plate, "700:meter/second^2", "2:minute")
protocol.uncover(diluted_oligo_plate)
# Anneal
annealing_plate = protocol.ref("annealing_oligo_plate", None, "384-pcr", storage="cold_20")
anneal_wells = annealing_plate.wells_from(0, len(params.mutants))
protocol.transfer(ssDNA_mix,
anneal_wells.wells,
"2.2:microliter",
dispense_speed="50:microliter/second",
**transfer_kwargs(7, True, True))
for i, oligo_reaction in enumerate(zip(diluted_oligo_wells.wells, anneal_wells.wells)):
protocol.transfer(oligo_reaction[0],
oligo_reaction[1],
"2:microliter",
aspirate_source=aspirate_source(depth("ll_bottom", distance=".001:meter")),
mix_after=True,
mix_vol="2:microliter",
flowrate="50:microliter/second",
repetitions=2,
new_group=det_new_group(i),
**transfer_kwargs(5))
protocol.seal(annealing_plate)
protocol.spin(annealing_plate, "700:meter/second^2", "2:minute")
protocol.thermocycle(annealing_plate, [{
"cycles": 1,
"steps": thermocycle_ramp("95:celsius", "25:celsius", "60:minute", "4:minute")
}],
volume="5:microliter",
dataref=None,
dyes=None)
# Step 4 - Polymerize
protocol.unseal(annealing_plate)
polymerize_MM = mix_plate.well(12)
reagents = {"buffer": ['rs17sh5rzz79ct', 0.6],
"t4ligase": ['rs16pc8krr6ag7', 0.4],
"t7polymerase": ['rs16pca2urcz74', 0.4],
"dntp": ['rs16pcb542c5rd', 0.4]
}
provision_reagents(reagents, polymerize_MM)
protocol.transfer(atp, polymerize_MM, "%s:microliter" % ((len(params.mutants) + 1) * 0.4 * add_mm), new_group=True)
for reaction in anneal_wells.wells:
protocol.transfer(polymerize_MM,
reaction,
"2.2:microliter",
mix_after=False,
**transfer_kwargs(10))
protocol.seal(annealing_plate)
protocol.incubate(annealing_plate, "ambient", "1.5:hour")
# Transformation using Zymo 10B Competent Cells
transformation_cells = []
for i in range(len(params["mutants"])):
transformation_cells.append(provision_to_tube(protocol, "cell_%s" % (i), "micro-1.5",
"rs16pbjc4r7vvz", 50))
num_colonies = params["num_colonies"]
assert len(params["mutants"]) * num_colonies <= 96, ("This protocol is limited to 96 sequenced colonies, please "
"submit additional runs if needed.")
transformation_plate = protocol.ref("transformation_plate", None, "96-pcr", discard=True)
protocol.incubate(transformation_plate, "cold_20", "10:minute")
transformation_wells = transformation_plate.wells_from(0, len(params.mutants), columnwise=False)
for i, tube in enumerate(transformation_cells):
protocol.transfer(tube, transformation_wells[i], "50:microliter", mix_after=False)
protocol.unseal(annealing_plate)
for i, rxt in enumerate(anneal_wells):
protocol.transfer(anneal_wells[i], transformation_wells[i], "2.0:microliter",
dispense_speed="10:microliter/second",
mix_after=False,
new_group=det_new_group(i))
protocol.cover(transformation_plate, lid="universal")
protocol.incubate(transformation_plate, "cold_4", "20:minute", shaking=False, co2=0)
protocol.uncover(transformation_plate)
agar_plates = []
assert len(mutants) == len(transformation_wells), ("Sanity check failed. There is an issue with the number of"
"mutants and the number of transformations.")
for well in range(0, len(transformation_wells), 6):
agar_plate = ref_kit_container(protocol,
"agar-%s_%d_%s" % (params["antibiotic"].split("_")[-1],
well + 1,
printdatetime(time=False)),
"6-flat",
return_media('solid')[params["antibiotic"]],
discard=False, store='cold_4')
agar_plates.append(agar_plate)
for i, well in enumerate(transformation_wells[well:well + 6]):
protocol.spread(well, agar_plate.well(i), "50:microliter")
protocol.incubate(agar_plate, "warm_37", "18:hour")
growth_plate = protocol.ref("growth_plate_%s" % printdatetime(time=False), None, "96-flat", discard=True)
cols = int(math.ceil(len(params.mutants) * num_colonies / float(8)))
columns = [{"column": i, "volume": "150:microliter"} for i in range(0, cols)]
protocol.dispense(growth_plate, return_media('liquid')[params["antibiotic"]], columns)
growth_wells = growth_plate.wells_from(0, num_colonies*len(params.mutants), columnwise=True)
i = 0
for k, plate in enumerate(agar_plates):
for j in range(6):
if plate.well(j).volume:
protocol.autopick(plate.well(j),
growth_wells[i:i+num_colonies],
min_count=1,
dataref=mutants[k * 6 + j]['mutant_label'])
i = i + num_colonies
protocol.cover(growth_plate, lid="low_evaporation")
protocol.incubate(growth_plate, "warm_37", "24:hour", shaking=True, co2=0)
protocol.uncover(growth_plate)
if params.t7pro:
seq_plate = protocol.ref("sequencing_plate_t7pro_%s" % printdatetime(time=False),
cont_type="96-pcr",
storage="cold_4")
seq_well_group = seq_plate.wells_from(0, num_colonies*len(params.mutants), columnwise=True)
t7pro_primer = protocol.ref("t7promoter",
cont_type="micro-1.5",
storage="cold_4")
protocol.provision("rs17tcpekfy7v9", t7pro_primer.well(0), "1:microliter")
protocol.provision("rs17gmh5wafm5p", t7pro_primer.well(0), "%s:microliter" % (num_colonies*(len(params.mutants)+2)))
protocol.transfer(growth_wells, seq_well_group, "30:microliter")
protocol.seal(seq_plate)
protocol.sangerseq(seq_plate, seq_well_group.indices(), "Seq_primer_T7promoter", type="rca", primer=t7pro_primer)
if params.t7term:
seq_plate = protocol.ref("sequencing_plate_t7term_%s" % printdatetime(time=False),
cont_type="96-pcr",
storage="cold_4")
seq_well_group = seq_plate.wells_from(0, num_colonies*len(params.mutants), columnwise=True)
t7term_primer = protocol.ref("t7terminator",
cont_type="micro-1.5",
storage="cold_4")
protocol.provision("rs17tcpwfbgzqd", t7term_primer.well(0), "1:microliter")
protocol.provision("rs17gmh5wafm5p", t7term_primer.well(0), "%s:microliter" % (num_colonies*(len(params.mutants)+2)))
protocol.transfer(growth_wells, seq_well_group, "30:microliter")
protocol.seal(seq_plate)
protocol.sangerseq(seq_plate, seq_well_group.indices(), "Seq_primer_t7terminator", type="rca", primer=t7term_primer)
if not (params.t7pro or params.t7term):
seq_plate = protocol.ref("sequencing_plate_%s" % printdatetime(time=False),
cont_type="96-pcr",
storage="cold_4")
seq_well_group = seq_plate.wells_from(0, num_colonies*len(params.mutants), columnwise=True)
j = 0
seq_primers = {}
mutant_well_table = {}
for seq_set in mutants:
seq_wells = WellGroup(seq_well_group[j:j+num_colonies])
protocol.transfer(growth_wells[j:j+num_colonies], seq_wells, "30:microliter")
if seq_set["sequencing_primer"] not in seq_primers:
seq_primers[seq_set["sequencing_primer"]] = seq_wells.indices()
mutant_well_table[seq_set["sequencing_primer"]] = {seq_set["mutant_label"]: seq_wells.indices()}
else:
seq_primers[seq_set["sequencing_primer"]].extend(seq_wells.indices())
if seq_set["mutant_label"] not in mutant_well_table[seq_set["sequencing_primer"]]:
mutant_well_table[seq_set["sequencing_primer"]].update({seq_set["mutant_label"]: seq_wells.indices()})
else:
mutant_well_table[seq_set["sequencing_primer"]][seq_set["mutant_label"]].extend(seq_wells.indices())
j += num_colonies
protocol.seal(seq_plate)
for primer, wells in seq_primers.iteritems():
dataref = "Seq_primer_%s" % (primer.container.name)
assert primer.volume - Unit("1.1", "microliter").__mul__(len(wells)) > Unit("0", "microliter"), "You must have at least 1.1uL of sequencing primer per reaction well."
protocol.sangerseq(seq_plate, wells, dataref, type="rca", primer=primer.container)
if __name__ == '__main__':
from autoprotocol.harness import run
run(kunkel_mutagenesis)