Greta_Tronberg_Simulation.py

#!/usr/bin/env python
# coding: utf-8

# In[ ]:


from opentrons import simulate
metadata = {'apiLevel': '2.8'}
protocol = simulate.get_protocol_api('2.8')


#Labware (keep aluminium block tuberack in freezer until setup)
tiprack1 = protocol.load_labware('opentrons_96_tiprack_20ul', 2) 
tiprack2 = protocol.load_labware('opentrons_96_tiprack_300ul', 3)
tiprack3 = protocol.load_labware('opentrons_96_tiprack_20ul', 5)
tiprack4 = protocol.load_labware('opentrons_96_tiprack_300ul', 6)

heatblock = protocol.load_module('tempdeck', 4)
plate1 = heatblock.load_labware('corning_96_wellplate_360ul_flat')

thermocycler = protocol.load_module('Thermocycler Module')
plate2 = thermocycler.load_labware('corning_96_wellplate_360ul_flat')

tuberack = protocol.load_labware('opentrons_24_aluminumblock_nest_1.5ml_snapcap', 1)

#pipette
p20 = protocol.load_instrument('p20_single_gen2', 'right', tip_racks=[tiprack1, tiprack3])
p300 = protocol.load_instrument('p300_single_gen2', 'left', tip_racks=[tiprack2, tiprack4])

promoters = 3
genes = 3

##commands

#set block to 4 degrees
heatblock.set_temperature(4)

# set the initial temperature of the thermocycler to 4
thermocycler.set_block_temperature(4)

##pipette into plate in thermocycler

columns = [['A1', 'B1', 'C1', 'D1', 'E1', 'F1', 'G1', 'H1'], ['A2', 'B2', 'C2', 'D2', 'E2', 'F2', 'G2', 'H2'], ['A3', 'B3', 'C3', 'D3', 'E3', 'F3', 'G3', 'H3'], ['A4', 'B4', 'C4', 'D4', 'E4', 'F4', 'G4', 'H4'], ['A5', 'B5', 'C5', 'D5', 'E5', 'F5', 'G5', 'H5'], ['A6', 'B6', 'C6', 'D6', 'E6', 'F6', 'G6', 'H6'], ['A7', 'B7', 'C7', 'D7', 'E7', 'F7', 'G7', 'H7'], ['A8', 'B8', 'C8', 'D8', 'E8', 'F8', 'G8', 'H8'], ['A9', 'B9', 'C9', 'D9', 'E9', 'F9', 'G9', 'H9'], ['A10', 'B10', 'C10', 'D10', 'E10', 'F10', 'G10', 'H10'], ['A11', 'B11', 'C11', 'D11', 'E11', 'F11', 'G11', 'H11'], ['A12', 'B12', 'C12', 'D12', 'E12', 'F12', 'G12', 'H12']]

rows = [['A1', 'A2', 'A3', 'A4', 'A5', 'A6', 'A7', 'A8', 'A9', 'A10', 'A11', 'A12'], ['B1', 'B2', 'B3', 'B4', 'B5', 'B6', 'B7', 'B8', 'B9', 'B10', 'B11', 'B12'], ['C1', 'C2', 'C3', 'C4', 'C5', 'C6', 'C7', 'C8', 'C9', 'C10', 'C11', 'C12'], ['D1', 'D2', 'D3', 'D4', 'D5', 'D6', 'D7', 'D8', 'D9', 'D10', 'D11', 'D12'], ['E1', 'E2', 'E3', 'E4', 'E5', 'E6', 'E7', 'E8', 'E9', 'E10', 'E11', 'E12'], ['F1', 'F2', 'F3', 'F4', 'F5', 'F6', 'F7', 'F8', 'F9', 'F10', 'F11', 'F12'], ['G1', 'G2', 'G3', 'G4', 'G5', 'G6', 'G7', 'G8', 'G9', 'G10', 'G11', 'G12'], ['H1', 'H2', 'H3', 'H4', 'H5', 'H6', 'H7', 'H8', 'H9', 'H10', 'H11', 'H12']]

#define all destination wells
TC_all = []
for i in range(genes):
    TC_all += columns[i][:promoters]

#define CDS and vector destination wells
TC_columns = []
for i in range(genes):
    TC_columns.append(columns[i][:promoters])

#define promoter destination wells
TC_rows = []
for i in range(promoters):
    TC_rows.append(rows[i][:genes])

#pipette MasterMix into all wells
p300.distribute(
    14,
    tuberack['A1'],
    [plate2.wells_by_name()[well_name] for well_name in TC_all],
    air_gap = 0)

#pipette promoters 
for i in columns[0][:promoters]:
    for j in TC_rows[columns[0].index(i)][:]:
        p20.transfer(
            2,
            plate1.wells(i),
            plate2.wells(j),
            air_gap = 2,
            blow_out = True)

#pipette CDS
for i in columns[1][:genes]:
    for j in TC_columns[columns[1].index(i)][:]:
            p20.transfer(
            2,
            plate1.wells(i),
            plate2.wells(j),
            air_gap = 2,
            blow_out = True)

#pipette vectors - vectors go into the same wells as CDS
for i in columns[2][:genes]:
    for j in TC_columns[columns[2].index(i)][:]:
            p20.transfer(
            2,
            plate1.wells(i),
            plate2.wells(j),
            air_gap = 2,
            blow_out = True)

#run the thermocycler for assembly
thermocycler.close_lid()
thermocycler.set_lid_temperature(100)

profile = [
    {'temperature': 37, 'hold_time_seconds': 300},
{'temperature': 16, 'hold_time_seconds': 300},]

thermocycler.execute_profile(steps=profile, repetitions=35, block_max_volume=20)

#heatshock for killing enzymes 
profile = [{'temperature': 70, 'hold_time_minutes': 10}]

thermocycler.execute_profile(steps=profile, repetitions=1, block_max_volume=20)

thermocycler.set_block_temperature(4)

thermocycler.open_lid()

#while the thermocycler is running, put the aluminium block in the freezer to keep the competent cells cold

#pipette 50 uL of competent cells into each construct
for j in TC_all:
    p300.transfer(
    50,
    tuberack['A2'],
    plate2.wells(j),
    air_gap = 2,
    blow_out = True)

#heat shock step
thermocycler.close_lid()
thermocycler.set_lid_temperature(37)

profile = [
    {'temperature':42, 'hold_time_seconds':45},
    {'temperature':4, 'hold_time_minutes':1}]
thermocycler.execute_profile(steps=profile, repetitions=1, block_max_volume=60)
thermocycler.open_lid()

#recovery - add SOC into each construct+cells 
for j in TC_all:
    p300.transfer(
    50,
    tuberack['A3'],
    plate2.wells(j),
    air_gap = 2,
    blow_out = True)

thermocycler.close_lid()
thermocycler.set_lid_temperature(37)

profile = [
    {'temperature': 37, 'hold_time_minutes': 60},]
thermocycler.execute_profile(steps=profile, repetitions=1, block_max_volume=60)
thermocycler.open_lid()

#plate cells out of opentrons onto agar plate
#put agar plate into incubator overnight

for line in protocol.commands(): 
    print(line)