TITLE: Loop assembly using Labcyte Echo 550
AUTHORS: Susana Sauret-Gueto, Anthony West

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Loop L1 and L2 type IIS assembly protocol, automated and minituarised for assembly of 500nl final volume reactions using the Labcyte Echo 550.</div></div>

[STEPS]
?. Next create LDV source plate that contain all plasmids needed but the APs.For L1 Loops, have a LDV source plate with the libary of L0 parts.For L2 Loops, have a LDV source plate with the library of L1 plasmids.Use Echo calibration 384LDV_AQ_B2 for these plates.LDV plates working range is~ 3-12 ul (maximum volume is 12 ul and dead volume is 3 ul).Thus, fill in the wells wtih 10 ul of a given plasmid.Source plasmids final concentration is 15 nM. For a final concentration of 15 nM, the concentration in [ng/ul]  equals N (the length in bp of the plasmid) divided by 110. This is an approximation of the formula:15∙10^(-9)mol/L x ((607.4 x N ) + 157.9)g/mol x 10^(-6)L/μL x 10^9ng/g = concentration (ng/μL)This plate can be sealed and kept at -20C, for reuse in the future as many times as necessary, and empty wells can be filled in with new plasmids.Keep a csv files with the LDV plate source well and sample name and update as you fill in new positions.
?. Next create PP source plate with the APs and MM and water.For L1 Loops, have a PP source plate with the pCk APs and L1-MM-BsaI.For L2 Loops, have a PP source plate with the pCs APs and L2-MM-SapI.Use Echo calibration 384PP_AQ_BP2 for these plates.PP plates working range is ~ 15-65 ul (maximum volume is 65 ul and dead volume is 15 ul).Thus, fill in the wells wtih ~30-50 ul of a given plasmid.MM contains enzymes, BSA and glycerol, and has a higher tendency to creep up the walls of the well than water, thus the dead volume is higher, at around 18 ul (always check the dead volume for your MM). The dead volume is the volume at which the transducer of the Echo can not detect the top of the meniscus anymore and thus can not focus to transfer droplets by accoustic energy.Prepare a source 384 PP plate with APs (pCks, pCs) at a final concentration of 7.5 nM and Loop plasmids with spacers (pCk1-spacer, pCk2-spacer,....) at a final concentration of 15 nM. Use two wells in this plate for water and for the master mix (MM).This plate can sealed, kept at -20C, and reused, and every time a new Loop-Echo assembly needs doing, choose a new pair of wells for water and MM. Alternatively have two different PP plates, one for the APs, and one for the water and MM.Keep a csv files with the PP plate source well and sample name and update as you fill in new positions.
?. Before using the Echo to do Loop assemblies, you need a list of all Loop reactions that needs doing, and all plasmids needed. Thus, make a csv files with all Loops that need doing. Separate Loop1 from Loop2. Examples:  ABCDEFG1L1 constructAccpetor plasmid (AP)L0 plasmid in position 1L0 plasmid in position 2L0 plasmid in position 3L0 plasmid in position 4L0 plasmid in position 52L1_001-Ck1pCk1PROM5_aCDS_a3TERM_a3L1_002-Ck2pCk2PROM5_aCDS12_bCTAG_b3TERM_a4L1_003-Ck2pCk2PROM5_bCDS12_bCTAG_b3TERM_a5L1_004-Ck2pCk2PROM_c5UTR_cCDS12_bCTAG_b3TERM_a  ABCDEF1L2 constructAccpetor plasmid (AP)L1 plasmid in position 1L1 plasmid in position 2L1 plasmid in position 3L1 plasmid in position 42L2_001-CsApCsAL1_001-Ck1L1_002-Ck2L1_005-Ck3L1_006-Ck43L2_002-CsApCsAL1_001-Ck1L1_003-Ck2L1_005-Ck3L1_006-Ck4
ABCDEFG1L1 constructAccpetor plasmid (AP)L0 plasmid in position 1L0 plasmid in position 2L0 plasmid in position 3L0 plasmid in position 4L0 plasmid in position 52L1_001-Ck1pCk1PROM5_aCDS_a3TERM_a3L1_002-Ck2pCk2PROM5_aCDS12_bCTAG_b3TERM_a4L1_003-Ck2pCk2PROM5_bCDS12_bCTAG_b3TERM_a5L1_004-Ck2pCk2PROM_c5UTR_cCDS12_bCTAG_b3TERM_a
ABCDEF1L2 constructAccpetor plasmid (AP)L1 plasmid in position 1L1 plasmid in position 2L1 plasmid in position 3L1 plasmid in position 42L2_001-CsApCsAL1_001-Ck1L1_002-Ck2L1_005-Ck3L1_006-Ck43L2_002-CsApCsAL1_001-Ck1L1_003-Ck2L1_005-Ck3L1_006-Ck4
?. For L2 Loops, prepare MM as follows:  ABC1Volume for 1 reaction in 1 well in a 384 PCR plateVolume for 96 reactions in a 384 PCR plate2Water100 nl9.6 ul310x Tango buffer (Thermo Fisher)50 nl4.8 ul4  1 mg/mL bovine serum albumin (NEB)25 nl2.4 ul510mM ATP (SIGMA)50 nl4.8 ul6T4 DNA ligase (5 U/µL) (Thermo Fisher)12.5 nl1.2 ul7SapI (LguI) (5 U/µL) (Thermo Fisher)12.5 nl1.2 ul8Final volume250 nl24 ulTo prepare the MM always consider the volume you need for the plate reactions plus the dead volume of the MM solution in a PP plate.Always prepare the MM in an eppendorf tube first, mix by pipetting and then transfer the MM to the PP-well. This is to ensure all components of the MM are well mixed.
ABC1Volume for 1 reaction in 1 well in a 384 PCR plateVolume for 96 reactions in a 384 PCR plate2Water100 nl9.6 ul310x Tango buffer (Thermo Fisher)50 nl4.8 ul4  1 mg/mL bovine serum albumin (NEB)25 nl2.4 ul510mM ATP (SIGMA)50 nl4.8 ul6T4 DNA ligase (5 U/µL) (Thermo Fisher)12.5 nl1.2 ul7SapI (LguI) (5 U/µL) (Thermo Fisher)12.5 nl1.2 ul8Final volume250 nl24 ul
?. Final volume of L2-Loop reactions at the Echo is 500 nl, with 250 ul of MM (see next step on preparing MM) and 250 ul of plasmids (50 ul of each one of the 5 plasmids), as follows:  AB1PlasmidVolume2pCk250 nl3L1_001-Ck150 nl4L1_002-Ck250 nl5L1_004-Ck350 nl6L1_005-Ck350 nl7L2_MM-SapI250 nl8Final volume500 nl
AB1PlasmidVolume2pCk250 nl3L1_001-Ck150 nl4L1_002-Ck250 nl5L1_004-Ck350 nl6L1_005-Ck350 nl7L2_MM-SapI250 nl8Final volume500 nl
?. Final volume of L1-Loop reactions at the Echo is 500 nl. In this case, because number of plasmids needed is variable (4-7) the MM volume needs to change depending on that. Thus, if we want to do different L1-Loops with different number of plasmids, we can assume a 6 plasmids for all, and use water for the positions without a plasmid as  whown below.  ABCDEF1L1-Loop with 6 plasmidsL1-Loop with 5 plasmidsL1-Loop with 4 plasmids2pCk250 nlpCk250 nlpCk250 nl3PROM_c50 nlPROM5_b50 nlPROM5_a50 nl45UTR_c50 nlCDS12_b50 nlCDS_a50 nl5CDS12_b50 nlCTAG_b50 nl3TERM_a50 nl6CTAG_b50 nl3TERM_a50 nlwater100 nl73TERM_a50 nlwater50 nl8L1_MM6-BsaI200 nlL1_MM6-BsaI200 nlL1_MM6-BsaI200 nl9Final volume500 nlFinal volume500 nlFinal volume500 nl10
ABCDEF1L1-Loop with 6 plasmidsL1-Loop with 5 plasmidsL1-Loop with 4 plasmids2pCk250 nlpCk250 nlpCk250 nl3PROM_c50 nlPROM5_b50 nlPROM5_a50 nl45UTR_c50 nlCDS12_b50 nlCDS_a50 nl5CDS12_b50 nlCTAG_b50 nl3TERM_a50 nl6CTAG_b50 nl3TERM_a50 nlwater100 nl73TERM_a50 nlwater50 nl8L1_MM6-BsaI200 nlL1_MM6-BsaI200 nlL1_MM6-BsaI200 nl9Final volume500 nlFinal volume500 nlFinal volume500 nl10
?. For L1 Loops, prepare MM for maximum of 6 parts as follows:  ABC1Volume for 1 reaction in 1 well in a 384 PCR plateVolume for 96 reactions in a 384 PCR plate2Water100 nl9.6 ul310x T4  ligase buffer (NEB)50 nl4.8 ul41 mg/mL bovine serum albumin (NEB)25 nl2.4 ul5T4 DNA ligase at 400 U/μL (NEB)12.5 nl1.2 ul610 U/μL BsaI (NEB)12.5 nl1.2 ul7Final volume200 nl19.2 ulTo prepare the MM always consider the volume you need for the plate reactions plus the dead volume of the MM solution in a PP plate.Always prepare the MM in an eppendorf tube first, mix by pipetting and then transfer the MM to the PP-well. This is to ensure all components of the MM are well mixed.
ABC1Volume for 1 reaction in 1 well in a 384 PCR plateVolume for 96 reactions in a 384 PCR plate2Water100 nl9.6 ul310x T4  ligase buffer (NEB)50 nl4.8 ul41 mg/mL bovine serum albumin (NEB)25 nl2.4 ul5T4 DNA ligase at 400 U/μL (NEB)12.5 nl1.2 ul610 U/μL BsaI (NEB)12.5 nl1.2 ul7Final volume200 nl19.2 ul
?. Generate a csv file to upload in the Labcyte cherry pick software with the following information:  ABCDEFGH1Source Plate NameSource WellDestination WellSample NameTransfer VolumeSource Plate TypeDestination Plate NameDestination Plate TypeGeneration of the csv files can be done as more convinient,  with an excel template or a phyton script, or ...Previous to running and Echo experiment, make sure the information in the csv file is correct and the Echo is running what you intend to run. You can simulate the running at the Echo.
ABCDEFGH1Source Plate NameSource WellDestination WellSample NameTransfer VolumeSource Plate TypeDestination Plate NameDestination Plate Type
?. Loop assembly using the Echo:Load csv file in the cherry pick.Get LDV and PP source plates:LDV plate:  defrost, spin down and keept at RT.PP plate: defrost, spin down, keep at RT. Prepare MM, spin and keep at RT.Get the destination plates: skirted 384-PCR plates Run the L1 or L2 Loop assemblies at the Echo.If there are any exceptons, proceed to re-run those dispensations.Seal the 384-PCR plate with a PCR foil seal and spin it down.
?. Place the  384-PCR plates at the termocycler and run the follwoing cycling conditions:Assembly: 26 cycles of  for  and for . Termination and enzyme denaturation:  for  and for .
37 °C
16 °C
50 °C
80 °C
?. Transform the Loop assemblies with highly competent cells (10^8) as follows:Note: we use homemade Top10 HS higly competent cells.But any other ones higly competent will work as well.Add 2 ul of cells to each wellIncubate  on iceHeat shock at  for  in a thermocyclerIncubate  on iceAdd 15 u of LB to each wellSeal the plate with a breath-easy sealing membraneIncubate the plate at on a shaker for Plate(*) the ~ 18ul fo the reaction in LB agar plates with with either:for L1 kanamycin 50 μg/mL and X-gal  40 ng/ mLfor L2 spectinomycin 100 μg/mL and X-gal  40 ng/ mLIncubate plates  at All white colonies have an insert inside the AP vector. The efficiency of the assemnmbly is really high, and we normally just check 2 colonies.(*) Alternatively, one can use the Echo to tranfer the transformed cells onto a plate. It needs transfering of the transformation to a PP plate and spotting with the Echo onto an omnitray.
42 °C
37 °C
[O/N]
37 °C