TITLE: Loop and uLoop assembly
AUTHORS: Bernardo Pollak

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is used for performing Type IIS assembly by either </span><span style = "font-style:italic;">BsaI</span><span> or </span><span style = "font-style:italic;">SapI</span><span>-mediated restriction/ligation using Loop assembly with either Loop or uLoop plasmids.</span></div><div class = "text-block"><span>Loop assembly comprises 8 receiver plasmids in odd and even levels (4 per level), which contain directional overhangs for higher level assembly. Odd and even level receiver plasmids are used in a recursive schema for assembly in iterative 'loops'. Any assembly (except for L0 part composition into a L1 transcriptional unit) requires the usage of 4 donor plasmids (each in a specific position) and one receiver plasmid. </span><span style = "font-style:italic;">BsaI</span><span> is used for assembly into odd levels using kanamycin selection, and </span><span style = "font-style:italic;">SapI</span><span> is used in even levels using spectynomycin selection. Loop odd receivers use the Common Syntax standard for L0 part assembly into TUs (Patron, </span><span style = "font-style:italic;">et al.</span><span> 2015 [10.1111/nph.13532]). Then, 4 L1 plasmids (positions 1-4) are assembled into an even receiver plasmid to provide a L2 assembly (4 TUs). For higher level assemblies the same assembly structure is followed, 4 L2 plasmids (positions 1-4) with an odd receiver to yield a L3 assembly (16 TUs).</span></div><div class = "text-block">The Loop type IIS assembly protocol is based on: </div><div class = "text-block">“Patron, NJ (2016) DNA Assembly for Plant Biology. Current Protocols in Plant Biology 1:1-13 [doi: 10.1002/cppb.20038]”, but with slight modifications for DNA concentrations:</div><div class = "text-block"><span style = "font-weight:bold;">This protocol uses a target amount of each donor DNA of 15 fmol and 7.5 fmol for receiver plasmid DNA in a 10 µL reaction. In order to perform accurate dispensing, the plasmid DNA is diluted to their corresponding concentration of 15 fmol/µL for donor parts and of 7.5 fmol/µL for the receiver plasmid, and then 1 µL of each plasmid is added to the DNA mix. The plasmid part target concentration (in ng/uL) equals to each donor plasmid length / 100, and of the receiver plasmid length / 200. </span><span></span></div><div class = "text-block"><span style = "font-weight:bold;">Volumes CAN be reduced by half, but expect more variability from aliquoting.</span></div></div>

[STEPS]
?. [Calculate plasmid target concentrations ]
The required concentration for each donor part in ng/μL is plasmid length / 100. That will yield a concentration of 15 fmol/µL. For the receiver plasmid the target concentration is length / 200, yielding 7.5 fmol/µL.
?. [Prepare DNA mixture]
Add into a PCR tube:1 µL of each donor part at 15 fmol/µL.1 µL of receiver plasmid at 7.5 fmol/µL.Add water up to 5 µL (assemblies from L1 and above do not need adjustment since 5 plasmids are used)
?. [Master mixes]
Odd and even level assemblies use different formulations due to specific requirements of the restriction enzymes used.For odd levels (BsaI or BsaI-HFv2), 10x T4 DNA ligase buffer is used.For even levels (SapI), 10x Cutsmart buffer is used mixed with 10x T4 DNA ligase buffer in a 1:1 ratio.For BSA (bovine serum albumin), check stock concentration. NEB 'molecular biology grade BSA' comes at 20 mg/mL, depreciated NEB purified BSA comes at 10 mg/mL. Prepare 20 µl of 1 mg/mL diluted BSA in water solution.Thaw buffers at RT, then place on ice.IMPORTANT:Do not use BsaI-HF, that enzyme will NOT work with this reaction. Use either BsaI or BsaI-HFv2.Use aliquoted volumes of T4 Buffer since ATP and DTT will degrade over short amounts of time (less than a month) affecting  the efficiency of the reaction.If low efficiency is seen, use double the restriction enzyme volumes.
?. [Prepare reaction master mix]
Calculate the number of reactions to be performed and include an extra reaction to account for pipetting errors.Per reaction: Odd level BsaI-Ligase 2x master mix:  ABC1ReagentConcentrationVolume (µL)2HPLC H20&nbsp;33T4 DNA ligase buffer10X14Diluted BSA (Bovine Serum Albumin)1 mg/mL0.55T4 DNA ligase400 U/µL0.256BsaI (or BsaI-HFv2)10 U/µL0.25Even level SapI-Ligase 2x master mix:  ABC1ReagentConcentrationVolume (µL)2HPLC H20&nbsp;3.53Cutsmart buffer10X0.54T4 DNA ligase buffer10X0.55T4 DNA ligase400 u/µL0.256SapI10 U/µL0.25
Mix by pipetting (enzymes in glycerol will be in the bottom of the tube). Then pipette from the top for accurate dispensing.
ABC1ReagentConcentrationVolume (µL)2HPLC H20&nbsp;33T4 DNA ligase buffer10X14Diluted BSA (Bovine Serum Albumin)1 mg/mL0.55T4 DNA ligase400 U/µL0.256BsaI (or BsaI-HFv2)10 U/µL0.25
ABC1ReagentConcentrationVolume (µL)2HPLC H20&nbsp;3.53Cutsmart buffer10X0.54T4 DNA ligase buffer10X0.55T4 DNA ligase400 u/µL0.256SapI10 U/µL0.25
?. [Mix DNA and reaction master mix]
Add the 5 µL of the corresponding reaction master mix to the 5 µL of DNA mix and mix thoroughly by pipetting.
?. [Incubate reaction]
Place the reaction on a thermocycler (PCR machine). And run the following program:Step 1 - 37 ºC for 3 minStep 2 - 16 ºC for 4 minRepeat 25x step 1 and 2 Step 3 - 50 ºC - 5 minStep 4 - 80 ºC - 10 minNote: number of cycles and incubation times can be adjusted but the reaction efficiency may decrease.
?. [Transform and plate]
Use 1 µL of reaction to transform chemically competent cells.Recover in SOC for 1 hour incubating at 37 ºC in a shaking incubator.Prepare plates with respective antibiotics with 50 ug/mL of X-GAL and 1 mM IPTG.
?. [Pick colony grow overnight and validate]
Pick 2 white colonies for each construction and grow in 10 mL of LB + respective antibiotic and perform plasmid purification. Validate assembly by means of restriction digest. Suggested enzymes: ThermoFischer XbaI and PstI FastDigest enzymes. If higher concentrations are required, increase inoculation volume and perform Midi prep as directed by manufacturer's instructions.