TITLE: Cloning of standardized L0 parts into pUAP4 for Loop type IIS assembly
AUTHORS: Eftychis Frangedakis, Susana Sauret-Gueto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for cloning of DNA L0 parts into pUAP4 using SapI type IIS assembly.</div><div class = "text-block"><span>A standarised L0 part has at its end 5' and 3' fusion sites that follow the </span><a href="https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.13532" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">common syntax</span></a><span>. </span></div><div class = "text-block">For Marchantia L0 parts, we commonly use eight positions and nine fusion sites, by combining positions A2-A3 for proximal promoter (PROMP), and B1-B2 for 5’ untranslated region (5UTR). The other types of parts are: A1 for distal promoter (PROMD), B3 for coding sequence with start codon and no stop codon (CDS1), B4 for coding sequence without start or stop codon (CDS2), B5 coding sequence without start codon and with stop codon (CTAG), B6 for 3’ untranslated region (3UTR) and C1 for transcription terminator (TERM). Parts can span multiple fusion sites, like A1-A3 for promoter (PROM), A1-B2 for promoter with 5’ UTR (PROM5), B3-B4 for coding sequence with start codon and no stop codon for N-terminal fusion with CTAG (CDS12), B3-B6 for coding sequence with start and stop codons (CDS), or B6-C1 for 3’ UTR with terminator (3TERM).</div></div>

[STEPS]
?. [Summary of primers and cloning into pUAP4]
?. [PCR amplification of DNA part]
Design primers as described in step 1 to add the SapI recognition site, a partial BsaI recognition site, and the common syntax fusion site.
?. [PCR amplification of DNA part]
L0 parts are PCR amplified from the source DNA part (e.g. plasmid DNA or genomic DNA), using a high fidelity DNA polymerase such as Phusion. Use 10 ng of DNA if the template is plasmid DNA and 100ng if the template is genomic DNA. The cycling conditions are: Denaturation at 98 °C for 30 s. 35 cycles of: denaturation at 98 °C for 10 s, annealing at primer annealing temperature for 30 s, and extension at 72 °C for 15 s/kb. Final extension at 72 °C for 10 min.
?. [PCR amplification of DNA part]
Run the PCR products on a 1.5% (w/v) agarose gel.
?. [PCR amplification of DNA part]
Gel extract the band that corresponds to the size of the amplified L0 part using a kit such as QIAquick Gel Extraction Kit.
?. [SapI assembly]
Determine L0 part and pUAP4 concentration with spectrophotometry (Nanodrop).
?. [SapI assembly]
Prepare aliquots at a concentration of 15 nM for the L0 part and of 7.5 nM for the pUAP4 acceptor vector.
?. [SapI assembly]
Set up a SapI Type IIS assembly reaction into a 0.2 mL tube according to Table:  AB1  Sterile water
  5  μL2pUAP41 μL3Amplified L0 part1 μL410x Tango buffer (Thermo Fisher)1 μL5  1 mg/mL bovine serum albumin (NEB)
  0.5  μL6  T4 DNA ligase (5 U/µL) (Thermo Fisher)
  0.25 μL7  10mM ATP (SIGMA)
  1 μL8  SapI (LguI) (5 U/µL) (Thermo Fisher)
  0.25 μL9  Final volume
  10 μL
AB1  Sterile water
  5  μL2pUAP41 μL3Amplified L0 part1 μL410x Tango buffer (Thermo Fisher)1 μL5  1 mg/mL bovine serum albumin (NEB)
  0.5  μL6  T4 DNA ligase (5 U/µL) (Thermo Fisher)
  0.25 μL7  10mM ATP (SIGMA)
  1 μL8  SapI (LguI) (5 U/µL) (Thermo Fisher)
  0.25 μL9  Final volume
  10 μL
?. [SapI assembly]
Place samples on a thermocycler and use the following program: Assembly: 26 cycles of 37​ °C for 3 min and 16 °C for 4 min. Termination and enzyme denaturation: 50 °C for 5 min and 80 °C for 10 min.
?. [SapI assembly]
Transform 20 μL of chemically competent E. coli cells (transformation efficiency of 1 × 107 transformants/µg plasmid DNA) using 2 μL of the assembly reaction and then spread on LB agar plates containing 25 μg/mL chloramphenicol and 40 μg/mL of X-gal for blue-white screening.
?. [SapI assembly]
Incubate overnight at 37 °C.
?. [SapI assembly]
Select white colonies for sequencing.
?. [SapI assembly]
Confirm the presence of the correct insert with Sanger sequencing using the primers UAP_F (CTCGAGTGCCACCTGACGTCTAAGAAAC) and UAP_R (CGAGGAAGCCTGCATAACGCGAAGTAATC) and any additional DNA part specific primers.
?. [Summary of primers and cloning into pUAP4]
Common syntax fusion sites.