uLoop

Domestication

This refers to the process of making DNA sequences usable with Type IIS assembly systems. The requirements are:
(1) that the sequence does not contain sites for the Type IIS restriction enzymes used in the assembly system, since this would interfere with the reaction. For Loop and uLoop. BsaI and SapI sites.
(2), that flanking overhangs are added to the sequence to make it a standard part. These flanking overhangs have been predefined to provide further functionality.

Syntax

Loop & uLoop follow the common syntax for standardised DNA components used in Type IIS assembly systems proposed in the plant field. This ensures that standard parts are interoperable with other Type IIS assembly systems as MoClo and GoldenBraid, given that no SapI sites are present.
The "common syntax" defines 12 overhangs for flanking subgenetic DNA sequences in accordance to a prototypical eukaryotic transcription unit.

Common Syntax


For uLoop we will be using the following specification according to the common syntax:

Common Syntax

Recode2S

To help with the domestication of sequences, we have developed Recode2S, an application to simplify searching for restriction sites and adding overhangs. Recode2S is also frame-aware, in order to avoid changing codons by selecting silent mutations if the site is not in the reading-frame of the CDS.
Important The output of Recode2S shows how your sequence should look like once domesticated as a L0 standard part. From this output, we will use the region between (and including) the flanking overhangs without the BsaI sites.
If the sequence does not contain internal BsaI and SapI sites continue to the next section. Otherwise, read through the rest of the document and go to the last section.

Domestication vector pL0R-lacZ

We have created a entry vector that enables domestication through overlap-dependent assembly as well as SapI-mediated restriction ligation. The pL0R-lacZ vector contains a lacZ negative selection marker, a spectynomycin resistance cassette and restriction sites for BsaI and SapI. SapI restriction releases lacZ for entry of domesticated sequences while at the same time reconstituting BsaI sites. Then, cloned sequences can be used in a L0 to L1 assembly. The vector contains unique nucleotide sequences (UNS) for sequencing and cPCR verification.

Domestication vector

To domesticate DNA sequences through SapI-mediated restriction/ligation, flanking sequences have to included in the DNA insert in order to enable SapI restriction, overhangs for reconstitution of the BsaI site in the L0 receiver and overhangs according to the common syntax. A specific set of 3 bp overhangs exposed by SapI digestion been designed for efficient assembly into the L0 receiver which contain distinct 5’ and 3’ overhangs. These 3 bp overhangs will reconstitute the BsaI site that are exposed in the receiver when the L0 receiver is digested by SapI along with the N nucleotide spacer between the BsaI restriction site and the common syntax overhangs.

SapI is shown in boldface, 3 bp overhangs for cloning into the pL0R-lacZ entry vector are underlined, NNNN corresponds to the 4 bp common syntax overhangs and XXX corresponds to the DNA module.

Domestication mechanism

If your sequence does not require removing BsaI and SapI sites, then add AAGCTCTTCATCC at the 5' end, and CGAAGAAGAGCAA at the 3' end through PCR. Domesticate design

Domesticating internal BsaI and SapI sites

Sites must be removed through mutation (PCR) or by synthesis (gBlocks). If few sites are present (2 or less), then PCR might be a suitable way of removing these sites unless the sequence contains repetitive sequences or low-complexity regions.

For each site present, the sequence will be split in that section to change a single nucleotide. Several techniques can be used to eliminate the sites (overlap-extension PCR, Gibson assembly, homologous recombination), but in this case we will use SapI-mediated directional assembly. To achieve this, every section will include flanking SapI sites included by PCR as in the previous section, however the main challenge will be to find unique overhangs that will enable the reaction to assemble the sections in the correct order. Since we will be using SapI, overhangs are 3 bp and they cannot be too similar (hopefully 2 or more dissimilar bases), be of low-complexity or either TCC, CGA, TCG or GGA. It does not matter, however, if the mutated restriction site base pair is any position of the overhang.

Domesticate multiple In this case, the primers used to create the SapI sites that would allow SapI to cut directly into the junction site. This is accomplished by adding AAGCTCTTCA to the 5' of the oligonucleotide (blue). The flanking oligonucleotides (black) still contain same sequence as described previously.