Part:BBa_K1636002

Linker

This linker was designed by the iGEM TecCEM team in order to retrieve single-stranded oligonucleotides when produced as single-stranded genetic material (e.g. plasmid). The linker should be flanking said oligonucleotides, and given the design of its sequence, it is able to form a hairpin-like secondary structure that allows the usage of a restriction enzyme (MlyI from New England BIolabs) with a blunt cut thereby releasing the oligos of interest.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

The linker was cloned into plasmid pSB1C3, previously cut with EcoRI and PstI, and then a purification was performed. Several clones were selected to isolate plasmid by miniprep (figure 1A). In order to observe the integrity of the four extracted plasmids an electrophoresis was run (figure 1B). 10 microliters were loaded into each lane of plasmid, loaded with 4 microliters of buffer.

Figure 1. A) Scheme of linker cloning in pSB1C3 plasmid and B) electrophoresis with agarose gel at 0.8% to analyse plasmid extraction from different clones.

Linker cloning in pSB1C3 further verified a PCR was performed using universal primers VF2 and VR. Those primers amplify a PCR product of 338 pb when the insert is present (figure 2A). Several plasmid from 4 different clones were tested and all of them had the PCR product of aproximately 338 bp corresponding to the plasmid with insert. These PCR reactions was be observed in figure 2B.

Figure 2. Analysis of linker cloning into pSB1C3 plasmid by PCR. A) Plasmid scheme where the position of the cloned linker is shown and is flanked by the restriction sites EcoRI and PstI, also the alignment position of primers VF2 and VR is indicated. B) Four plasmids from different clones were used as templates for PCR with universal primers VF2 and V (lanes 2-5). Lane 1 contains Quick-Load® 2-Log DNA Ladder(0.1-10.0 kb), NEB.

Additionally two PCR products were selected to cut with MlyI (figure 4) in order to verify the presence of the linker. MlyI has several recognition sites inside the PCR product (figure 3 A-B) and an electrophoresis was performed in order to verify the digestion (figure 3C: 8 microliters of PCR product were loaded with 4 microliters of buffer). Several bands are expected to be observed with the complete digestion (124, 91, 56 and 67 bp). We observed a partial digestion of the PCR product only in one of the candidates, a band with less than 100 bp is observed. This band could correspond to the fragments 56 and 67 bp. This result suggests that the linker was cloned and that the design was successful, for it is functional.

Figure 3. Analysis of amplification product for MlyI restriction. A) Identification of MlyI restriction sites in product sequence. B) In silico analysis for product digestion with MlyI. C) Experimental restriction with MlyI of the products: lane 2 and 3 were loaded with 8 microliters of restricted linker candidates and 4 microliters of loading buffer. Lane 1 contains Quick-Load® 2-Log DNA Ladder(0.1-10.0 kb), NEB.