Part:BBa_K4390031

L2NC Silica Tag (N-terminal)

This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard which is also accepted by iGEM.

This is a Level 0 part of type N part generating the following 4 base overhangs at upstream (AATG) and downstream (AGCC) ends.

L2NC is a silica tag, attach this to a protein to immobilise on silica beads.

Usage and Biology

L2NC is a 130 amino acids peptide, a truncated version of the L2 ribosomal protein from E. coli (N-terminal 1-60 and C-terminal 203-273 amino acids of L2), which can fuse to N-terminal of a functional enzyme using JUMP assembly. This tag contains just the N and C-terminal regions of L2 which were shown to have silica binding capacity, allowing the use of a smaller tag without compromising on binding affinity. From literature, the dissociation constant between L2NC silica tag and silica beads is 1.7nM. Therefore, this tag facilitates immobilisation to silica surfaces, enabling enzyme immobilisation or purification using silica-based spin columns (Kim et al., 2020). The N-terminal L2NC silica tag is inspired by 2021 Edinburgh OG teams (BBa_K3946002).

Design

Inspired by iGEM 2021 Edinburgh OG, we design the L2NC silica tag which is suitable for the N-terminal tagging based on BioBrick and JUMP assembly.

Characterization

We tried to move the L2NC DNA fragment from plasmid containing C-terminal-L2NC sequence to new plasmids as N-terminal-L2NC. However, we failed to characterize this part since the primers designed by us can’t amplify the DNA from C-terminal-L2NC silica tag.

Figure 1. Agarose gel shows the PCR result of L2NC silica tag (agarose concentration 1.2%). The left lane was loaded with L2NC PCR product. The ladder used on the right lane was: 1 kb DNA Ladder from NEB (N3232S)

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 38
  
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

Kim S, Joo K Il, Jo BH, Cha HJ. Stability-Controllable Self-Immobilization of Carbonic Anhydrase Fused with a Silica-Binding Tag onto Diatom Biosilica for Enzymatic CO2 Capture and Utilization. ACS Appl Mater Interfaces. 2020 Jun 17;12(24):27055–63.