Ideas

This page lists a number of ideas, primarily molecular tools, which I would like to encourage others to pursue. Our lab is not working on any of these, nor plans to anytime in the near future, but we’d be happy to discuss these further. Please steal these ideas and work on them, but note that I’m not the first person to think of these (and in some cases people have been working on these for decades, without success).

Site-specific Protease with C-terminal recognition site

Engineer a site-specific protease with specificity determining residues only on C-terminal side of the scissile bond. Why? Scarless C-terminal tagging, particularly for libraries where you want complete freedom over all residues on the N-terminal side of the scissile bond. Looking at the MEROPS db for proteases with specificity residues only on C-term side of scissile bond with 3 residues or more, we see only:

  • Papain of IgG -> Fab fragments fame
  • Leishmanolysin, but its specificity is not great (aliphatic/basic/basic)
  • a few more options if you’re willing to reduce specificity to 2 residues

Bacterial conjugation into liposomes

Bacteria have conjugation systems which facilitate the transfer of genetic material between two cells. These systems have an unknown mechanism to prevent conjugation into “dead” cells. Previous attempts to conjugate DNA from bacteria into liposomes have failed, likely due to this mechanism (which may involve membrane energetics to determine if the recipient cell is alive). Determine how to disable or bypass this system to allow conjugation into liposomes or lipid bilayers. Why? Facilitate genetic transfer from cells to in-vitro systems. Note that phages have no such mechanism and can readily inject their genome into liposomes containing the appropriate membrane protein receptor.

Triple-coding reading frame reporter gene

A nucleotide sequence which exploits codon degeneracy such that three different regulatory proteins are encoded on each of the three fully-overlapping reading frames present on the same sequence. Even a (dual-coding) two frame version would be useful. Why? Determine which frame the ribosome is translating and couple the answer to an transcriptional output. Phages often use RNA pseudoknots to cause frameshifts leading to multiple different translation products. Phage genomes often have overlapping coding sequences.