Team:UNSW Australia/Parts

The parts we've submitted to the registry are all about OMVs, whether for stimulation of production, or tailoring of function. See Table 1 below for a full list, but beneath that we'll discuss them in more detail

Table 1: All parts submitted to the registry


Parts for hypervesiculation

Hypervesiculation is a state in which a cell (e.g. E. coli) is overproducing OMVs. In the literature, a wide variety of proteins and peptides have been shown induce hypervesiculation, and part of our project was to take a curated selection of these and characterise them as best we could. These are: BBa_K2022000 (henceforth referred to as "g3p"), and BBa_K2022002 ("tolR"), for which we also made respective His-tagged versions in BBa_K2022001 and BBa_K2022003.

We also began work on a part called alMGS (BBa_2022004), which is known to catalyse the insertion of lipids into the inner-membrane of E. coli. Doing so causes membrane expansion, and, in turn, stress; this stress is released by the blebbing off of vesicles, that form in the cytoplasm. There was evidence to suggest that alMGS can localise to the outer-membrane (Ge et al., 2013), and so we were eager to see if it could help OMV formation. We didn't get too far on this part, and encourage future teams to make it RFC10 compatible, and test to see if it helps make inner- or outer-membrane vesicles!

Fluorescent Parts

One way to better visualise OMVs would be to package them with fluorescent proteins. Typically, though, classic reporters like eGFP (BBa_E0040) or RFP (BBa_E1010) fill the entire cell; our intention was to modify these proteins to bias them towards OMVs, and, in doing so, make the job of visualing tiny OMVs easier by increasing their fluorecent signal relative to that from cells. We achieved this through the use of tags, fusing them to the front of fluorescent proteins

The first tag we used was intended to direct our fluorescent proteins to the periplasm - the space between the two outer membranes of gram-negative bacteria. We used the pelB leader tag (Lei et al., 1987), which guides any protein to which it is tag into the sec translocation pathway.

A second tag we tried was INPNC (see part BBa_K811005), which acts as a membrane anchor.

Contributions to Existing Registry Parts

As we harp on about a lot, one of our project's key aims was to provide a platform upon which future teams could build, in particular for synthetic biologists who would want to use OMVs. That's what the registry is for - so that no project starts from scratch, with uncharacterised tools and ill-optimised protocols. We decided to use the registry to its full extent by, where possible, using and modifying existing parts, and adding to the characterisation there. Below we list all our contributions:

tolR: Previously, Paris 2009 had submitted a domain of the E. coli protein TolR to registry as BBa_K257005. We intended to use part, only to find that it lacked a start codon, as well as a second stop codon (a common feature to better terminate translation). The big issue, then, is that for this part to be expressed it needs an external start codon, which is often fine if being cloned into an expression vector. However, commonly 3A assembly doesn't add a start codon, as coding parts tend to begin with an ATG. To thus make this part more usable in 3A assembly, we submitted a new construct to the registry (BBa_K2022002) with the above additions, and went so far as to characterise it and confirms its role in the cell! All the above is also documented on Paris 2009's part page

INPNC: We fused this part (BBa_K811005) to GFP and RFP, which can help to cheaply visualise its efficacy as a membrane-anchor.

pelB Leader Sequence: This part (BBa_J32015) is a tag that can be affixed to the N-terminus of a protein, to guide the fusion product to the periplasm of E. coli. We fused it to RFP and GFP to guide fluorescence to that region

Green- and Red-Fluorescent Protein: We expanded the capabilities of these parts (BBa_E0040 and BBa_E1010, respectively) by fusing to their N-termini either a pelB leader (for periplasm-localisation), or INPNC (see above)