Promoter Format

The majority of C. elegans promoters used in research don’t draw a distinction between the cis-acting recognition sites used during the RNA transcription process and the upstream region of the transcript used during polypeptide translation, instead combining the two and labelling that the promoter. [http://worfdb.dfci.harvard.edu/promoteromedb/ Promoterome], for example, goes as far as including the start codon itself in its definition of the promoter. While this is simplistic for synthetic biology’s purposes, for the time being we have largely chosen to keep this format, although to cope with the scarring caused by ligation, we do not include the start codon in the promoter, and so typically remove the three final nucleotides of the promoter sequence.

Coding Brick Format

The coding brick, in our format, extends from the start of a synthetic Kozak sequence, past the start codon, through the coding DNA itself, and ends with the stop codon. It may contain introns. The Kozak sequence we typically use are the nucleotides GCCGCCACC placed immediately before the start codon. This improves translational efficiency, and can be altered to provide the additional flexibility of controlling expression from within the protein-coding component of a complete gene. Note: under certain circumstances in C. elegans, the UGA codon does not stop translation, but instead appends the rare amino acid selenocysteine. While this has not impeded us so far, it may be preferable to use UAA or UAG instead to be safe.

3' UTR Format

These are taken verbatim from available databases and laboratories after checking for cut sites. Mutagenesis of these sites typically has few ramifications. See the article on 3' UTRs for more information on why.

Future Project Ideas

We realize that the idea of working with an organism other than the familiar E. coli chassis is probably an challenging prospect for a lot of teams, especially when it comes to the design phase. We came up with a lot of ideas that only seemed apparent after we had done a great deal of research on the worm; many of them are foundational advances that are analogous to things that can be done currently in bacteria, and would be crucial to making C. elegans a fully-viable system. We've put the most lucid bunch of these ideas here, with the hope that it would help jump-start other teams and make their work less daunting.

"Backward compatibility" with E. coli

• Exosymbiosis. Attaching the bacteria to the outside of the worm (using lectins?) on a surface-disrupted mutant. This may require bacteria that don't emit the usual nutritive signal. The BactoBlood team went to some length to try and perfect such a bacterium, and since we're not talking about putting things in people, their work may be applicable here. An extension of this might be found in enabling the bacteria to attach or detach from the worms under certain circumstances, creating a bacteria distribution and relocation mechanism. Such a project would need to look out for the potential of covering the amphid or mouth, which some bacteria do naturally as a means of killing worms.
• Enterosymbiosis. There are cases in nature where nematodes and bacteria have learned to work more or less together, in which the bacteria reside in the worm's gut. Studying Xenorhabdus nematophilia, Photorhabdus luminescens, and the Heterorhabditidae worms and trying to create something similar between E. coli and C. elegans; also look into bus mutants.

Messaging in the worm

• Inter-worm signalling. The C. elegans excretory gland normally produces some pheromones that are used for communication between worms; so do anus and vulva. Could we engineer these organs to produce and excrete some new molecule? The potential secondary applications are extensive. We haven't been able to find much literature on these topics, but the prospect is alluring.
• Hormones. The pseudocoelom bathes most tissues, and it's believed that the worm has a number of hormones that it uses to communicate between tissues. It might be possible to transmit stuff in here of our own devising. As of yet, we don't have a strategy for receiving molecules from the pseudocoelom by a cell, but a literature search helped us find a tag that works to introduce molecules into the pseudocoelom. You can find it in our pseudocoelom article. We have not personally tested this technique.

Genetics

• Self-censorship. C. elegans supports RNAi, a technique that leverages machinery which enables complementary RNA molecules to destroy each other. RNAi is generally used to knock out genes by feeding the worm bacteria that produce the complementary RNAs, or soaking the worm in a large volume of these RNAs. Since integrating new DNA into the C. elegans chromosomes is difficult and time-consuming, it may make more sense to give the worm a plasmid that knocks out the mRNAs autonomically. To restore functionality of a protein knocked out in this way, especially a mutated version of such a protein, consider employing heavy use of synonyms (alternative codons for the same amino acid). See our article on RNAi.
• Variable UTRs. We've only provided a single 3' UTR in our kit (a synthetic modification of unc-54's 3' UTR). However, it's known that UTRs play a role in tissue specificity and expression strength by binding various factors. A better understanding of these UTRs, or even just a larger variety of them, stands to be a very useful project.
• Modular promoters. Heidelberg stole the show in 2009 by creating a detailed set of promoter elements that could be arranged to produce predictable and precise variations in strength for HeLa cells. It would be very cool if something like this could be done for worms, especially if this could also include tissue specificity. The potential for doing something similar for 3' UTRs is also alluring.

Biosynthesis

• The hindgut (intestine) of C. elegans can be targeted with expression patterns that place emphasis on the front and back regions. In combination with the intestinal export tags used for secreting proteases and other digestive enzymes, this provides an environment of approximately pH 5 in which multi-step biosynthesis could be possible.

Fundamental Tools

• One thing we were hoping to make this summer but never got around to was a multi-species plasmid: a new BioBrick backbone vector that would express a fluorescent protein in both C. elegans and E. coli. This would make it possible to eliminate the coinjection marker from the microinjection process, and guarantee that the DNA was functional in the worm if an injection were successful. In order to be grown up in E. coli, however, it would need to reuse the coding sequence for both eukaryotic and prokaryotic translation in order to save space. Implementing this might require the crafty use of introns.