Part:BBa_K1112000

fadB promoter + flp-21 iRNA

Description

This composite part consists of the antisense sequence of flp-21 transcript of C.elegans, placed under the control of the fatty acid- sensitive promoter fadB of E. coli.

Usage and Biology

The biobrick promoter mentioned above (fadBp) is induced by fatty acids, but not all the fatty acids are able to induce the same level expression (Figure 1). Thus, as can be seen in Figure 1, the oleic acid produce the highest activation of the promoter.

Figure 1. Effect of chain length upon induction. Strain DC530 0 (fadB-lacZ) was grown in minimal medium with acetate as the carbon source and with various fatty acids present at 2 mM. The induction ratio is the β-galactosidase activity, relative to the control culture containing no fatty acid. Fatty acids are designated by “n:k”, where “n” is the number of carbon atoms and “k” is the number of double bonds.

OLE, Oleate; CVC, cis-vaccenate; 16:1, palmitelaidate.

Adaptation from Clark's paper figure, 1981.

So, what's the role of the flp21 iRNA? This iRNA regulates the behaviour of Caenorhabditis elegans, specifically social behaviour when they feed, also known as “clumping" (Figure 2).

Figure 2. Social and solitary behaviour of the nematode C. elegans ilustrated with the molecular mechanim used in our project: iRNA mediated.

Universality

BBa_K1112000 can be used in any microbial species that may constitute the nematode nourishment: yeasts as S. cerevisiae, different bacterial genus(despite of E. coli is the main bacteria for C. elegans feeding in the laboratory, it is not the only one that the nematode is able to digest) as Bifidobacterium (Komura et al., 2013), Burkholderia (Cooper et al., 2009), Bacillus (Abada et al., 2009; Bichai et al., 2009; Shtonda et al., 2006), Cellulomonas, Corynebacterium, Erwynia, Pseudomonas, Mycoplana, Variovorax (Abada et al., 2009) , etc.

Some considerations about the biobrick:

- The fadB promoter, sensitive to fatty acids, owns to Escherichia coli. This fact ensures its correct operation in a group of closed related species but not to the wide range of microorganism. Moreover, according to the sustrate of interest (we used fatty acids for PHA production but this detection and bacteria concentration system is not limited to this process but can be used ofr any activity of interest such as the degradation of recalcitrant compounds), we can change the fadB promoter to any other sensitive to hotspots composition.

- Also be taken into account the limitations that the plasmid, where the construction is cloned, imposed. I mean, the backbones Ori is not universal, so it would be necessary to check that if Ori is compatible with the organism we want to transform.

Compatibility with current standards and systems (Modularity)

Being a small size (200 nt aprox) non-coding RNA the problem of toxicity due to the synthesis and subsequent translation: inclusion bodies formation, impact on cell structures, cellular process interference, etc., does not even exist.

The cell consumption is also minimum compared to other processes where proteic products have to been synthesized. This allows a development of the cell nearly to the optimal one.

Innovative point

The present BioBrick represent the first one that enables modify the behaviour of a higher eukariotic organism in a directed way.

Figure 3. Clumping SEM picture. Narrow marks the presence of a large worm surrounded by small ones. By the way, the zoomed part shows a bacterial lawn where clumping occurs (concentration effect).