Team:INSA-Lyon/Project/Stage2/Strategy

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Strategy

Storage lipids

Many lipids are nowadays well-known to be very useful for health as DHA and EPA.
Granules are inclusion bodies of PHB and then a storage lipids drop.


We wanted to test the hypothesis: if there are overproduced lipids inside the bacteria, they would be stored inside the PHB granules.It is less demanding for the bacteria, from an energetic point of view, to store it inside the PHB granules than to create another specific inclusion body.


B-carotene (red-orange) and lycopene(red) are carotenoids pigments which are made of 40 carbons with isoprene units. Then we can observe the position of those proteins into the bacteria, thanks to the pigment coloration. Moreover, those parts are already ligate as composite parts in the registry under the control of the pBad/araC promoter which is induced by arabinose. (I0500). We use the plasmids BBa_K274220 and BBa_K274120.


Beta-carotenes and lycopenes are molecules chemically really different from the PHB.
The first are long carbon chain with isoprene units ( polyisoprenes) whereas PHB are polyesters.

Lycopene

Beta-carotene

The properties that their structure confers to the molecule could explain that carotenoids cannot enter into the granules, but if our hypothesis is borne out, we have good hope that smaller carbon chains as DHA and EPA would be stored into the granules as well.

In order to realize this experiment, we co-transformed a pili1 plasmid producing PHB granules and a carotenoid plasmid.

Purification of protein

We wanted to use the PHA granules as a mean of purification for molecules produces in E.coli.

Based on Banki 2005 we designed a self cleavable fusion protein that we aim to insert in the granules’ membrane. To achieve our goal, we used the protein phasin which is present constitutively in the original bacterial operon. The publication emphasized that the addition of a second phasin increase the rate of purification. As lipid bodies, the granules can be extracted from the bacteria quite easily with the phasins attached on it. Then to recuperate the molecule we want to purify, we added behind the phasins a self cleavable protein which breaks under pH 6.

To design the part, we chose to use the sequence of phasin already designed by the team of Utah state in 2009 (Part:BBa_K208001). And we found the sequence of the self cleaving protein delta.I-CM mini intein from mycobacterium tuberculosis described in the publication in a patent. There was no restriction site forbidden by iGEM on the sequence so we used it directly. We linked those sequences using the same intersequences than in the article. At the ends of the whole sequence, we added the fusion standart biobrick sequence.

We sent this sequence to Mr gene. Unfortunately because of the repetition of the phasin sequence, Mr gene could not syntetize the right sequence. So we have to redesign our sequence in two parts: one phasin and one phasin+ intein. Each part have fusion biobrick standart.