Team:INSA-Lyon/Project/Stage2/Strategy

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.