INSA LYON TEAM First ¡par)cipa)on ¡in ¡iGEM ¡
THE ¡ PROJECT ¡
Hey Doc, what’s the problem? THE E DR DROPPY Y PROJEC JECT: TWO CHALLEN ENGES ES… … ONE E SOLUTION Larg Large e scale scale lipid lipid producti tion Larg Large e scale scale prote tein purificati tion Unsoluble molecules Multistep process with expensive materials Hard to store for a cell (affinity columns) No convenient biological system How to to pu purif rify to produce lipids prote tein in in a a ch cheaper eaper & faste & ter wa way? ? How to to produ produce ce lipids lipids in E. E.coli?
The te team asks asks f for or mo molecul ular cu cuis isin ine e ch chef ef’s ’s adv advice ice (VIDEO DEO 1). « I am a very famous french molecular cuisine chef. When I have to deal with protein purification for my dishes, I prefer to use PolyHydroxyalcanoate granules as self-cleaving micro-beads, instead of expensive affinity columns. PHA are common prokaryotic storage molecules. Their lipidic nature makes them hard to store in a free state in the cell. Thus, a large range of bacteria developped PHAs storage structure known as granule. Granules are not only lipids, various proteins associated with the granules are involved in its synthesis and its regulation. To my knowledge, Escherichia coli does not naturally develop granules which raised the question of its ability to make them from recombinant genes. But, in 1988 Steven Slater and collaborators managed to clone genes responsible for Polyhydroxybutyrate synthesis from Ralstonia eutropha in E. coli . They also observed that E. coli stored PHB in granules. Earlier in the year, I made synthesized a plasmid similar to Slater’s one. I can give it to you. If you analyse it, you will find how to produce granules. It would be the first step in your production of self- cleaving micro-beads. »
pILI1: a synthetic plasmid which makes E.Coli produce granule ¡Genes: ¡ ¡ ¡ phaCAB ¡operon ¡from ¡ Ralstonia ¡eutropha ¡ ¡ • ¡PhaA, ¡B ¡and ¡C: ¡3 ¡enzymes ¡involved ¡in ¡PHB ¡ • synthesis ¡in ¡ Ralstonia ¡eutropha ¡ ¡ ¡T ranscrip=on ¡ under ¡the ¡control ¡of ¡its ¡na=ve ¡ • promoter ¡responsive ¡to ¡glucose ¡ PHB ¡biosynthesis ¡pathway ¡ piLI 1 8,4kb
pILI1: a synthetic plasmid which makes E.Coli produce granule Test on plates with nile red dye Test by microscopy with nile red dye + 7% glucose Without glucose Visible light Fluorescence Observation of stained lipid droplets in presence of glucose Cloning of Cloning of ph phaC aCAB in in a a plas plasmid id allow allows us to to produ produce ce PHB PHBs granul gr nules s in n E. E.coli
PhaCAB : new parts more adapted for iGEM community One part sent to registry: PhaC Two parts under construction : Pha A and B Improvements ts to to existi ting parts ts (Pha PhaC): ): • phaC gene from R. eutropha : an organism closer to E.coli, a sequenced part, a functional part because it allows the production of granules in pILI1 transformed bacteria. How did we design it? t? • Use of silent mutations to remove iGEM restriction sites in R. eutropha phaC gene. Design of phaA and B is easier: no iGEM restriction site within the sequence. (PCR derived biobricks)
The te team asks asks f for an or an anoth ther ch chef ef’s ’s adv advice ice (VIDEO DEO 2). « How to associate proteins to the surface of a granule ? hum…. I usually use a recipe designed by Mr Banki . Phasins are proteins that insert themselves into the granule to regulate its size. Mr Banki used this feature to create an engineered protein made up of two phasins and one intein fused to a protein of interest. Both phasins allow a strong association of the protein to the granule. Then a Switch of pH to an acide value induces the cleavage of the intein sequence and the release of the protein of interest from the granule… It’s purification. If you manage to express a phasin-phasin- intein fusion in the same cell as PHB granules, you should obtain functional self-cleaving micro-beads. »
The Phasin-Phasin-Intein fusion protein Prefix with strong RBS pH Silver fusion suffix for E.coli Molecule of interest + Phasin ¡ Phasin ¡ Intein ¡ Silver fusion prefix Intergenic region, which facilitates a correct folding of the fusion protein.
The te team asks asks th the chef for a last t adv advice ice (VIDEO DEO 3). « You have to produce the granule before the expression of the phasin-phasin-intein fusion. » « But how ? » « I’m just a cooker. But like every other cooker, when I am preparing my dishes, I change temperature of the oven or the speed of my shaking. So, let’s see what you can do with this.»
The natural curli promoter in E.Coli Temperature Intergenic ¡region ¡with ¡ Shaking Speed Transcrip=on ¡Factor ¡Boxes ¡ csgD Curli promoter + Osmotic Pressure CsgD CsgD OmpR an activator of csgD transcription • ompR234 mutation increases the CsgD production •
The Curli promoter: our design In ¡E.Coli ¡ csgD genome ¡ Acti tivati tion of transcripti tr tion CsgD CsgD + ompR234* Our parts Curli promoter The part we submitted is the mutated ompR gene The part we submitted only contains the csgD regulatory region. Experiments have been done with the complete intergenic region.
The curli promoter: Experiment 1 Sensitive to Temperature The ¡ac=va=on ¡of ¡curli ¡promoter ¡is ¡op=mal ¡at ¡ 28°Celsius . ¡
The curli promoter: Experiment 2 Sensitive to Osmotic Pressure The ¡ac=va=on ¡of ¡curli ¡promoter ¡is ¡op=mal ¡at ¡ low ¡osmolarity . ¡
The curli promoter: Experiment 3 Sensitive to Shaking Speed The ¡ac=va=on ¡of ¡curli ¡promoter ¡is ¡op=mal ¡at ¡100 ¡rpm. ¡
Thermoregulation 28°C: • Curli promoter is ON Production of PHB granules • RNAth is OFF. Temperatu ture Switc tch 37°C: • Curli promoter is OFF • RNAth is ON : Translation of molecules of interest.
Conclusion We succeed in producing PHB granules in E.Coli We designed new parts and sent it to the registry Sequences have been confirmed by alignement. Design and origin have been documented in the Registry.
Achievements in the manufacturing area A new useful silver-fusion part, phasin-phasin-inte tein: : • quick purification of proteins well documented in the literature A new promoter, cu curli rli: • efficient response to different parameters parameters easy to control in industrial production more common: temperature uncommon: shaking speed
Further directions (1) Further characterisation of our parts • Prove the economic advantage of the • purification strategy Study the addressing of other lipids in granules •
Further directions (2) FAS enzymes conserved domains:
iGEM team collaborations: Team Team Wars Warsaw aw: we are the team with the most answers to their survey ! Team Ed Edinburgh: we have answered to their survey. Team MET ETU-Turkey: we have answered to their survey. Team Team Hon Hong-Kon ong CUHK CUHK: we have answered to their survey. Aknowledgement: Biosciences Department of INSA Lyon, Sandrine, UMR5240 CNRS, Véro, Jean- Michel, Corinne, Sylvie, Agnès, Joël Kuiper, David W. Wood, Bruno, Adrien
THANK YOU !!
Recommend
More recommend
