INSA LYON TEAM First par)cipa)on in iGEM THE PROJECT Hey - - PowerPoint PPT Presentation

INSA LYON TEAM First ¡par)cipa)on ¡in ¡iGEM ¡

PROJECT ¡ THE ¡

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 prote tein purificati tion Larg Large e scale scale lipid lipid producti tion

Multistep process with expensive materials (affinity columns) Unsoluble molecules Hard to store for a cell No convenient biological system to produce lipids

How to to produ produce ce lipids lipids in E. E.coli? How to to pu purif rify prote tein in in a a ch cheaper eaper & & faste ter wa way? ?

The te team asks asks f for

• r 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

• f 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 ¡

• ¡ ¡Transcrip=on ¡under ¡the ¡control ¡of ¡its ¡na=ve ¡

promoter ¡responsive ¡to ¡glucose ¡

piLI 1

8,4kb PHB ¡biosynthesis ¡pathway ¡

Cloning Cloning of

• f ph

phaC aCAB in in a a plas plasmid id allow allows us to to produ produce ce PHB PHBs gr granul nules s in n E. E.coli

+ 7% glucose Without glucose Test on plates with nile red dye Test by microscopy with nile red dye

Visible light Fluorescence Observation of stained lipid droplets in presence of glucose

pILI1: a synthetic plasmid which makes E.Coli produce granule

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

• r 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

• btain functional self-cleaving micro-beads. »

The Phasin-Phasin-Intein fusion protein

Phasin ¡ Phasin ¡ Intein ¡

Molecule of interest

Prefix with strong RBS for E.coli

Intergenic region, which facilitates a correct folding

• f the fusion protein.

pH Silver fusion suffix Silver fusion prefix +

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.»

+

CsgD CsgD

Temperature Shaking Speed csgD Curli promoter

The natural curli promoter in E.Coli

Osmotic Pressure

• an activator of csgD transcription
• mpR234 mutation increases the CsgD production

Intergenic ¡region ¡with ¡ Transcrip=on ¡Factor ¡Boxes ¡ OmpR

+

CsgD CsgD

csgD Curli promoter In ¡E.Coli ¡ genome ¡

Our parts

The Curli promoter: our design

• mpR234*

The part we submitted only contains the csgD regulatory region. Experiments have been done with the complete intergenic region. The part we submitted is the mutated ompR gene

Acti tivati tion of tr transcripti tion

The curli promoter: Experiment 1

Sensitive to Temperature

The ¡ac=va=on ¡of ¡curli ¡promoter ¡is ¡op=mal ¡at ¡28°Celsius. ¡

The ¡ac=va=on ¡of ¡curli ¡promoter ¡is ¡op=mal ¡at ¡low ¡osmolarity. ¡ The curli promoter: Experiment 2

Sensitive to Osmotic Pressure

The curli promoter: Experiment 3

Sensitive to Shaking Speed

The ¡ac=va=on ¡of ¡curli ¡promoter ¡is ¡op=mal ¡at ¡100 ¡rpm. ¡

Thermoregulation

37°C:

• Curli promoter is OFF
• RNAth is ON:

Translation of molecules of interest.

28°C:

• Curli promoter is ON

Production of PHB granules

• RNAth is OFF.

Temperatu ture Switc tch

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.

• 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

Achievements in the manufacturing area

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

• ng 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 !!