User:IGEM Paris Bettencourt/ToDoList

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General

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Chitin project

Questions already answered

  • Yield: 50g/L with Monomer production (see article x in Dropbox); Polymer production depend on Synthase.
  • Increase the yield: UDP-GlcNac is a precursor for chitin synthesis.there are four genes(GFA1,AGM1,GNA1 and UAP1) encoding enzymes that are responsible for the synthesis of UDP-GlcNac and all genes are up-regulated during meiosis and sporulation.Treatment of yeast cells with α-factor(eg. glucosamine) causes a three to five fold increase in the chitin level associated with the increase in the level of the UDP-GlcNac pool( see article in drop box:chitin synthesis). so we can add glucosamine to the growth media to have an increase in the monomer pool responsible for the synthesis of chitin
  • Effect on cells: Chitin can asphyxiate cells (nutriments, not oxygen) but unlikely, monomers are rejected by E.Coli (no problem then).
  • Polymer Caracteristics : strong C-O ... NH hydrogen bond, distance 0,47 nm. Article in dropbox, crystal structures and conformations
  • Yield:UDP-GlcNac is a precursor for chitin synthesis.there are four genes(GFA1,AGM1,GNA1 and UAP1) encoding enzymes that are responsible for the synthesis of UDP-GlcNac and all genes are up-regulated during meiosis and sporulation.Treatment of yeast cells with α-factor(eg. glucosamine) causes a three to five fold increase in the chitin level associated with the increase in the level of the UDP-GlcNac pool( see article in drop box:chitin synthesis). so we can add glucosamine to the growth media to have an increase in the monomer pool responsible for the synthesis of chitin.
  • Polymer purification: from an old patent but i'm not sure of the efficiency. chitin is purified by adding 1 part of the chitin to 12 parts of 2-chloroethanol and 16 parts of 73% sulfuric acid.mix and heat below the boiling point of 2-chloroethanol.the chitin is dissolved in a relatively low viscosity solution and then precipitated by addition of water,methanol and aqueous ammonium hydroxide(see chitin purification patent in drop box). Recently chitin is dissolved when an alkaline suspension(eg. dissolution in NaOH ) is mixed with ice.so i think from this step we can finish up with the purification ( see drop box:solubility of chitin).
  • Test Chitin production:
  1. we can perform staining test for chitin using Calcofluor White stain (Fluorescent Brightener M2R, price:36.60Euro from Sigma), which shows enhanced fluorescence when it binds to chitin.confocal laser scanning microscopy is required for observation (see drop box: chitin evidence).But i guess we still need a way to quantify it. for quantification, i saw some guys dry and weigh their chitin after purification.

Questions to be answered

  1. Interest of leaving the Synthase on the yeast ?
  2. How to start polymerization ?
  3. What are genes compatibility ?
  4. How to extract Chitin afterward ?
  5. What is the biochemistry of the pathway of Chitin's production ?

Nanotubes project

Step 0

  • Check diffusion with fluorescence microscope/ with antibiotic resistance.

Experience is in progress for the fluorescence test.

  • Check if our proteins don't diffuse through the membrane.
  • Check diffusion of our parts.

We need to test the reliability of our Biobricks and check if all proteins diffuse independantly in Nanotubes.

  • Check gene activation through Nanotubes.

We are going to check the activation of genes in E.Coli which has been expressed by Basillus.

  • Create a library of all parts.

Step 1

Questions already answered
  • LacI repressor protein is composed of four identical subunits. Each subunit is about 360 aa in size (1440 aa). tetR can only dimerize (wich is a good news) Each subunit is about 220 aa in size (450 aa).
Missions
  • Find all parts and check their reliability.
  • Modeling with TinkerCell.
  • Start plasmid genetic circuit in vitro and integrate it into Bacillus.
  • Make quantitative measures and adapt the model depending on results.
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