Imperial College/21 July 2008

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Contents

Briefing

3 Subgroups to research on their specific areas: Alternative photoreceptors to YtvA-σB activation pathway, Biomaterial synthesis and B.Subtilis protocols/promoters/rbs etc.


Debriefing

Each Group gave a summary of their sections, followed by a discussion on any potential problems:

B.subtilis

  • The knock out of espE is in a wild-type strain as opposed to lab based stains. Group was unclear on what a lab stain actually was and the problems of not using them.
  • Wild type strains have differing motility from lab strains.
  • Look at degradation tags to add to the EspE, this could help tune our design after the initial testing and the modeling.
  • Need to identify promoters including - inducible, constitutive and possibly repressible.
  • Need to identify sequences for integration.
  • Use of integration method to introduce BioBricks into B.Subtilis, a.k.a. Integrated BioBricks
  • Plasmids will be grown in E.Coli so as to facilitate genetic manipulation and maintainance before transferring any genetic material into B.Subtilis.
  • LacZ, IPTG inducible promoters are most commonly used in B.Subtilis.

Light Induction

  • Pursue the possibility of synthesising two constructs,
  1. The natural YtvA can be used, all we have to do is to use a σB promoter that can be put upstream of our constructs.
  2. Investigate an heterogeneous pathway in B.subtilis. One suggestion has been to investigate the use of a previously engineered part from iGEM competition were EnvZ-OmpR was made photosensitive.
  3. Rhodopsin is membrane bound, might not work on Gram positive bacteria.
  • Need to find out any parameters such as time dependence, threshold of response.
  • Check on the wavelength of light which affects B.Subtilis.
  • Find out what kind of data can be collected on bacteria motility. Should try to look at the motility of E.Coli first, then swap over to B.Subtilis.

Biomaterials

  • Need to firstly identify a list of biomaterials with relevant features, i.e. concentration dependence of assembly, no. of subunits.
  • Need to investigate the types of secretion we can pursue, what sequence do we need and what machinery do we need.

Focus on three main points:

  • Self Assembly, might want to look at amyloid peptides,
  • Conc dependence,
  • One or multiple subunits needed,
  • More details are required on the in-vitro properties of biomaterials.
  • Find out the protein coating on spores.

Summary

  • Need to get into construct, part design. Draw out design diagrams, Bioengineers to help with conceptual framework and modelling i.e. responsiveness of signal to clutch and other parameters.
  • Look at websites for culturing B.Subtilis.
  • Identify other companies which do DNA sequencing, should not rely entirely on GeneArt.
  • Need for timetable and project management, milestones on design, modelling, protocols etc.
  • Next Monday and Wednesday, demonstration on lab use.
  • 1 August Team project descriptions due (Late summer teams)!!!
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