Team:BCCS-Bristol/Notebook

From 2009.igem.org

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==Parts Submitted to The Registry==
==Parts Submitted to The Registry==
 +
===Carrier Proteins===
*[http://partsregistry.org/Part:BBa_K259000 FhuA(BBa_K259000)] -  Iron Chelator
*[http://partsregistry.org/Part:BBa_K259000 FhuA(BBa_K259000)] -  Iron Chelator
*[http://partsregistry.org/Part:BBa_K259001 Fiu (BBa_K259001)] -  Iron Chelator
*[http://partsregistry.org/Part:BBa_K259001 Fiu (BBa_K259001)] -  Iron Chelator
 +
 +
===BioScaffold Linkers===
 +
*[http://partsregistry.org/Part:BBa_K259002 BpuEI-BseRI (BBa_K259002)] -  BioScaffold Linker Transformer
 +
*[http://partsregistry.org/Part:BBa_K259003 BpuEI-CspCI (BBa_K259003)] -  BioScaffold Linker Transformer
==Outline of Project Work==
==Outline of Project Work==

Revision as of 19:25, 1 August 2009

BCCS-Bristol
iGEM 2009

Contents

Parts Submitted to The Registry

Carrier Proteins

BioScaffold Linkers

Outline of Project Work

Week Zero

  • Familiarising with standard lab procedures for the past week (bacterial culture growth, restriction enzyme usage, agarose gel electrophoresis).
  • Will start designing some biobricks for the project today.

Week 1

Canditate Proteins for Biobricks

  • Isolated 3 canditate proteins to act as carriers for our biobricks. These are FhuA,Fiu & OsmE. Started to design primers to amplify the selected genes via PCR.
  • Primers designed and ordered. Waiting for their arrival to do PCR! :D

Reporters,RBS,Backbones

  • Decided to use 3 reporter genes, 1 RBS, 1 High Copy plasmid backbone for now.
Reporters
*RFP(Bba_E1010)
*GFP(Bba_E1040)
*LacZ(Bba_I732005)
RBS
*Bba_J61100 - From Anderson Family
Plasmid Backbone
*BBa_J04450 ; pSB1A3
  • Tried to extract DNA from the iGEM biobricks and transfrom into bacteria.
  • Transformations do not work properly with non-commercial E.coli strain (XL-1).
  • Transformations worked the 2nd time round with commercial Nova Blue E.coli Strain. DNA samples in toolkit must be of low concentrations!
  • Regrew bacterial colonies to amplify DNA of reporters,RBS,backbone.
  • Miniprepped the DNA of Reporters,RBS,plasmid backbone and made glycerol stocks.
  • Realised that we are faced with a problem when wanting to assemble biobricks for protein fusions.

Week 2

PCR

  • Primers finally arrived. Did PCR to amplify carrier genes.
  • PCR worked. PCR products (3 candidate proteins)ligated onto biobrick backbones pSB1A3 and pSB1A2 (contains RBS BBa_J61100).
  • The plasmid backbones with the genes of interest inserted into them were used to transform the XL1-BLUE E.coli strain (although not competent enough compared to NovaBlue cells they are much cheaper!!)
  • Most transformations are successful. Used transformed colonies to prepare liquid cultures so that we can proceed with minipreping them.

In frame protein fusions

  • Started working on finding an easy assembly method for in-line protein fusions.
  • Developed the design for a Bioscaffold-Linker transformer family (inspired by Bioscaffolds). Should allow fusions of proteins and all RFC10 biobricks in-frame after using Bioscaffold specific restriction enzymes.
  • Prepared different versions of this Bioscaffold to be ordered and tested in the lab for actual functionality.


Week3

  • Finalised designs for the Bioscaffold-Linker biobrick and ordered from GeneART/Mr.Gene the construct.
  • Finding canditate primers for sequencing the first 3 carrier biobrick proteins (fhuA/osmE/fiu).
  • Started thinking of a quick and dirty in-frame fusion for testing functional carrier-reporter gene fusions.
  • Found easy fusion way for in line testing. Will take out end of FhuA and scar formed after RFC10 assembly of FhuA-GFP using RE's to take out FhuA end ( including TAA TAA SCAR) and part of GFP start. Will replace lost coding sequences with PCR.
  • Ordered primers for the quick-n-dirty assembly method.
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