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Team:BCCS-Bristol/Notebook
From 2009.igem.org
BCCS-Bristol
iGEM 2009
iGEM 2009
Contents |
Parts Submitted to The Registry
- [http://partsregistry.org/Part:BBa_K259000 FhuA(BBa_K259000)] - Iron Chelator
- [http://partsregistry.org/Part:BBa_K259001 Fiu (BBa_K259001)] - Iron Chelator
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.
