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| - | {{:Team:BCCS-Bristol/Header}}
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| - | ==Parts Submitted to The Registry==
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| - | *[http://partsregistry.org/Part:BBa_K259000 FhuA(BBa_K259000)] - Iron Chelator
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| - | *[http://partsregistry.org/Part:BBa_K259001 Fiu (BBa_K259001)] - Iron Chelator
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| - | ==Outline of Project Work==
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| - | ===Week Zero===
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| - | *Familiarising with standard lab procedures for the past week (bacterial culture growth, restriction enzyme usage, agarose gel electrophoresis).
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| - | *Will start designing some biobricks for the project today.
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| - | ===Week 1===
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| - | ====Canditate Proteins for Biobricks====
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| - | *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.
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| - | *Primers designed and ordered. Waiting for their arrival to do PCR! :D
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| - |
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| - | ====Reporters,RBS,Backbones====
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| - | *Decided to use 3 reporter genes, 1 RBS, 1 High Copy plasmid backbone for now.
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| - | =====Reporters=====
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| - | *RFP(Bba_E1010)
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| - | *GFP(Bba_E1040)
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| - | *LacZ(Bba_I732005)
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| - | =====RBS=====
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| - | *Bba_J61100 - From Anderson Family
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| - | =====Plasmid Backbone=====
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| - | *BBa_J04450 ; pSB1A3
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| - |
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| - | *Tried to extract DNA from the iGEM biobricks and transfrom into bacteria.
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| - | *Transformations do not work properly with non-commercial E.coli strain (XL-1).
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| - | *Transformations worked the 2nd time round with commercial Nova Blue E.coli Strain. DNA samples in toolkit must be of low concentrations!
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| - | *Regrew bacterial colonies to amplify DNA of reporters,RBS,backbone.
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| - | *Miniprepped the DNA of Reporters,RBS,plasmid backbone and made glycerol stocks.
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| - | *Realised that we are faced with a problem when wanting to assemble biobricks for protein fusions.
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| - | ===Week 2===
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| - | ====PCR====
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| - | *Primers finally arrived. Did PCR to amplify carrier genes.
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| - | *PCR worked. PCR products (3 candidate proteins)ligated onto biobrick backbones pSB1A3 and pSB1A2 (contains RBS BBa_J61100).
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| - |
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| - | *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!!)
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| - | *Most transformations are successful. Used transformed colonies to prepare liquid cultures so that we can proceed with minipreping them.
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| - |
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| - | ====In frame protein fusions====
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| - |
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| - | *Started working on finding an easy assembly method for in-line protein fusions.
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| - |
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| - | *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.
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| - |
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| - | *Prepared different versions of this Bioscaffold to be ordered and tested in the lab for actual functionality.
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| - | ===Week3===
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| - | *Finalised designs for the Bioscaffold-Linker biobrick and ordered from GeneART/Mr.Gene the construct.
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| - |
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| - | *Finding canditate primers for sequencing the first 3 carrier biobrick proteins (fhuA/osmE/fiu).
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| - |
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| - | *Started thinking of a quick and dirty in-frame fusion for testing functional carrier-reporter gene fusions.
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| - |
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| - | *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.
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| - | *Ordered primers for the quick-n-dirty assembly method.
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