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Team:BCCS-Bristol/Notebook/Week 2
From 2009.igem.org
BCCS-Bristol
iGEM 2009
iGEM 2009
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.
Novel protein additions were cloned from the MG1655 genome with PCRt. Lane 1 shows the presence of fhuA, lane 2 fiu and lane 3 osmE. The ladder is Lambda-DNA HindIII digest from NEB (In descending order; 23130, 9416, 6557, 4361,2322, 2027. Carrier proteins were then placed onto pSB1A3 plasmid backbones to create biobricks novel protein carriers.
