Team:BCCS-Bristol/Notebook

From 2009.igem.org

(Difference between revisions)
(→Week3)
Line 73: Line 73:
===Week3===
===Week3===
 +
*Ligation successful only for the OsmE and FhuA inserts on pSb1A3 backbone. Unfortunately we couldn't get any of the 3 proteins on the pSB1A2 backbone.
 +
 +
*Seems that overxpression of these proteins on pSB1A2 is unfavourable for the cells. Hence decided to replace the existing promoter with an inducible one of our choice such  as the AraC promoter.
 +
 +
*Promoter removed from pSB1A2 and religated. Fiu still not able to be ligated on any of the two backbones.
 +
 +
*In the meantime the two parts comprising of the AraC promoter were extracted from the iGEM kit plates and used to transform NovaBlue competent cells. Also liquid cultures of the transformed cells were prepared.
 +
   
 +
*Finalised designs for the Bioscaffold-Linker biobrick and ordered from GeneART/Mr.Gene the construct.
*Finalised designs for the Bioscaffold-Linker biobrick and ordered from GeneART/Mr.Gene the construct.

Revision as of 15:53, 7 August 2009

BCCS-Bristol
iGEM 2009

Contents

Parts Submitted to The Registry

Carrier Proteins

  • [http://partsregistry.org/Part:BBa_K259000 FhuA(BBa_K259000)] - 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_K259004 BpuEI-CspCI (BBa_K259004)] - BioScaffold Linker Transformer

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

  • Ligation successful only for the OsmE and FhuA inserts on pSb1A3 backbone. Unfortunately we couldn't get any of the 3 proteins on the pSB1A2 backbone.
  • Seems that overxpression of these proteins on pSB1A2 is unfavourable for the cells. Hence decided to replace the existing promoter with an inducible one of our choice such as the AraC promoter.
  • Promoter removed from pSB1A2 and religated. Fiu still not able to be ligated on any of the two backbones.
  • In the meantime the two parts comprising of the AraC promoter were extracted from the iGEM kit plates and used to transform NovaBlue competent cells. Also liquid cultures of the transformed cells were prepared.


  • 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.

Week 4

  • Grew colonies for FhuA and OsmE midi preps.
  • Carried out midi preps for FhuA and OsmE ( [FhuA]=378.9 ng/ul, [OsmE]=113.2 ng/ul).
  • Transformed Nova Blue with biobrick part BBa_B0014 (double terminator)
  • Culture colonies of BBa_B0014 transformants to get ready for mini prep.
  • PCR FhuA and GFP for assembling together a quick and dirty fusion to be ready prior to BioScaffold arrival.
  • Carried out colony growth (agar & liquid) for the AraC promoter (2 parts BBa_R0080 (lacking O2 region) and BBa_R0081 (the O2 region).
  • Miniprepped DNA from cultures of BBa_R0080 and R0081.
  • Attempted a 2-way ligation and of the BBa_R0080 and R0081 parts. The final product should be a 2-component biobrick on a plasmid in 5'-R0080-R0081-3' direction which can be transformed into cells.
Retrieved from "http://2009.igem.org/Team:BCCS-Bristol/Notebook"