Team:Imperial College London/Lab Diaries/XylE team


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Lab Diaries Overview | Surface Protein Team | XylE Team | Vectors Team | Modelling Team
Here are the technical diaries for our project. We've split them up into three lab teams and the modelling team. We think it's really important that absolutely anyone can find out what we've been doing. For a really detailed look at what we did, and when, you've come to the right place!
Team XylE
XylE team Lab Objectives
  • construction of XylE fusion protein
  • Testing expression of XylE in E.coli and characterization under the control of a constitutive promoter
  • Construction of the -ComE promoter/XylE fussion protein- expression system
  • Construction of the -LacI promoter/XylE fusion construct- expression system
Lab notes and schedule
==Week 6==

Thursday, 12-Aug-2010

  • anealing DNA strands of J23101 promoter in a waterbath

we constructed the standard E.coli promoter J23101 with sticky ends. These ends are complementary to restriction sites made by EcoRI and SpeI enzyme. This promoter will be later used in 3A assemply to construct a promoter-RBS-XylE design in a psB1C3 vector. E.coli will be transformed with this final construct plasmid to assess XylE activity and characterization. It will also be one of the submitted biobricks.

  • prepared two overnight cultures of XylE transormed E.coli (one 50microliters and one of 450 microliters)

these cultures are going to be used tomorrow for mini-prepping. Miniprep will allow us to isolate E.coli's plasmid DNA(which contains the XylE gene).

Friday, 13-Aug-2010

  • mini-prep of XylE transformed E.coli

Mini-prep is usually used to confirm that our gene of interest has not been changed in any way, as the isolated plasnid id sent for sequencing. However, since XylE was taken from the registry, we assume that it is fine and no sequencing is required. The mini-prep will later be used for the midi-prep (that gives out higher yeilds of DNA needed for cloning).

  • gel analysis of plasmid DNA retreived from mini-prep of XylE transformed E.coli, cut with restriction enzymes. From light to the left, 50micrograms digested DNA : 50 undigested DNA : 450 digested DNA : 450 undigested DNA. In lanes 1 and 3 the smaller band has a size of about 1kB which corresponds to RBS-XylE gene. The bigger bands are the cut vectors. In lanes 2 and 4 is the uncut biobrick from the registry. It appears smaller on the gel than it actually is as circular DNA travels faster through the pores of agarose gel rather than linearised DNA.