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From 2010.igem.org

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

Contents 1 Week 6 1.1 Thursday, 12-Aug-2010 1.2 Friday, 13-Aug-2010 2 Week 7 2.1 Monday, 16-Aug-2010 2.2 Tuesday, 17-Aug-2010 2.3 Thursday, 19-Aug-2010 2.4 Friday, 20-Aug-2010 3 Week 8 Week 6 9.8.10-13.8.10 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.

Week 7

Monday, 16-Aug-2010

• midi-prepped the XylE-transformed E.coli. The DNA yeild from the midi-prep was 134micrograms as determined by spectrophotometry. This is the XylE that is going to be used for all further experiments.
• restriction digestion of midi-prepped XylE by Xbal and PstI to prepare it for 3A assemply. (with J23101 promoter and PSB1C3 vector)
• gel analysis of the restriction digestion mixture to isolate XylE gene

Midi-prep XylE digestion with xbaI and PstI The smaller size bands at lanes 2 & 3 are the ones that are going to be cut out and used in gel purification to extract the XylE gene (with sticky ends for XbaL and PstI). ▪ I made an overnight culture of Bacillus

Tuesday, 17-Aug-2010

• using the Gel Extraction Kit, we isolated the restriction enzyme cut XylE gene from the agarose gel lamp.
• gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine their ratios for 3A assemply ligation
• 3A assemply of XylE, J23101 promoter and pSB1C3 vector.
• Transformation of XL-Blue competent E.coli with the above construct.

gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine the volume ratios of samples to be used for 3A assemply ligation

• I followed Chris's Bacillus transformation protocol to transform Bacillus with constitutive GFP and RFP DNA as well as a control without DNA

Thursday, 19-Aug-2010

• We run the XylEGFP1 PCR reaction to construct the His-GFP-Flag and Linker-XylE-Spe construct.

Friday, 20-Aug-2010

The J23101 gene in a biobrick vector containg RFP gene

• we run a gel on XylEGFP1 PCR reaction. Results: GFP was extended successfully, XylE extension FAILED (too much non-specific annealing)
• catechol assay on 2hrs bench ligation of promoter,xylE and vector failed.
• two replica plates of overnight ligated J23101, XylE and pSB1C3 transformed E.coli (one for catechol assay)
• the His-GFP-Flag DNA was gel purified
• transformation of XL-1Blue cells with J23101 in J62001 vector from the registry. One more attemp to construct a successful promoter-xylE ligation, since we believe that the strand annealed promoter was of bad quality

Week 8