IGEM:Imperial/2010/XylE team

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

Schedule

Week 6	Monday	Tuesday	Wednesday	Thursday	Friday	Weekend
MORNING					mini-prep kit of XylE-transformed E.coli (already overnight grown)
lunch break
AFTERNOON				Starting of “Testing expression of XylE in E.coli” objective 1)Annealing EcoRI and speI oligos to J23101 promoter which will be annealed later in front of the RBS-XylE registry gene (overnight	gel analysis of mini-prep derived XylE plasmid.(requires first digestion of the vector with restriction enzymes)

Week 7	Monday	Tuesday	Wednesday	Thursday	Friday	Weekend
MORNING	3A assemply: ligation of RBS-XyL, J23101 promoter and PSB1C3 vector (overnight)	*estimated date of primers delivery 3A assemply: transformation of E.coli with XylE construct (overnight)	3A assemply: make replica plates (overnight) Catechol assay of E.coli	Mini-prep XylE E.coli	Midi-prep XylE E.coli
lunch break
AFTERNOON		Starting construction of XylE fusion protein

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.

Monday, 16-Aug-2010

• midi-preped the XylE-transformed E.coli. The DNA yeild from the midi-prep was 134ng/ml 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+terminator vector)