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 |
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Week 7 | Monday | Tuesday | Wednesday | Thursday | Friday | Weekend |
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*estimated date of primers delivery
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lunch break | ||||||
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Lab Diary
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).