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Team:Groningen/Notebook/28 September 2009
From 2009.igem.org
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===Metal Accumulation=== | ===Metal Accumulation=== | ||
| - | *'''Redo As(III) uptake experiment for ''E. coli'' WT (pSB1A2) and ArsR-RFP''' | + | *'''Redo [https://2009.igem.org/Team:Groningen/Notebook/25_September_2009 As(III) uptake experiment] for ''E. coli'' WT (pSB1A2) and ArsR-RFP''' |
** Take only the concentration range (0-100uM As(III) | ** Take only the concentration range (0-100uM As(III) | ||
Latest revision as of 20:19, 30 September 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
TODO Restrict GVP biobrick with XbaI/PstI and purify both vector and GVP fragment
TODO Restict GVP fragment and pMA-gvpL with MvaI/XhoI and purify 310bp. 2200bp, and 3300bp fragments
TODO Ligate the three fragments into the vector and transform E.coli DB3 cells
TODO Find out wath the problem is with RBS??
- Is it one of the restriction enzymes (SpeI/PstI) failing work
- Something wrong with biobrick in the sense that there is a red colour where none was expected (where did the RFP come from, and why is it activated so strongly)
- → From left to right: 1kB ladder, Sample Steven, SpeI/PstI control with BBa_K190017, Empty Slot, GVP (6x)
- → For GVP two fragments of ~6000bp and ~3000bp are expected, and for BBa_K190017 fragments of ~900bp and ~2000bp are expected.
- The sample of Steven-Jelle did not contain any DNA visible on gel!
- The GVP lanes showed the expected fragment sizes, with the addition of single cut vector at ~9000bp. The fragments were cut from the gel for purification.
- The restriction test to check SpeI/PstI confirmed the normal funtioning of the enzymes. This makes the RBS plasmid from -80 stock less attractive, because the unwanted RFP is not located behind the RBS to be cut out!
- To check another posibility, the stored plasmids from June were cut with SpeI/PstI and the stock plasmid was cut with EcoRI/XbaI to check if the RBS is placed infront of RBS.
- → From left to right: 1kB ladder, RBS (EcoRI/XbaI), RBS (EcoRI/PstI), Empty Slot, RBS no.1, RBS no.2, RBS no.3
Transporters
Metal Accumulation
- Redo As(III) uptake experiment for E. coli WT (pSB1A2) and ArsR-RFP
- Take only the concentration range (0-100uM As(III)
Vectors
Dry
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