Team:Groningen/Notebook/28 September 2009

GVP Cluster
Restrict GVP biobrick with XbaI/PstI and purify both vector and GVP fragment

Restict GVP fragment and pMA-gvpL with MvaI/XhoI and purify 310bp. 2200bp, and 3300bp fragments

Ligate the three fragments into the vector and transform E.coli DB3 cells

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

Metal Accumulation

 * Redo As(III) uptake experiment for E. coli WT (pSB1A2) and ArsR-RFP
 * Take only the concentration range (0-100uM As(III)