Team:Groningen/Notebook/7 September 2009

GVP Cluster

 * → Check transfer of pArsR-GVP, pZntR-GVP, pCueO-GVP, and pLacI-GVP from J61035 and pSB1A2 to pSB2K3. (✅, isolation, and restriction (the colonies all grew on kanamycin plates with IPTG, this is the first selection, because the parts come from an ampicillin plasmid))


 * → ✅ Cut out GVP cluster from biobrick vector (can be used for fragment formation)
 * → Create fragments for 400bp repeat removal from the gvpL gene. (restriction, gel purification)


 * → Perform ligation of all fragments into pSB1AC3 vector. (the pSB1AC3 vector has been the most succesfull vector to use in assemblies)


 * → Grow L-GVP, M-GVP, and pLacI-GVP (with IPTG) on plates for test of buoancy in Saline solution. (as control use pNL29 with IPTG, and J61002-pCueO)(✅, plates)


 * → ✅ Order synthetic DNA for GVP
 * → ✅ Order primer for PstI site removal


 * → Check Saline solutions and take pictures!

Repeat Removal

The first attempts at removing the 400bp repeat from the gvpL gene will take place during this week.


 * → The GVP part from the registry (BBa_I750016) will be cut by EcoRI and PstI to obtain the biobrick part.


 * → After purification the biobrick will be cut with XhoI and MvaI to cut out the repaet part and purify the parts on both sites, one with the EcoRI site and the other with PstI site.


 * → The part with repeat will be replaced by the original part from the pNL29 plasmid, also cut out with XhoI and MvaI. The replacement will reintroduce the PstI site removed by Melbourne 2007, but will remove the unwanted mutation.


 * → With PCR and the correct primer the PstI site will be removed after succesfull ligation.


 * → The part between restriction sites MvaI and XhoI in gvpL has been ordered to be synthesized by Mr.Gene, with the biobrick pre- and suffix at the ends. This may be used to clone the part into a vector for duplication.

Restriction for Assembly

The biobrick plasmid BBa_I750016 was cut twice with PstI and EcoRI to create correct ends for insert into a standard plasmid from the registry.



Purification




 * → From left to right: 1kB ladder, Empty Slot, BBa_I750016 (EcoRI,PstI), Empty Slot, BBa_I750016 (EcoRI,PstI)


 * → The fragments were not as expected, but carefull examination let to the conclusion the SEQcentral vector map was not carefully constructed and missed the part between EcoRI and XbaI with gentamycin resistance.


 * → The fragments of GVP with addition of gentamycin part were used for purification and to cut it wit XbaI to create the GVP only fragment.

Restriction for Assembly (2)

The purified GVP fragment was cut with XbaI to create correct end for insert into a standard plasmid from the registry, and to loose the gentamycin part.



Purification




 * → From left to right: 1kB ladder, Samples from Frans (2-9), GVP (XbaI,PstI)


 * → The fragments were as expected.

Transporters
HmtA ON pcr F1 rev worked. 6 ng/ul purified from gel. This is used as template for an other PCR to again more DNA to work with.