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Team:Groningen/Notebook/8 September 2009
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Wet
GVP Cluster
- → DONE Check transfer of pArsR-GVP, pZntR-GVP, pCueO-GVP, and pLacI-GVP from J61035 and pSB1A2 to pSB2K3. (preculture growth, 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))
- → DONE Cut out GVP cluster from biobrick vector (can be used for fragment formation)
- → TODO Create fragments for 400bp repeat removal from the gvpL gene. (restriction, gel purification)
- → TODO Perform ligation of all fragments into pSB1AC3 vector. (the pSB1AC3 vector has been the most succesfull vector to use in assemblies)
- → DONE 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)(precultures, plates)
- → TODO Check Saline solutions and take pictures!
O.n. precultures
- → All tubes for plasmid isolation (pArsR-GVP, pZntR-GVP, pCueO-GVP, and pLacI-GVP in pSB2K3, and pSB2K3 (2x)) showed growth. The pArsR-GVP in pSB2K3 had less growth (and smaller pellet) than the other tubes, and pSB2K3 no.1 was red coloured.
- → All tubes for Saline Test (for plating on LB-amp100 and LB-amp100-IPTG) showed growth and 300uL was plated.
Restriction for Control
The plasmids from the o.n. precultures were cut with PstI and EcoRI to cut out the entire part between the pre- and suffix.
| Plasmid | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | XbaI fast digest enzyme | SpeI fast digest enzyme | PstI fast digest enzyme |
| pArsR-GVP | 10.0 | 5.0 | 3.0 | 1.0 | x | x | 1.0 |
| pZntR-GVP | 5.0 | 10.0 | 3.0 | 1.0 | x | x | 1.0 |
| pCueO-GVP | 4.0 | 11.0 | 3.0 | 1.0 | x | x | 1.0 |
| pLacI-GVP | 4.0 | 11.0 | 3.0 | 1.0 | x | x | 1.0 |
| pSB2K3 no.1 | 10.0 | 5.0 | 3.0 | 1.0 | x | x | 1.0 |
| pSB2K3 no.2 | 4.0 | 11.0 | 3.0 | 1.0 | x | x | 1.0 |
_2.jpg)
Purification
- → From left to right: 1kB ladder, pArsR-GVP, pZntR-GVP, pCueO-GVP, pLacI-GVP, Empty Slot, pSB2K3 no.1 (2x), Empty Slot, pSB2K3 no.2
- → The fragments were as expected, but
Tranformation
- add 4uL of the dissolved registry plasmid to 50uL competent E.coli TOP10 cells and 1uL of HmtA to 50uL competent E.coli TOP10 cells.
Incubate:
- 30 min @ ice
- 90 sec 42°C
- 2 min @ ice
- add 800uL LB-medium
- incubate for 1 h at 37°C
- plate on LB-amp100/LB-kan50/IPTG plates
7K BBa_P1010 pSB2K3 Plasmid pSB2K3 with part BBa_P1010
QC: Sequence (A) InconsistentResistance: /KGel: Q: None
http://partsregistry.org/assembly/plates.cgi?id=681
Transporters
Metal Accumulation
Vectors
Dry
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