Team:Groningen/Notebook/14 September 2009

GVP Cluster
Observations


 * Plasmids with a inducible copy number are best grown on agar plates without IPTG (probably the high amount of plasmid to be produced is to much), and growth in cultures is best done with induction by IPTG (better growth, higher density was observed).


 * The test of both the new competent cells and ampicillin batch seem to be positive. On the negative plates no/small growth was observed, and on the positive plates large number of expected red colonies.



Plasmid isolation

Plasmid isolation was performed on the pellets of E.coli TOP10 plasmids (made on saterday and stored in -20box) with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".


 * From each tube 5mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
 * Plasmids were eluted with 30μL MQ and stored in the fridge

Concentrations

Restriction for Assembly

The plasmids from the o.n. precultures of pZntR-GVP and pCueO-GVP were cut with EcoRI/PstI to cut out the entire part between the pre- and suffix, which should give bands of ~6000bp and ~4450bp as a control. The GVP BioBrick plasmid was cut with XbaI/PstI to create fragment of GVP for further work in repeat removal.




 * → From left to right: 1kB ladder, pZntR-GVP no.1, pZntR-GVP no.2, pCueO-GVP no.1, pCueO-GVP no.2, Empty Slot, GVP (3x)


 * → The fragments of pZntR-GVP and pCueO-GVP are at the expected position of ~6000bp, and the vector is at the expected position of ~4400bp (above 4000bp and well beneath 5000bp). From the restriction a positive movement of the contrust from J61035 to pSB2K3 can be concluded. Plates for glycerol stock will be made for both no.1 tubes, and isolated plasmid form the cultures for the stocks will be send for sequencing.


 * → The three lanes for GVP show the expected fragments at ~6000bp and ~3000bp, but also contain an additional band for single cut plasmid. The GVP can be used for purification of that fragment.

Purification




 * → In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use.


 * → The concentration of GVP fragment with X/P sticky ends was 81.9 ng/μL, and was stored in fridge for restriction by MvaI/XhoI on Tuesday.

Tranformation

The failed transformations of last week will be done a second time with the remaining ligation products (between 5 and 10uL). The ampicillin should work this time, giving colonies with the wanted plasmids of GlpF in pSB1AC3 (2x), pLacI in pSB1AC3, pLacI-GVP in pSB1A2 (no.1), and pLacI-GVP in pSB1A2 (no.2).


 * add the ligation product 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 plates


 * → Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ.

Cultures

Overnight cultures of E.coli TOP10 with pArsR-GVP (4x), GVP (2x, no.1), and pNL29 were made in 6mL LB-amp100 medium for plasmid isolation and inoculation of bigger cultures (two of the pArsR-GVP tubes). The cultures were placed in a 37°C waterbath.

Plates

For a new Saline test (place cells on top of chilled Saline column, see if it stays on top or sinks into the column) 200mL culture of E.coli TOP10 with pNL29, pLacI-GVP, J61002-J23101 (control), J23106-GVP, and J23109-GVP were spread on LB-amp100-agar plates. The plates were placed in a 37°C stove.

For glycerol stocks, the E.coli TOP10 cultures with pZntR-GVP and pCueO-GVP in pSB2K3 no.1 were striked in straight lines in LB-amp100 plates.

Metal Accumulation
HmtA New pcr attemt. PCR1 PCR2 F1 and 1850 and 1820

Creating heavy water, Floating preparations
Since seawater has a different density than drinking water we need to conduct a buoyancy test with seawater as well. Our marine biology department was so kind to provide us with seawater with 33‰ and 42‰ salt, measured with a atago refractometer. Water has been sterilized.

Water Densety check mass per ml (measurements from)