Team:Groningen/Notebook/15 September 2009

GVP Cluster
Planning


 * → work out the wiki page for GVP
 * → ✅ make a doodle for presentation planning (1-19 oct.)
 * → media attention
 * → ✅ place an ethics survey link on twitter



Plates


 * → The plates showed a low amount of colonies, and also red in the case of the positive plate.


 * → The two plates with stripes of pZntR-GVP (pSB2K3) and pCueO-GVP (pSB2K3) culture for glycerol stocks showed growth and some single colonies.


 * → All plates were stored in the fridge for further use.

Cultures

The overnight cultures with LB-amp100 medium of colonies E.coli TOP10 with pArsR-GVP (4x), GVP (2x), pNL29 (2x), and HmtA (1-3) all showed expected growth of bacteria.


 * Two of the pArsR-GVP tubes were handed over to Steven-Jelle for use of inoculating bigger cultures, the remaining tubes were used for plasmid isolation.



Plasmid isolation

Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".


 * From each tube 5 to 10mL of culture was collected in a 2.0mL cup (tubes from pArsR-GVP, GVP and pNL29 were combined), 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, and Control (HmtA)

The plasmids from the o.n. precultures of pArsR-GVP and pSB2K3 (earlier last week) were cut with PstI and EcoRI to cut out the entire part between the pre- and suffix. The plasmids with PstI were cut with PstI for control.




 * → From left to right: 1kB ladder, HmtA no.1, HmtA no.2, HmtA no.3, Empty Slot, pArsR-GVP, Empty Slot, pSB2K3

Purification




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

Concentrations

Ligation

A total amount of vector of 100ng was used in a 1:3 ratio with insert.
 * 3 uL Ligase buffer
 * 1 uL T4 Ligase
 * 9 uL MQ
 * 4 uL plasmid pSB2K3 EcoRI/PstI
 * 3 uL GVP restricted with EcoRI/PstI

Incubate:
 * 25°C 50min.
 * kept on ice for 10min.

Tranformation
 * add 10uL of 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-kan50 plates


 * → Negative control was MQ.

Saline Test

The o.n. plates for Saline test (5 in total) were taken from the stove and 4mL Saline solution was added to dissolve the colonies. The volume of dissolved cells was adjusted to 8mL, and a ten times dilution was made for OD600 measurements.

The tubes were diluted to adjust the OD600 to the lowest sample (1.86) of M-GVP. After adjustment the volumes were reduced to 8mL and an additional 3mL of Saline was added. The OD600 of all tubes was 1.3.

For the test a number of 5 tubes was filled with 10mL Saline and chilled in the fridge for 4 hours. From each solution of cells 1mL was carefully loaded on top of the column. With a bit of luck, a difference will appear between the cells with and without gas vesicles.

For the test a number of 5 tubes was filled with 5mL Saline, and 5mL of the cell solutions was added to create an OD600 of 0.650.


 * In the previous test an OD600 of 0.300 was to low to observe any differences, and an OD600 of 1.5 was so high a difference appeared on a small scale (tough this showed a difference between the low and medium constitutive promoter).


 * The tubes were placed in the same place as before, and at time zero the first picture was taken.