Team:Groningen/Notebook/7 July 2009

GVP Cluster
Plating of E.coli TOP10 cells

The plates with transformed E.coli TOP10 cells contained between 5-50 colonies for the non-diluted plates, depending on which plasmid was transformed into the cells. To increase the number and size of the colonies, the plates were stored at 37°C for an additional 24 hours.

Plasmid Purification

Plasmid isolation was performed on the cultures of GVP and Terminator containing cells with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".
 * From each tube 2mL of culture was collected in a 2mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
 * Cells were resuspended in 200μL Resuspension Solution by up and down pipetting.
 * To the mixture 200μL Lysis Solution was added, mixed by inverting the cup, and stored at room temperature for 5 minutes.
 * 350μL of Neutralisation Solution was added and the tubes inverted.
 * Cell debri was pelleted by centrifugation at full speed for 1 min.

Concentration of Plasmids

The concentration of isolated plasmid was determined with the use of a nano-drop.

GVP eluted in MQ
 * 88.2 ng/μL
 * 1.83 (260/280)
 * 2.08 (260/230)

GVP eluted in elution buffer
 * 92.2 ng/μL
 * 1.91 (260/280)
 * 2.78 (260/230)

Terminator eluted in MQ
 * 35.3 ng/μL
 * 1.74 (260/280)
 * 1.97 (260/230)

Terminator eluted in elution buffer
 * 38.8 ng/μL
 * 1.89 (260/280)
 * 1.93 (260/230)

Restriction analysis

The plasmids containing GVP were cut with EcoRI and XbaI fast digest enzymes. The double digestion should result in two fragments of 1400 and 8000 bp in size.

The cups were incubated in a heat block at 37°C for 15 min. followed by adding 4μL 6x loading buffer for gel electroforesis.
 * 6μL MQ
 * 10μL plasmid in MQ
 * 2μL Fast digest buffer
 * 1μL EcoRI fast digest enzyme
 * 1μL XbaI fast digest enzyme

Gel electroforesis

10μL of each sample was loaded on a 2% agarose gel with EtBr and a 1kb ladder was used (see picture).

Transporters
Purification of HmtA (PA Q9I147 6xHis Tag)

Growing cells and purification
 * Add 2ml preculture to 200ml TY+amp
 * Grow culture (37°C with shaking 200rpm) Measure OD660=0,6 (2h)
 * Add arabinose (final con. Of 0,05%)
 * Incubate for 6 h (37°C with shaking 200rpm)
 * Cool culture on ice
 * Spin down 10 min at 8000 rpm 4°C (GSA)
 * Wash pellet in 30ml ice cold KPi (50mM, pH 7,0)
 * Spin down 10 min at 8000 rpm 4°C (SS34)
 * Resuspend pellet in 2ml KPi (50mM pH 7,0)
 * Sonicate cells 15 sec, 45 sec break for 9 cycles
 * Spin down 5 min 9000rpm
 * Collect supernatant to new centrifuge tubes (TLA 100.4)
 * Spin down 25 min (TLA 100.4 90 000 rpm)
 * Store pellet overnight in -20°C

Vectors

 * Inoculated 10 mlm of Ty with top10 pSB3K3 and pSB1AC3


 * grow on 37&deg; 200rpm shaking

Dry
Most of the time was spent on searching for parameters for metal uptake. A database with characteristics of E coli was mentioned by one of last years Igem team members who came to visit.

Furthermore we have been busy linking all different types of units together in order to end up with some useful data in relation to Arsenic up-/intake.

Our wiki achievement of the day was a menu where you can calculate how many grams of arsenic is taken out of the water per cubic meter of living cells. We also tried to find out what the maximum pollution level is in order for our bacteria to still be able to get the concentration of Arsenic under the 10ppb (US safety standerd since 2001).