Team:Groningen/Notebook/7 July 2009

From 2009.igem.org

Wet

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.

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

Gel electroforese

Dry

Most of the time was spent on searching for parameters for metal uptake. [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi 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).