Difference between revisions of "Part:BBa K300007:Experience"

UNIPV-Pavia iGEM 2010

Sequencing of this part showed some (negligible) difformities when compared with the submitted sequence: see

TU Munich iGEM 2012

Idea

For proof of principle, we want to insert a functional mOrange cassette (BBa K300007), cut the integrative vector with SbfI in order to linearize it and transfect it into yeasts. After we've shown the general functionality, we'll work on and integrate one gene after another until all of our biobricks are integrated. In order to do that, we have to engineer new homologous regions to enable multiple transgenic integrations.

To show, that the stable integration worked, we picked 36 clones from the plates with yeasts transformed with the linearized integration vector plasmid (insert: BBa K300007[1]). These 36 clones were put into non-selective YPD medium and passaged 4 times. After 48 hours, the last culture was used to inoculate an overnight culture. The OD of all candidates was measured. We had 5 negatives, probably due to mistakes in pipeting. The rest of the cultures were diluted to OD 0.4 and 100 ml of it were used to plate on selective (meaning G418+) petri dishes.

Results

• 3
• 8
• 12
• 49
• 68
• 282
• >1000
• >10 000
• >10 000
• >10 000

The plan is to PCR at least two of the clones to gain knowledge why there's been such drastic differences in between the different clones regarding the in the integration efficiency and endurance.

As of the mOrange cassette (BBa K300007[2]), we couldn't show any fluorescence which suggests a corrupt insert.

BBa_K300006 for details.