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Applications of BBa_K300001
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UNIQa28b571152718c33-partinfo-00000000-QINU
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UNIPV-Pavia iGEM 2010
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The integration capability of this vector has been tested in S288C S. cerevisiae strain (BBa_K300979). Here is reported the followed protocol and the obtained results.
Protocol:
- S288C strain (Open Biosystems) was inoculated in 5 ml of YPD from a long term 15% glycerol stock and grown for 24h (30°C, 200rpm).
- The culture was diluted 1:10 in 50 ml of pre-warmed YPD in a 250 ml flask and was grown for additional 4 hours under the same conditions as before.
- Cells were pelleted (4000 rpm, 5 min) and resuspended in 25 ml of deionized water.
- Cells were pelleted (4000 rpm, 5 min), the supernatant was discarded and the pellet was resuspended in 1 ml of deionized water and transferred into a 1.5 ml tube.
- Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded and the pellet was resuspended in deionized water to a final volume of 1 ml (vortex mix vigorously).
- Three 100 ul aliquots were transferred into 1.5 ml tubes, while the remaining 600 ul of cells were not used in this protocol.
- The three tubes were centrifuged (4000 rpm, 30 sec) and the supernatant discarded.
- Each of the three pellets were resuspended (vortex mix vigorously) in 360 ul of transformation mix (240 ul of PEG 3350 50% w/v, 36 ul of LiAc 1.0 M, boiled salmon sperm DNA, 34 ul of linearized plasmid DNA plus water). The salmon sperm DNA was boiled for 5 min and pre-chilled before adding it in the transformation mix. The plasmid DNA was previously digested with SbfI (Fermentas), purified with the NucleoSpin Extract II kit (MN) and quantified with the NanoDrop in order to add 1 ug of DNA to the transformation mix.
- The tubes were heated at 42°C for 40 min.
- Cells were pelleted (4000 rpm, 30 sec), the supernatant was removed by pipetting and the pellet was gently resuspended in 1 ml of deionized water.
- Cells were pelleted (4000 rpm, 30 sec), the supernatant was discarded, the pellet was resuspended in 1 ml of YPD and incubated at 30°C, 200 rpm for 3 hours.
- Cells were pelleted (4000 rpm, 30 sec), resuspended in 200 ul of YPD and plated on a YPD agar plate with G418 antibiotic at 200 ug/ml.
- The plates were incubated at 30°C for about 3 days until colonies appeared.
The integration efficiency was estimated as the colony forming units (CFUs) yielded for each ug of DNA.
Protocol references:
[1] http://openwetware.org/wiki/High_Efficiency_Transformation
[2] Guldener U, Heck S, Fiedler T, Beinhauer J, Hegemann JH (1996), A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research, Vol. 24, No. 13 2519–2524.
Results:
The transformed inserts and their integration efficiency in S288C are listed here:
These results suggest that the integrative vector actually works and that the selection marker is highly specific (no colonies appeared on the "no DNA" plate).
The correct phenotype of the S288C bearing these parts has still to be validated (by mOrange fluorescence measurement for the BBa_K300007 part), as well as the actual integration position (by PCR).
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UNIQa28b571152718c33-partinfo-00000008-QINU
