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Materials and Methods
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4. pRS305, Ga11P/Adh1t | 4. pRS305, Ga11P/Adh1t | ||
| - | In some cases, finished constructs were subcloned as cassettes (using Kpn1 or PspOMI and SacI sites) into various pRS3__ series vectors to swap the | + | In some cases, finished constructs were subcloned as cassettes (using Kpn1 or PspOMI and SacI sites) into various pRS3__ series vectors to swap the markers. |
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| + | The regional silencing construct was constructed by PCR amplification of a Cyc1P-mCherry-Adh1t cassette with Kpn1 ends, which was cloned into the Kpn1 site upstream of the 5' LexA operators in the GFP reporter plasmid. | ||
Revision as of 06:49, 28 October 2008
Materials and Methods
1. General Cloning/Molecular biology
Constructs were cloned using the AarI method, developed by Sergio Pesajovich the 2007 UCSF iGEM team. To learn more about AarI cloning:
Everything you ever wanted to know about AarI
In brief: yeast genomic DNA was used as a template to PCR amplify "parts". Parts were TOPO cloned, validated by sequencing, and then combinatorially cloned into the following AarI adapted acceptor vectors:
To make the silencers:
1. pRS315, Adh1P/Adh1t
2. pRS315, Cyc1P/Adh1t
3. pRS315, Fig1P/Adh1t
4. pRS305, Ga11P/Adh1t
To make the reporters: (same plasmids as above, with a 5' or 3' 8X LexA operator array)
1. pRS315, Adh1P/Adh1t
2. pRS315, Cyc1P/Adh1t
3. pRS315, Fig1P/Adh1t
4. pRS305, Ga11P/Adh1t
In some cases, finished constructs were subcloned as cassettes (using Kpn1 or PspOMI and SacI sites) into various pRS3__ series vectors to swap the markers.
The regional silencing construct was constructed by PCR amplification of a Cyc1P-mCherry-Adh1t cassette with Kpn1 ends, which was cloned into the Kpn1 site upstream of the 5' LexA operators in the GFP reporter plasmid.
Yeast Strains
SF992/W303 or CB008: W303 MATa, Ste5::KanR, bar1::NatR, far1D, his 3, trp1, leu2, ura3 (for pheromone experiments) were transformed with finished plasmids using standard protocols. The dual-tagged strain used for the cooperativity experiment was built in SF992, and was gal2::NatR, allowing graded activation of galactose inducible promoters (Media:Hawkins and Smolke.pdf).
2. Silencing Assay In cases where galactose induction was required, yeast were re-streaked on complete or dropout synthetic Raffinose plates, plus or minus 2% galactose. Overnight cultures in the same media were diluted in the morning (typically 1:50 or 1:100), and 3 hours later, at OD600 0.05-0.1 range, flow cytometry measurements were taken using a BD LSR-II flow cytometer (BD Biosciences). For each sample, 10,000 cells were counted, and GFP fluorescence was measured by exciting at 488 nm with a 100 mW Coherent Sapphire laser. For experiments with pheromone-inducible promoters, cultures were grown to early log phase (OD600=0.1-0.3) in complete synthetic dropout media and then treated with 1 μM α-factor (Zymo Research) to activate the pathway. Cultures were analyzed after 3 hours of growth.
Representative flow cytometery plots are shown for all experiments, but each experiment was conducted in triplicate (using different clones) with consistent results.
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