Team:UCSF/Materials and Methods

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Materials and Methods

1. General Cloning/Molecular biology

Constructs were cloned using the AarI method, developed by Sergio Pesajovich and 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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