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Materials and Methods
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== Materials and Methods == | == Materials and Methods == | ||
| - | '''1. Cloning''' | + | '''1. General Cloning/Molecular biology''' |
Constructs were cloned using the AarI method, developed by Sergio Pesajovich for the 2007 iGEM competition. To learn more about AarI cloning: | Constructs were cloned using the AarI method, developed by Sergio Pesajovich for the 2007 iGEM competition. To learn more about AarI cloning: | ||
| Line 7: | Line 7: | ||
[[Everything you ever wanted to know about AarI]] | [[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 acceptor vectors: | + | 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 marker. | ||
| + | |||
| + | 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. | ||
| + | |||
| + | '''2. Silencing Assay''' | ||
| + | In cases where galactose induction was required, yeast were re-streaked to S-Raffinose plates, plus or minus 2% galactose. Overnight cultures in the same media were diluted in the am (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. | ||
Revision as of 16:52, 22 October 2008
Materials and Methods
1. General Cloning/Molecular biology
Constructs were cloned using the AarI method, developed by Sergio Pesajovich for the 2007 iGEM competition. 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 marker.
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.
2. Silencing Assay In cases where galactose induction was required, yeast were re-streaked to S-Raffinose plates, plus or minus 2% galactose. Overnight cultures in the same media were diluted in the am (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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