Materials and Methods

From 2008.igem.org

(Difference between revisions)
(Materials and Methods)
(Materials and Methods)
Line 1: Line 1:
== 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.


Home The Team The Project Parts Submitted to the Registry Modeling Human Practices Notebooks