Materials and Methods

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== Materials and Methods ==
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== General Cloning/Molecular biology ==
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'''1. Cloning'''
 
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Constructs were cloned by the AarI method. This a multi-part/combinatorial cloning method that is particularly well suited to shuffling protein domains with various promoters and terminators. The key to this approach is the Type II-S restriction enzyme, AarI, a rare (7-cutter) that cuts 4bp offset from its binding site. Thus, '''AarI can generate four base overhangs of any sequence'''.  
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Constructs were cloned using the AarI method, developed by Sergio Pesajovich and the 2007 UCSF iGEM team.  
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To learn more about AarI cloning: '''[[Everything you ever wanted to know about AarI]]'''
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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 pRS series AarI-adapted acceptor vectors.
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[[Image:AarI fig1b.png]]
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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.
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The distant silencing (250, 500, 1000, 2000 and 3000 bp variants) were constructed using spacer fragments of corresponding length that were PCR amplified from the coding region of the mammalian PI3K gene, which we reasoned was likely to be relatively free of regulatory sites that would be recognized in yeast. The spacer fragments were subcloned into the SacI site between the Adh1 terminator and LexA operator of the 3' silencing construct:
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Since the user can specify the overhangs, this method can be used to "stitch-together" fragments '''without a scar''', which is sometimes necessary to preserve protein function. More importantly, these overhangs can be '''non-palindromic''', which solves the biggest problem faced when trying to do multipart ligations using standard restriction enzymes, illustrated here:
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Cyc1P-GFP-Adh1t-SPACER-8X LexA Ops
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Yeast were transformed using standard procedures (LiOAc method).
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== Yeast Strains ==
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[[Image:AarI fig2.png]]
 
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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]]).
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By contrast, AarI cloning allows high efficiency ligations using up to 4 parts (vector plus 3 inserts). While parts can be made with any 4 base overhang (end), we chose a standard set, termed A, B, C, and D. This allows parts to be traded between researchers. We are building a lab database of parts.
 
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== Silencing Assay ==
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In the standard silencing assay, overnight cultures 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. GFP fluorescence was measured by exciting at 488 nm with a 100 mW Coherent Sapphire laser. mCherry fluorescence was measured by excitation at 532 nm with a 50-150 mW Coherent Compass laser.
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[[Image:AarI fig3.png]]
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In cases where galactose induction was required, yeast were re-streaked on complete or dropout synthetic Raffinose plates, plus or minus 2% galactose, and grown in liquid media of the same formulation.  
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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.
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For the caloric restriction experiments, cells were plated/cultured on 0.5% glucose SD media, and compared to the standard 2% glucose plates.
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These ends yield 3 possible parts: AB, BC and CD. For two part ligations, we use AB and BD parts. Parts are typically generated by PCR from a genomic DNA or plasmid template, then TOPO cloned and sequenced.  
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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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{| style="color:#333333;background-color:#cccccc;" cellpadding="3" cellspacing="3" border="0" bordercolor="#231f26" width="99%" align="center"
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!align="center"|[[Team:UCSF|Home]]
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!align="center"|[[Team:UCSF/Team|The Team]]
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!align="center"|[[Team:UCSF/Project|The Project]]
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!align="center"|[[Team:UCSF/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:UCSF/Modeling|Modeling]]
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Digestion yields parts with standard, non-palindromic, overhangs.
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!align="center"|[[Team:UCSF/Human Practices|Human Practices]]
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!align="center"|[[Team:UCSF/Notebook|Notebooks]]
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|}
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'''AarI Shuttle Vector'''
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To facilitate exchange of parts between AarI users and the biobricks community, we are offering a Shuttle Vector. This vector accepts AarI parts in the AB or BD format (as were used in our 2008 project). These parts can then be cut out of the vector, with in-frame biobrick ends.
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Latest revision as of 23:50, 29 October 2008

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 pRS series AarI-adapted acceptor vectors.

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.

The distant silencing (250, 500, 1000, 2000 and 3000 bp variants) were constructed using spacer fragments of corresponding length that were PCR amplified from the coding region of the mammalian PI3K gene, which we reasoned was likely to be relatively free of regulatory sites that would be recognized in yeast. The spacer fragments were subcloned into the SacI site between the Adh1 terminator and LexA operator of the 3' silencing construct:

Cyc1P-GFP-Adh1t-SPACER-8X LexA Ops

Yeast were transformed using standard procedures (LiOAc method).

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).


Silencing Assay

In the standard silencing assay, overnight cultures 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. GFP fluorescence was measured by exciting at 488 nm with a 100 mW Coherent Sapphire laser. mCherry fluorescence was measured by excitation at 532 nm with a 50-150 mW Coherent Compass laser.

In cases where galactose induction was required, yeast were re-streaked on complete or dropout synthetic Raffinose plates, plus or minus 2% galactose, and grown in liquid media of the same formulation.

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.

For the caloric restriction experiments, cells were plated/cultured on 0.5% glucose SD media, and compared to the standard 2% glucose plates.

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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