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, developed by Sergio Pesajovich for the 2007 iGEM competition. 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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[[Image:AarI fig1b.png]]
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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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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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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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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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[[Image:AarI fig2b.png]]
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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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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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Cyc1P-GFP-Adh1t-SPACER-8X LexA Ops
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[[Image:AarI fig3.png]]
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Yeast were transformed using standard procedures (LiOAc method).
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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 could be promoters, protein domains, or terminators, and are typically generated by PCR from a genomic DNA or plasmid template, then TOPO cloned and sequenced. These '''donor vectors''', once validated, can be shuffled with other validated parts, into '''acceptor vectors''', creating large combinatorial libraries of constructs that do not require further sequencing.
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== Yeast Strains ==
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While any vector can be adapted to be an acceptor for AarI cloning, we have been working with the yeast pRS__ series of vectors ([http://www.genetics.org/cgi/reprint/122/1/19.pdf]), and we provide to the registry several types of acceptor vectors built in the pRS315 or 305 backbone. When necessary, markers exchanged by one-piece subclones of the completed cassettes into alternative pRS vectors, using the Kpn1/PspOMI and SacI sites.
 
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For more information on how to clone with the UCSF AarI parts, or better yet, to design your own AarI parts, check out the following protocols.  
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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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== 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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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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'''AarI Shuttle Vector'''
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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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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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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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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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!align="center"|[[Team:UCSF/Human Practices|Human Practices]]
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!align="center"|[[Team:UCSF/Notebook|Notebooks]]
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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.


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