Team:Queens-Canada/parts

From 2010.igem.org

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{{:Team:Queens-Canada/head}}
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=Part Structure=
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<h1>WormWorks Parts List</h1>
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<div class="box">
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Below is a complete list of all of the constructs and parts which we extracted and assembled for use in ''C. elegans'' this summer. It is much longer of a list than most projects, which is to be expected since we sought to build a chassis and not merely a BioDevice.
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<html><a href="#" onclick="toggle_visibility('groupparts');">Show/hide parts</a></html> as entered in the [http://partsregistry.org Registry].
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<div id="groupparts" style="display: none;">
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<groupparts>iGEM010 Queens-Canada</groupparts>
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</div>
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</div>
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'''Note:''' The following list is tentative, and better reflects our goals rather than our current accomplishments. We are still in the process of isolating and characterising these components.
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<html><div class="section"><h2>Regulatory</h2></html>
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=General=
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All but one of the regulatory elements we isolated are promoters, and are described in brief detail below, with more elaborate information on their parts registry pages. The remaining BioBrick is the 3' UTR from ''unc-54'', which is roughly equivalent to a bacterial terminator and performs a number of important regulatory functions in complement to the promoter. All complete ''C. elegans'' constructs must include some form of functional 3' UTR. Promoters were selected based on their utility, strength of expression, and ease of avoiding potentially harmful cutsites.
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==Promoters==
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<h3>Constitutive</h3>
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* '''pGpd-2''': ''gpd-2'' is part of the glycolysis pathway. The gpd-2 promoter thus expresses at a very high level—AceView <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?exdb=AceView&db=worm&term=gpd-2&submit=Go">says 43.5 times the average</a></html> for a ''C. elegans'' constitutive gene.
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* '''pSip-1''': ''<html><a target="_new" href="http://wormbase.org/db/gene/gene?name=WBGene00004798;class=Gene">sip-1</a></html>'' encodes a member of the heat shock family of proteins. Accoring to AceView, ''sip-1'' is expressed at a level <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?exdb=AceView&db=worm&term=sip-1&submit=Go">17.6 times the average</a></html>, at all levels of development.
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* '''pRab-7''': ''rab-7'' expresses a GTPase involved in endosome trafficking, and expresses at <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?exdb=AceView&db=worm&term=rab-7&submit=Go">4.3 times the average</a></html>.
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{|
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<h3>Inducible</h3>
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!Name
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!Description
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!Strength
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|-
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|''sip-1''
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|Heat shock
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|
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|-
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|''rab-7''
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|Constitutive
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|high
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|-
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|''mec-7''
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|Mechanosensory neurons
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|
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|-
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|''hsp-3''
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|Heat shock
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|-
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|''gpd-2''
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|Constitutive
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|very high
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|-
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|''unc-47''
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|GABA-using (gabergic) neurons
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|
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|}
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==Coding Sequences==
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* '''pHsp-3''': HSP-3 is a protein involved in the heat shock response pathway, and is expressed constitutively. AceView <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?exdb=AceView&db=worm&term=hsp-3&submit=Go">says this is 22.9 times the average</a></html>. However, <html><a target="_new" href="http://www.wormbase.org/db/gene/gene?name=WBGene00002007;class=Gene">WormBase asserts that</a></html> transcriptional levels can be enhanced by the presence of diothiothreitol or tunicamycin.
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{|
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<h3>Tissue-Specific</h3>
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!Name
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!Description
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|-
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|GFP
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|Enhanced Green Fluorescent Protein. From biobrick BBa_I714891. Proof of concept to see if genes can be imported from bacteria.
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|eGFP
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|Enhanced Green Fluorescent Protein. From Chin-Sang plasmid pIC691.
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|mCherry
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|Red Fluorescent Protein. From Chin-Sang plasmid.
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|-
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|YFP
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|Yellow Fluorescent Protein. From Chin-Sang plasmid.
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|-
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|eCFP
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|Enhanced Cyan Fluorescent Protein. From Chin-Sang plasmid.
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|}
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==Backbones==
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These all target different sensory neurons. For more information on what most of these neurons do, see [[Team:Queens-Canada/nervous#The Amphid|our section on the amphid]].
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* Since it is possible to microinject plasmids into ''C. elegans'', we're working on a combination backbone that will produce GFP in both ''E. coli'' and ''C. elegans'', as well as providing antibiotic selection markers for ''E. coli'', and the cutsites from Standard 10.
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* '''pMec-7''': This targets the mechanoreceptor neurons. We were able to use it in a construct successfully with eCFP and our 3' UTR brick: see <html><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K309032" target="_new">its page on the parts registry</a></html>.
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* Another project we're pursuing is the possible usage of double-stranded RNA as a liaison for DNA import. If this works, we'll have hybrid strand backbones for that method of transformation as well.
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* '''pOdr-1''': This targets the AWC sensory neuron.
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* '''pStr-1''': This targets the AWB sensory neuron.
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* '''pOsm-10''': This targets the ASH and ASI sensory neurons, as well as the PHA and PHB phasmid neurons.
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* '''pFlp-1''': This targets the AVA sensory neuron.
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* '''pSra-10''': This targets the AVB sensory neuron.
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* '''pStr-220''': This targets the AWC sensory neuron.
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=Optogenetics (R.''C. elegans'')=
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<html></div><div class="section"><h2>Reporters</h2></html>
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==Promoters==
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* '''eGFP''': excitation: 395 nm; emission: 509 nm.
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* '''eCFP''': excitation: 433 nm; emission: 475 nm.
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* '''eYFP''': excitation: ~485 nm; emission: ~700 nm.
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* '''mCherry''': excition: ~580 nm; emission: ~620 nm.
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{|
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<html></div><div class="section"><h2>Optogenetics Proteins</h2></html>
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!Name
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!Description
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!Strength
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|-
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|}
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==Coding Squences==
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* '''ChR2''' (channelrhodopsin-2): A surface protein. Excitation by light at 460 nm causes the admission of cations into the cell. These cations are nonspecific (H<sup>+</sup>, K<sup>+</sup>, Na<sup>+</sup>, Ca<sup>2+</sup>), but can directly trigger the depolarization of a neuron, forcing it into a firing state where it will remain until the light source is removed.
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* '''NpHR''' (halorhodopsin): A surface protein. Excitation by light at 580 nm causes the admission of chloride anions into the cell. If used in a neuron, this can prevent it from firing as long as the light source is present.
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* ''Fusions'': ChR2::eYFP and NpHR::eCFP are both also provided. These are useful for ensuring that the channels localized successfully to their target.
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The excitation wavelengths of these two proteins are different enough that they can actually be implemented in the same organism and triggered separately, with very little cross-talk.  This means that it is possible to control two different neural inputs in the same worm at the same time.  Such an arrangement opens up many possibilities, such as using one protein to convince the worm that it needs to go forward, and another that that it needs to go backwards.  Similarly, one protein could be used to trigger the worm to turn, and another to keep it going straight.
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<html></div><div class="section"><h2>Constructs</h2></html>
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<html>
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<img src="http://2010.igem.org/wiki/images/7/72/Qgem_content_mec7_ecfp.jpg" style="float: right; max-width: 800px; width: 40%; box-shadow: 1px 2px 3px #808080; margin-left: 10px;" title="Our mec-7::eCFP::unc-54 3' UTR construct in action" alt="Our mec-7::eCFP::unc-54 3' UTR construct in action">
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</html>
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Unlike the fusions listed under Optogenetics Proteins, these were assembled through BioBrick digestion/ligation.
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* '''pStr-220::eCFP::unc-54 3' UTR'''
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* '''pOdr-10::eCFP::unc-54 3' UTR'''
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* '''pGpd-2::eCFP::unc-54 3' UTR'''
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* '''pSip1::eCFP::unc-54 3' UTR'''
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* '''pFlp1::eCFP:unc-54 3' UTR'''
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* '''ChR2::eYFP::unc-54 3' UTR'''
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* '''pHsp3::eCFP::unc-54 3' UTR'''
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* '''pOsm-10::eCFP::unc-54 3' UTR'''
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* '''mCherry::unc-54 3' UTR'''
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* '''eYFP::unc-54 3' UTR'''
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* '''pOdr-1::eCFP::unc-54 3' UTR'''
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* '''pMec-7::eCFP::unc-54 3' UTR'''
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<html></div><div class="section"><h2>Getting the Parts</h2></html>
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You can get our special worm parts through the standard iGEM distribution channel: the <html><a href="http://partsregistry.org" target="_new">Parts Registry</a></html>. Click on a part number below to be taken to the relevant description page.
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<html><script type="text/javascript">
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function toggle_visibility(id) {
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el = document.getElementById(id);
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if(el.style.display == "none") {
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el.style.display = "block";
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} else {
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el.style.display = "none";
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}
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}</script>
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<div class="box">
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<div id="groupparts" style="display: block;">
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</html><groupparts>iGEM010 Queens-Canada</groupparts><html>
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</div>
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</div>
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{|
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</div></html>
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!Name
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!Description
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|-
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|ChR2
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|Channelrhodopsin-2. Permits entry of positive ions in response to 460 nm light. Can be used to trigger nerve cells.
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|NpHR
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|Halorhodopsin. Permits entry of chloride ions in response to 580 nm light. Can be used to block nerve cells from firing.
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|}
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{{:Team:Queens-Canada/foot}}
{{:Team:Queens-Canada/foot}}

Latest revision as of 00:24, 28 October 2010

WormWorks Parts List

Below is a complete list of all of the constructs and parts which we extracted and assembled for use in C. elegans this summer. It is much longer of a list than most projects, which is to be expected since we sought to build a chassis and not merely a BioDevice.

Regulatory

All but one of the regulatory elements we isolated are promoters, and are described in brief detail below, with more elaborate information on their parts registry pages. The remaining BioBrick is the 3' UTR from unc-54, which is roughly equivalent to a bacterial terminator and performs a number of important regulatory functions in complement to the promoter. All complete C. elegans constructs must include some form of functional 3' UTR. Promoters were selected based on their utility, strength of expression, and ease of avoiding potentially harmful cutsites.

Constitutive

  • pGpd-2: gpd-2 is part of the glycolysis pathway. The gpd-2 promoter thus expresses at a very high level—AceView says 43.5 times the average for a C. elegans constitutive gene.
  • pSip-1: sip-1 encodes a member of the heat shock family of proteins. Accoring to AceView, sip-1 is expressed at a level 17.6 times the average, at all levels of development.
  • pRab-7: rab-7 expresses a GTPase involved in endosome trafficking, and expresses at 4.3 times the average.

Inducible

Tissue-Specific

These all target different sensory neurons. For more information on what most of these neurons do, see our section on the amphid.

  • pMec-7: This targets the mechanoreceptor neurons. We were able to use it in a construct successfully with eCFP and our 3' UTR brick: see its page on the parts registry.
  • pOdr-1: This targets the AWC sensory neuron.
  • pStr-1: This targets the AWB sensory neuron.
  • pOsm-10: This targets the ASH and ASI sensory neurons, as well as the PHA and PHB phasmid neurons.
  • pFlp-1: This targets the AVA sensory neuron.
  • pSra-10: This targets the AVB sensory neuron.
  • pStr-220: This targets the AWC sensory neuron.

Reporters

  • eGFP: excitation: 395 nm; emission: 509 nm.
  • eCFP: excitation: 433 nm; emission: 475 nm.
  • eYFP: excitation: ~485 nm; emission: ~700 nm.
  • mCherry: excition: ~580 nm; emission: ~620 nm.

Optogenetics Proteins

  • ChR2 (channelrhodopsin-2): A surface protein. Excitation by light at 460 nm causes the admission of cations into the cell. These cations are nonspecific (H+, K+, Na+, Ca2+), but can directly trigger the depolarization of a neuron, forcing it into a firing state where it will remain until the light source is removed.
  • NpHR (halorhodopsin): A surface protein. Excitation by light at 580 nm causes the admission of chloride anions into the cell. If used in a neuron, this can prevent it from firing as long as the light source is present.
  • Fusions: ChR2::eYFP and NpHR::eCFP are both also provided. These are useful for ensuring that the channels localized successfully to their target.

The excitation wavelengths of these two proteins are different enough that they can actually be implemented in the same organism and triggered separately, with very little cross-talk. This means that it is possible to control two different neural inputs in the same worm at the same time. Such an arrangement opens up many possibilities, such as using one protein to convince the worm that it needs to go forward, and another that that it needs to go backwards. Similarly, one protein could be used to trigger the worm to turn, and another to keep it going straight.

Constructs

Our mec-7::eCFP::unc-54 3' UTR construct in action

Unlike the fusions listed under Optogenetics Proteins, these were assembled through BioBrick digestion/ligation.

  • pStr-220::eCFP::unc-54 3' UTR
  • pOdr-10::eCFP::unc-54 3' UTR
  • pGpd-2::eCFP::unc-54 3' UTR
  • pSip1::eCFP::unc-54 3' UTR
  • pFlp1::eCFP:unc-54 3' UTR
  • ChR2::eYFP::unc-54 3' UTR
  • pHsp3::eCFP::unc-54 3' UTR
  • pOsm-10::eCFP::unc-54 3' UTR
  • mCherry::unc-54 3' UTR
  • eYFP::unc-54 3' UTR
  • pOdr-1::eCFP::unc-54 3' UTR
  • pMec-7::eCFP::unc-54 3' UTR

Getting the Parts

You can get our special worm parts through the standard iGEM distribution channel: the Parts Registry. Click on a part number below to be taken to the relevant description page.

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