http://2010.igem.org/wiki/index.php?title=Special:Contributions/Glh&feed=atom&limit=50&target=Glh&year=&month=2010.igem.org - User contributions [en]2024-03-28T20:24:25ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Queens-Canada/contributingTeam:Queens-Canada/contributing2010-10-28T03:34:01Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>Contributing</h1><br />
<br />
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border: 1px #808080 solid;<br />
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<div class="asideR"><p>A simple worm construct:</p><table id="magic_table"><tr><td align="center">Promoter Brick<td align="center">Protein-Coding Brick<td align="center">3' UTR Brick</table><p></p></div></html><br />
<br />
We expect that future worm BioBricks will be submitted to the Parts Registry just like every other standard biological part, and use the conventional ligation standards such as Standard 10, which we utilized for our project. In the future, this will make sense to extend so that it fully supports protein fusions through the use of [[Team:Queens-Canada/transcripts|introns]] to conceal scars by placing intron splice sequences at the starting and finishing parts of fusion-compatible bricks, or by using special primers to insert the intron sequences during PCR to provide more control over which ends of the bricks are concealed. For now, however, this article will concentrate on the format we’ve put into practice.<br />
<br />
<html><div class="section"><h2>Promoter Format</h2></html><br />
<br />
The majority of ''C. elegans'' promoters used in research don’t draw a distinction between the cis-acting recognition sites used during the RNA transcription process and the upstream region of the transcript used during polypeptide translation, instead combining the two and labelling that the promoter. [http://worfdb.dfci.harvard.edu/promoteromedb/ Promoterome], for example, goes as far as including the start codon itself in its definition of the promoter. While this is simplistic for synthetic biology’s purposes, for the time being we have largely chosen to keep this format, although to cope with the scarring caused by ligation, we do not include the start codon in the promoter, and so typically remove the three final nucleotides of the promoter sequence.<html></div></html><br />
<br />
<html><div class="section"><h2>Coding Brick Format</h2></html><br />
<br />
The coding brick, in our format, extends from the start of a [[Team:Queens-Canada/promoter|synthetic Kozak sequence]], past the start codon, through the coding DNA itself, and ends with the stop codon. It may contain introns. The Kozak sequence we typically use are the nucleotides '''GCCGCCACC''' placed immediately before the start codon. This improves translational efficiency, and can be altered to provide the additional flexibility of controlling expression from within the protein-coding component of a complete gene. ''Note'': under certain circumstances in ''C. elegans'', the UGA codon does not stop translation, but instead appends the rare amino acid selenocysteine. While this has not impeded us so far, it may be preferable to use UAA or UAG instead to be safe.<html></div></html><br />
<br />
<html><div class="section"><h2>3' UTR Format</h2></html><br />
<br />
These are taken verbatim from available databases and laboratories after checking for cut sites. Mutagenesis of these sites typically has few ramifications. See the [[Team:Queens-Canada/rnai|article on 3' UTRs]] for more information on why.<html></div></html><br />
<br />
<html><div class="section"><h2>Future Project Ideas</h2></html><br />
<br />
We realize that the idea of working with an organism other than the familiar E. coli chassis is probably an challenging prospect for a lot of teams, especially when it comes to the design phase. We came up with a lot of ideas that only seemed apparent after we had done a great deal of research on the worm; many of them are foundational advances that are analogous to things that can be done currently in bacteria, and would be crucial to making ''C. elegans'' a fully-viable system. We've put the most lucid bunch of these ideas here, with the hope that it would help jump-start other teams and make their work less daunting.<br />
<br />
<h3>"Backward compatibility" with E. coli</h3><br />
<br />
* '''Exosymbiosis'''. Attaching the bacteria to the outside of the worm (using lectins?) on a surface-disrupted mutant. This may require bacteria that don't emit the usual nutritive signal. The [https://2007.igem.org/Berkeley_UC BactoBlood] team went to some length to try and perfect such a bacterium, and since we're not talking about putting things in people, their work may be applicable here. An extension of this might be found in enabling the bacteria to attach or detach from the worms under certain circumstances, creating a bacteria distribution and relocation mechanism. Such a project would need to look out for the potential of covering the amphid or mouth, which some bacteria do naturally as a means of killing worms.<br />
* '''Enterosymbiosis'''. There are cases in nature where nematodes and bacteria have learned to work more or less together, in which the bacteria reside in the worm's gut. Studying ''Xenorhabdus nematophilia'', ''Photorhabdus luminescens'', and the ''Heterorhabditidae'' worms and trying to create something similar between ''E. coli'' and ''C. elegans''; also look into ''bus'' mutants.<br />
<br />
<h3>Messaging in the worm</h3><br />
<br />
* '''Inter-worm signalling'''. The ''C. elegans'' excretory gland normally produces some pheromones that are used for communication between worms; so do anus and vulva. Could we engineer these organs to produce and excrete some new molecule? The potential secondary applications are extensive. We haven't been able to find much literature on these topics, but the prospect is alluring.<br />
* '''Hormones'''. The [[Team:Queens-Canada/pseudocoelom|pseudocoelom]] bathes most tissues, and it's believed that the worm has a number of hormones that it uses to communicate between tissues. It might be possible to transmit stuff in here of our own devising. As of yet, we don't have a strategy for receiving molecules from the pseudocoelom by a cell, but a literature search helped us find a tag that works to introduce molecules into the pseudocoelom. You can find it in our [[Team:Queens-Canada/pseudocoelom|pseudocoelom article]]. We have not personally tested this technique.<br />
<br />
<h3>Genetics</h3><br />
<br />
* '''Self-censorship'''. ''C. elegans'' supports [[Team:Queens-Canada/rnai|RNAi]], a technique that leverages machinery which enables complementary RNA molecules to destroy each other. RNAi is generally used to knock out genes by feeding the worm bacteria that produce the complementary RNAs, or soaking the worm in a large volume of these RNAs. Since integrating new DNA into the ''C. elegans'' chromosomes is difficult and time-consuming, it may make more sense to give the worm a plasmid that knocks out the mRNAs autonomically. To restore functionality of a protein knocked out in this way, especially a mutated version of such a protein, consider employing heavy use of synonyms (alternative codons for the same amino acid). See our article on RNAi.<br />
* '''Variable UTRs'''. We've only provided a single 3' UTR in our kit (a synthetic modification of unc-54's 3' UTR). However, it's known that UTRs play a role in tissue specificity and expression strength by binding various factors. A better understanding of these UTRs, or even just a larger variety of them, stands to be a very useful project.<br />
* Modular promoters. Heidelberg [https://2009.igem.org/Team:Heidelberg stole the show in 2009] by creating a detailed set of promoter elements that could be arranged to produce predictable and precise variations in strength for HeLa cells. It would be very cool if something like this could be done for worms, especially if this could also include tissue specificity. The potential for doing something similar for 3' UTRs is also alluring.<br />
<br />
<h3>Biosynthesis</h3><br />
<br />
* The [[Team:Queens-Canada/digestive|'''hindgut''']] (intestine) of ''C. elegans'' can be targeted with expression patterns that place emphasis on the front and back regions. In combination with the intestinal export tags used for secreting proteases and other digestive enzymes, this provides an environment of approximately pH 5 in which multi-step biosynthesis could be possible.<br />
<br />
<h3>Fundamental Tools</h3><br />
<br />
* One thing we were hoping to make this summer but never got around to was a '''multi-species plasmid''': a new BioBrick backbone vector that would express a fluorescent protein in both ''C. elegans'' and ''E. coli''. This would make it possible to eliminate the coinjection marker from the microinjection process, and guarantee that the DNA was functional in the worm if an injection were successful. In order to be grown up in ''E. coli'', however, it would need to reuse the coding sequence for both eukaryotic and prokaryotic translation in order to save space. Implementing this might require the crafty use of [[Team:Queens-Canada/transcripts|introns]].<br />
<html></div></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/contributingTeam:Queens-Canada/contributing2010-10-28T03:29:18Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>Contributing</h1><br />
<br />
<html><style type="text/css"><br />
#magic_table td {<br />
border: 1px #808080 solid;<br />
}<br />
</style><br />
<div class="asideR"><p>A simple worm construct:</p><table id="magic_table"><tr><td align="center">Promoter Brick<td align="center">Protein-Coding Brick<td align="center">3' UTR Brick</table><p></p></div></html><br />
<br />
We expect that future worm BioBricks will be submitted to the Parts Registry just like every other standard biological part, and use the conventional ligation standards such as Standard 10, which we utilized for our project. In the future, this will make sense to extend so that it fully supports protein fusions through the use of [[Team:Queens-Canada/transcripts|introns]] to conceal scars by placing intron splice sequences at the starting and finishing parts of fusion-compatible bricks, or by using special primers to insert the intron sequences during PCR to provide more control over which ends of the bricks are concealed. For now, however, this article will concentrate on the format we’ve put into practice.<br />
<br />
<html><div class="section"><h2>Promoter Format</h2></html><br />
<br />
The majority of ''C. elegans'' promoters used in research don’t draw a distinction between the cis-acting recognition sites used during the RNA transcription process and the upstream region of the transcript used during polypeptide translation, instead combining the two and labelling that the promoter. [http://worfdb.dfci.harvard.edu/promoteromedb/ Promoterome], for example, goes as far as including the start codon itself in its definition of the promoter. While this is simplistic for synthetic biology’s purposes, for the time being we have largely chosen to keep this format, although to cope with the scarring caused by ligation, we do not include the start codon in the promoter, and so typically remove the three final nucleotides of the promoter sequence.<html></div></html><br />
<br />
<html><div class="section"><h2>Coding Brick Format</h2></html><br />
<br />
The coding brick, in our format, extends from the start of a [[Team:Queens-Canada/promoter|synthetic Kozak sequence]], past the start codon, through the coding DNA itself, and ends with the stop codon. It may contain introns. The Kozak sequence we typically use are the nucleotides '''GCCGCCACC''' placed immediately before the start codon. This improves translational efficiency, and can be altered to provide the additional flexibility of controlling expression from within the protein-coding component of a complete gene. ''Note'': under certain circumstances in ''C. elegans'', the UGA codon does not stop translation, but instead appends the rare amino acid selenocysteine. While this has not impeded us so far, it may be preferable to use UAA or UAG instead to be safe.<html></div></html><br />
<br />
<html><div class="section"><h2>3' UTR Format</h2></html><br />
<br />
These are taken verbatim from available databases and laboratories after checking for cut sites. Mutagenesis of these sites typically has few ramifications. See the [[Team:Queens-Canada/rnai|article on 3' UTRs]] for more information on why.<html></div></html><br />
<br />
<html><div class="section"><h2>Future Project Ideas</h2></html><br />
<br />
We realize that the idea of working with an organism other than the familiar E. coli chassis is probably an challenging prospect for a lot of teams, especially when it comes to the design phase. We came up with a lot of ideas that only seemed apparent after we had done a great deal of research on the worm; many of them are foundational advances that are analogous to things that can be done currently in bacteria, and would be crucial to making ''C. elegans'' a fully-viable system. We've put the most lucid bunch of these ideas here, with the hope that it would help jump-start other teams and make their work less daunting.<br />
<br />
<h3>"Backward compatibility" with E. coli</h3><br />
<br />
* '''Exosymbiosis'''. Attaching the bacteria to the outside of the worm (using lectins?) on a surface-disrupted mutant. This may require bacteria that don't emit the usual nutritive signal. The [http://parts.mit.edu/igem07/index.php/Berkeley_UC BactoBlood] team went to some length to try and perfect such a bacterium, and since we're not talking about putting things in people, their work may be applicable here. An extension of this might be found in enabling the bacteria to attach or detach from the worms under certain circumstances, creating a bacteria distribution and relocation mechanism. Such a project would need to look out for the potential of covering the amphid or mouth, which some bacteria do naturally as a means of killing worms.<br />
* '''Enterosymbiosis'''. There are cases in nature where nematodes and bacteria have learned to work more or less together, in which the bacteria reside in the worm's gut. Studying ''Xenorhabdus nematophilia'', ''Photorhabdus luminescens'', and the ''Heterorhabditidae'' worms and trying to create something similar between ''E. coli'' and ''C. elegans''; also look into ''bus'' mutants.<br />
<br />
<h3>Messaging in the worm</h3><br />
<br />
* '''Inter-worm signalling'''. The ''C. elegans'' excretory gland normally produces some pheromones that are used for communication between worms; so do anus and vulva. Could we engineer these organs to produce and excrete some new molecule? The potential secondary applications are extensive. We haven't been able to find much literature on these topics, but the prospect is alluring.<br />
* '''Hormones'''. The [[Team:Queens-Canada/pseudocoelom|pseudocoelom]] bathes most tissues, and it's believed that the worm has a number of hormones that it uses to communicate between tissues. It might be possible to transmit stuff in here of our own devising. As of yet, we don't have a strategy for receiving molecules from the pseudocoelom by a cell, but a literature search helped us find a tag that works to introduce molecules into the pseudocoelom. You can find it in our [[Team:Queens-Canada/pseudocoelom|pseudocoelom article]]. We have not personally tested this technique.<br />
<br />
<h3>Genetics</h3><br />
<br />
* '''Self-censorship'''. ''C. elegans'' supports [[Team:Queens-Canada/rnai|RNAi]], a technique that leverages machinery which enables complementary RNA molecules to destroy each other. RNAi is generally used to knock out genes by feeding the worm bacteria that produce the complementary RNAs, or soaking the worm in a large volume of these RNAs. Since integrating new DNA into the ''C. elegans'' chromosomes is difficult and time-consuming, it may make more sense to give the worm a plasmid that knocks out the mRNAs autonomically. To restore functionality of a protein knocked out in this way, especially a mutated version of such a protein, consider employing heavy use of synonyms (alternative codons for the same amino acid). See our article on RNAi.<br />
* '''Variable UTRs'''. We've only provided a single 3' UTR in our kit (a synthetic modification of unc-54's 3' UTR). However, it's known that UTRs play a role in tissue specificity and expression strength by binding various factors. A better understanding of these UTRs, or even just a larger variety of them, stands to be a very useful project.<br />
* Modular promoters. Heidelberg [https://2009.igem.org/Team:Heidelberg stole the show in 2009] by creating a detailed set of promoter elements that could be arranged to produce predictable and precise variations in strength for HeLa cells. It would be very cool if something like this could be done for worms, especially if this could also include tissue specificity. The potential for doing something similar for 3' UTRs is also alluring.<br />
<br />
<h3>Biosynthesis</h3><br />
<br />
* The [[Team:Queens-Canada/digestive|'''hindgut''']] (intestine) of ''C. elegans'' can be targeted with expression patterns that place emphasis on the front and back regions. In combination with the intestinal export tags used for secreting proteases and other digestive enzymes, this provides an environment of approximately pH 5 in which multi-step biosynthesis could be possible.<br />
<br />
<h3>Fundamental Tools</h3><br />
<br />
* One thing we were hoping to make this summer but never got around to was a '''multi-species plasmid''': a new BioBrick backbone vector that would express a fluorescent protein in both ''C. elegans'' and ''E. coli''. This would make it possible to eliminate the coinjection marker from the microinjection process, and guarantee that the DNA was functional in the worm if an injection were successful. In order to be grown up in ''E. coli'', however, it would need to reuse the CDS for both eukaryotic and prokaryotic translation in order to save space. Implementing this might require the crafty use of [[Team:Queens-Canada/transcripts|introns]].<br />
<html></div></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/rnaiTeam:Queens-Canada/rnai2010-10-28T03:24:29Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<html><div class="section"><h1>RNA Interference and the 3' UTR</h1></div><br />
<br />
<div class="section"><h2>RNAi</h2></html><br />
<br />
'''RNA interference''' is a laboratory technique wherein a natural gene-silencing mechanism is exploited to suppress undesired gene activity, by targeting messenger RNAs with their complementary sequences. It was discovered in ''C. elegans'', and has been used in many other organisms since.<br />
<br />
Like DNA, RNA can form a stable duplex, although it is much more delicate in vitro as a result of the abundant ribonucleases (RNases) produced by higher animals. This double-stranded RNA ('''dsRNA''') is used as the storage format for the genomes of Group III viruses, and in the laboratory is typically formed using viral RNA polymerase, either ''in vitro'' like a single-step PCR reaction, or ''in vivo'' using engineered ''E. coli'', which are then fed to the worm. The double-stranded RNA enters the worm’s tissues via the '''SID-2''' channels located in the intestine, where it is then detected by a type of RNase III, called '''Dicer'''.<br />
<br />
Dicer is an endonuclease that cuts dsRNA into 20–25 bp-long fragments, with 2 nt overhangs. These fragments, called small interfering RNAs (siRNAs), are responsible for the job of actually censoring messenger RNAs: they can fit through another channel called '''SID-1''', which allows the RNA interference effect to spread throughout the rest of the worm’s body, except neurons, and bind to RDE-4 to create the RNA-induced silencing complex (RISC), which catches messenger RNAs that match the siRNA, and then cuts them up. In addition, the siRNA is amplified directly by an RNA-dependent RNA polymerase.<br />
<br />
<html><a name="mirna"></a></html>The RNAi mechanism most likely evolved to stop dsRNA viruses, and plays an important role in restraining the activity of cancer-fighting interferon proteins, as well as transposons (genetic elements that use transcriptases and endonucleases to move their sequences around the genome.) However, it is also used natively as a gene-silencing mechanism, and a gene-moderating mechanism; there are 175 known '''miRNAs''' ('''MicroRNAs''') in ''C. elegans'', which in some cases may be more sensitive and not induce full suppression due to imperfect base-pairing. miRNAs often target sequences in the 3' untranslated region of messenger RNAs rather than within the CDS (coding sequence), as these may more readily be selected to contain a distinct sequence without affecting protein structure or function.<br />
<br />
Protocols for working with RNAi can be found can be found at <html><a target="_new" href="http://cshprotocols.cshlp.org/cgi/search?tocsectionid=Protocol&tocsectionid=Topic+Introduction&tocsectionid=Information+Panel&tocsectionid=Recipe&tocsectionid=Product+Protocol&tocsectionid=Kit&tocsectionid=Emerging+Model+Organisms&fulltext=RNAi+elegans&Go.x=0&Go.y=0">Cold Spring Harbor Protocols</a></html>. <br />
<br />
RNAi offers synthetic biologists the opportunity to selectively silence gene expression, and may in the future allow for finer tuning of these effects (i.e. dampening expression to varying degrees as opposed to abruptly silencing it). RNAi is a versatile technique in that its effects can be induced by ingestion of RNAi (through feeding E. coli that have been engineered to express it, or through direct exposure) or by expression of RNAi by the worm. The latter offers especially interesting opportunities, as the expression can be put under an inducible promoter, which allows a gene to be silenced as a result of an external factor or input. For example, a temperature-regulated promoter could be used to induce silencing of a gene.<br />
<br />
<html><div class="asideL"><p><b>More on RNAi</b></p><ul><br />
<li><a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed?term=rnai%20mec-8">This paper</a> outlines a method for the inducible expression (in this case by temperature) of RNAi that results in the inhibition of mechanosensation by a worm.<br />
<li><a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed/18443013">Here</a> is a fantastic overview of the major RNAi techniques used by researchers that study gene expression. Many of these techniques could be useful tools to a synthetic biologist working with C. elegans.<br />
<li>As mentioned above, the neurons in the N2 strain which we use as a standard chassis lack SID-1 (the receptor that allows siRNAs to proliferate from one cell to another) rendering them invulnerable to the effects of ingested RNAi. <a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed?term=enhanced%20neuronal%20rnai%20sid-1">This paper</a> demonstrates that neurons can be made susceptible to RNAi by engineering them to express SID-1.<br />
</ul><br />
</div></div><br />
<br />
<div class="section"><h2>The 3' UTR</h2></html><br />
<br />
In eukaryotes, there is typically a large amount of RNA in a messenger RNA that is not part of the coding sequence itself. The portion that precedes the start codon is called the 5' untranslated region, and the portion that follows it is called the '''3' untranslated region'''. Both of these are generally hundreds of basepairs in length, which often inflates the apparent size of ''C. elegans'' promoters, as worm biologists typically measure up to the start codon, rather than drawing a distinction between the transcription and translation steps.<br />
<br />
The 3' UTR, however, extends in the opposite direction, after the stop codon, and provides binding sites for moderating proteins and miRNAs (see <html><a href="#mirna">above</a></html>), as well as stability and protection against RNA-degrading proteins, due to polyadenylation. (This is contrary to bacteria, where poly(A) sequences promote degradation for coding RNAs instead of preventing it.) 3' UTRs can greatly extend the lifespan of a messenger RNA transcript in the cell, as well as select for tissue and growth phase specificity. It is only in combination with 3' UTRs that promoters are able to target individual cells with the incredible precision necessary for the minute detail of the nematode’s structures.<br />
<br />
The most important element of the 3' UTR is the polyadenylation signal, which also serves as the effective transcriptional terminator, even though the stop site of transcription may vary by up to 50 bases. The most common polyadenylation signal is AAUAAA, which is recognized in the transcript by a protein called CstF. The poly(A) sequence produced by the AAUAAA sequence is 250 nt long, which is sufficient to last several hours under ideal circumstances, and gradually shortens (variations on this signal yield shorter tails.) In addition to limiting degradation, poly(A) sequences are involved in nuclear export and translational regulation.<br />
<br />
For the original WormWorks chassis set, only the 3' UTR from the gene ''unc-54'' is included. This 3' UTR is the one most frequently used in ''C. elegans'' research when constructing transgenes, and gives good, constitutive expression in all tissues, allowing selection to be done by promoters only. However, this also limits the precision with which we can target and differentiate between worm tissues. Future work in ''C. elegans'' synthetic biology may benefit greatly from the ability to design and assemble 3' UTRs with predictable, flexible, quantified characteristics. Work towards this may benefit from the knowledge gathered at <html><a target="_new" href="http://128.122.61.5/cgi-bin/UTRome/utrome.cgi">UTRome</a></html> and <html><a target="_new" href="http://utrdb.ba.itb.cnr.it/">UTRdb</a></html>.<br />
<br />
'''[[Team:Queens-Canada/guide|Return to the Guide Hub]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/promoterTeam:Queens-Canada/promoter2010-10-28T03:22:50Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>The Promoter and 5' UTR</h1><br />
<br />
<html><div class="section"><h2>The Eukaryotic Promoter</h2></html><br />
<br />
The promoter is a stretch of DNA anywhere from a hundred to two thousand bases in length which prefaces the segment of DNA that will be transcribed. The promoter serves as one of the largest centers of regulatory activity; many different cofactors are typically involved in determining whether or not a transcript should be created for a gene. For a long time, it was presumed to be the only point in the central dogma at which regulation occurs, but it has since been determined that each step of the DNA-to-RNA-to-protein-to-destination has some amount of control in establishing the fate of a gene product.<br />
<br />
In C. elegans, the promoter has not been studied very extensively. Because of the efficiency of <html><a href="https://2010.igem.org/Team:Queens-Canada/transcripts#%3Ci%3ETrans%3C/i%3E-splicing%20(and%20operons)"><i>trans</i>-splicing</a></html>, it has been impossible to determine the actual start of transcription for most genes from pre-mRNA using traditional techniques from molecular genetics. It is generally assumed that the majority of steps closely reflect those of other, better-studied eukaryotes like yeast, humans, and fruitflies, and even WormBook falls back on material from these sources in <html><a target="_new" href="http://www.wormbook.org/chapters/www_transcriptionmechanisms/transcriptionmechanisms.html">its description of the pre-initiation process</a></html>, which is a must-read for those interested in synthetic worm biology and cannot be usefully summarized here.<br />
<br />
Regulation of transcription in C. elegans is a messy subject, as it is with many eukaryotic genes. Regulatory elements may be located in introns, several kilobases upstream, or, as in the case of ''egl-1'', downstream, on the other side of an unrelated gene. ''lin-39'' required 30 kb of surrounding DNA to reproduce its expression pattern faithfully—but these are exceptional cases. The majority of ''C. elegans'' protein-coding genes occur in tightly-packed groups, where there is no room for unusual structures such as these, and their promoters, including remote elements, can be adequately contained within less than 2000 bases; some are less than a hundred.<br />
<br />
Engineering a synthetic promoter or promoter system for ''C. elegans'' has the potential to be quite a substantial and worthwhile project, but the amount of work involved at present may be prohibitive. See <html><a target="_new" href="http://www.wormbook.org/chapters/www_transcriptionalregulation/transcriptionalregulation.html">“Transcriptional regulation” in WormBook</a> for a starting point on information of what is known of promoter elements in the worm.<br />
<br />
<html></div><div class="section"><h2>Operons</h2></html><br />
<br />
An operon is a genetic structure in which one promoter is followed by multiple coding sequences. These sequences are transcribed as one, and then typically separated by splicing the mRNA transcript. Operons are used and studied extensively in prokaryotes, with the lac operon of ''E. coli'' serving as a standard introduction to gene structure in second-year genetics courses.<br />
<br />
<html><div class="asideR" style="max-width: 40%"></html><br />
While at WormGuide we generally try to use “gene” to mean a structure that includes the promoter, the 5' UTR, the CDS, introns, and the 3' UTR, nomenclature often breaks down when this structure is violated. Geneticists traditionally have meant specifically the protein-coding (or functional RNA transcript) sequence, and sometimes surrounding the sequence that is specific to it. This definition has constantly been a matter of confusion over the years, and the issue has only gotten worse with the blossoming of genomics.<br />
<html></div></html><br />
<br />
Up until the early nineties, it was believed that eukaryotes had fully rejected operons in favor of more elaborate control mechanisms, but <html><a href="http://www.ncbi.nlm.nih.gov/pubmed/8098272" target="_new">Spieth <i>et al</i>.</a></html> determined this was not the case in 1993. There are approximately 1000 operons in ''C. elegans'', containing more than a tenth of the worm’s genes. In general, these operons contain proteins that must be expressed in controlled ratios to each other and interact with one another, especially essential genes such as mitochondrial, transcriptional, splicing, and translational machinery.<br />
<br />
Operons have proven themselves to be immensely useful in the construction of BioDevices in ''E. coli'', and will likely prove to be just as valuable in ''C. elegans''. They also must be taken into consideration when searching for promoters, as they mean that the region upstream of the protein sequence may not actually contain any regulatory or polymerase-binding sites, especially for high-activity genes. For information on constructing operons, see <html><a href="https://2010.igem.org/Team:Queens-Canada/transcripts#%3Ci%3ETrans%3C/i%3E-splicing%20(and%20operons)">the section on <i>trans</i>-splicing</a></html>.<br />
<br />
<html></div><div class="section"><h2>The Kozak Consensus Sequence</h2></html><br />
<br />
In prokaryotes such as ''E. coli'', the Shine-Dalgarno sequence, located just before the translational start, acts as a ribosomal binding site (RBS). In absence of the SD sequence, expression is greatly reduced or eliminated.<br />
<br />
There is no direct analog of this in eukaryotes. The ribosome latches onto a structure of cofactor proteins that have bound to certain features at the start of the transcript, and then scans down the mRNA until it finds a start codon. A ‘naked’ start codon is sufficient to start translation, but it is very weak, and it is likely that the ribosome will skip it over and not express the gene.<br />
<br />
To improve the efficacy of translational initiation, a larger sequence is used. The more precisely this matches certain well-known patterns, the more likely it is that the ribosome will stop scanning at the correct point and begin translation.<br />
<br />
The canonical pattern for a Kozak sequence is as follows:<br />
<br />
<blockquote>(GCC)GCCRCC<u>AUG</u>G</blockquote><br />
<br />
Where R is A or G (more commonly A), <u>AUG</u> is the start codon, and the final G is the first nucleotide of the first codon of the gene itself. <html><a target="_new" href="http://en.wikipedia.org/wiki/Kozak_consensus_sequence">Wikipedia has some examples</a></html> of weaker consensus sequences.<br />
<br />
<html></div><div class="section"><h2>The Promoter in <i>C. elegans</i> Research</h2></html><br />
<br />
In most research biology studies of ''C. elegans'', the focus is on physiological and developmental processes, not the central dogma or transcriptional regulatory mechanisms. As a consequence, although transcriptional start sites are sometimes known, when assembling constructs, biologists typically elide the difference between the upstream portion of the transcript and the promoter itself—in fact, for many genes, even the start of the promoter is not known, and a large portion of the intergenic region is used instead. While messy, this saves time and is effective as a safeguard against accidentally losing promoter elements. In combination with a protein’s original 3' UTR, exact replication of an expression pattern can be ensured.<br />
<br />
''Note'': in order to be compatible with [[Team:Queens-Canada/parts|our BioBricks]], modification to these promoter sequences is required. Specifically, we remove the start codon from the <html><a href="http://worfdb.dfci.harvard.edu/promoteromedb/" target="_new">Promoterome</a></html> record and place it with an artificial Kozak sequence at the start of the protein in order to conceal ligation scars. As a result, following our standard exactly will typically produce a higher-than-normal level of expression. See [[Team:Queens-Canada/contributing|Contributing]] for more information.<html></div></html><br />
<br />
'''[[Team:Queens-Canada/transcripts|Continue to Introns and Transcripts]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/transcriptsTeam:Queens-Canada/transcripts2010-10-28T03:21:17Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<html><div class="section"><h1>Transcripts and Introns</h1></html><br />
<br />
<html><center><img src="http://upload.wikimedia.org/wikipedia/commons/thumb/a/a7/Gene2-plain.svg/708px-Gene2-plain.svg.png" style="max-width: 708px; width: 50%;"><center><br><br />
<a href="http://en.wikipedia.org/wiki/Gene#Functional_structure_of_a_gene">From <i>Wikipedia</i></a></html><br />
<br />
The transcribed region contained in the worm’s DNA is not final. During the creation of the messenger RNA that leaves the nucleus, segments of RNA marked by certain specific sequences are removed. The parts that get removed are called '''introns''', and their surroundings, the parts of the sequence that stay, are called '''exons'''. Introns aren’t common in prokaryotes, but they do exist, particularly in the form of '''self-splicing''' introns, which are also found in eukaryotes, but aren’t very common in either case.<br />
<br />
Eukaryotic genes with certain introns in them experience a significantly higher rate of expression than genes without introns; as a result, adding these introns to DNA can often improve the rate of production of imported proteins, and may prove useful to those looking to import BioBricks from the Registry of Standard Biological Parts. They may also be useful as a method for concealing ligation scars, promoters intended for other organisms, or other genetic elements that would benefit from being within the mRNA, but would run the risk of causing a frameshift mutation or producing an undesirable amino acid sequence.<br />
<br />
Self-splicing introns are possible because normal intron removal is accomplished by a set of RNA molecules that act catalytically (ribozymes). In normal splicing, these are found in the nucleus, in a complex called the '''spliceosome'''. This binds to the exposed pre-messenger RNA by complementary base-pairing and then twists it into the correct shape, snapping off the intron. In order for this to succeed, the middle of the intron must include at least one adenosine. One example of an intron sequence is:<br />
<br />
<blockquote>cag<b><u>gt</u>aagt … <u>a</u> … ttttgttt<u>cag</u></b>g</blockquote><br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Introns</b></p><ul><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Intron">Learn more about introns</a><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/RNA_splicing">Learn more about the splicing process</a></ul><br />
</div></html><br />
<br />
The parts in bold will be removed completely. The unbolded parts of this are not necessary, but appear in a substantial portion of ''C. elegans'' introns, especially the final G. Underlined text is absolutely or almost absolutely necessary; non-underlined text merely helps, although it is very common that the region just upstream from the end of the intron is pyrimidine-rich (lots of C and U/T). Again, this sequence does not require any specific reading frame to function, as intron removal occurs prior to translation. The rather short and simple content of the underlined text (which is the minimum required to define an intron) means that it is sometimes surprisingly easy for a point mutation to trigger a deletion by creating an intron, and this should be considered.<html></div></html><br />
<br />
<html><div class="section"><h2><i>Trans</i>-splicing (and operons)</h2></html><br />
<br />
The nuclear splicing machinery starts to assemble itself at the 5' splice site (the GU... at the start of the intron) and then works its way down, generally to the first 3' splice site (the ...AG at the end) that it can recognize. If a messenger RNA contains an unpaired 3' splice site, however, then a different ribonucleoprotein will catalyze at it instead. Such a site is called an '''outron site'''. When an outron is spliced at, the upstream piece of mRNA is lost, and replaced instead with a leader sequence. In ''C. elegans'', there are two such leaders, SL1 and SL2, which function as both catalytic agents and final components, being consumed in the process. They contain regulatory information and typically replace most of the 5' UTR in the transcript. About half of all genes in ''C. elegans'' use the SL1 leader sequence, 20% use SL2, and only 30% go unspliced.<br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Trans-splicing and Operons</b></p><ul><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_transsplicingoperons/transsplicingoperons.html">WormBook on trans-splicing and operons</a><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_mechregultranslation/mechregultranslation.html">WormBook on mechanism and regulation of translation</a><br />
</ul></div></html><br />
<br />
For the time being, applications of trans-splicing are limited in synthetic biology; it is known that different 5’ leader sequences have different regulatory effects, but not exactly what those effects are. However, there is one particular usage which may prove to be of substantial interest: [[Team:Queens-Canada/promoter|'''operons''']]. These use SL2 to separate their transcripts by placing an intron 3' splice site a small distance (typically about 100 nt) after the polyadenylation signal (AAUAAA). The protein that binds to the poly(A) signal, CstF, appears to recruit SL2 to perform the cut and splice itself in. This mechanism is more efficient if the interim sequence is U-rich.<br />
<br />
<br />
'''[[Team:Queens-Canada/rnai|Continue to RNA Interference and the 3' UTR]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/teamTeam:Queens-Canada/team2010-10-28T03:19:35Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
<h1>Students</h1><br />
<html><br />
<div><br />
<img style="box-shadow: 1px 2px 3px #808080" src="https://static.igem.org/mediawiki/2010/3/36/Queens_Group.JPG" width="100%"></div><br />
</html><br />
<p>'''Everyone who showed up on May 17th'''. From left: Nelson Yao, Yuli Zhang, Chris Palmer, Steve Goldie, Geoff Halliday, Thai Phi, Hao Shi, Kevin Hong.</p><br />
<br />
<html><style class="text/css"><br />
div.person {<br />
box-shadow: 1px 2px 3px #808080;<br />
border-left: 1px #e9e9e9 solid;<br />
border-top: 1px white solid;<br />
border-right: 1px #787878 solid;<br />
border-bottom: 1px #3b3b3b solid;<br />
background: #dcdcdc;<br />
margin-left: auto;<br />
margin-right: auto;<br />
width: 80%;<br />
padding: 5px;<br />
margin-bottom: 15px;<br />
}<br />
span.name {<br />
display: block;<br />
float: right;<br />
font-size: 140%;<br />
color: #808080;<br />
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float: left;<br />
border-radius: 4px;<br />
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border-left: 1px #787878 solid;<br />
border-top: 1px #3b3b3b solid;<br />
margin-right: 10px;<br />
}<br />
</style><br />
<br />
<div class="person"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/f/f8/Qgem_team_lb.png"><br />
<span class="name">Louis Briggs</span><br />
<a href="mailto:louis.briggs@qgemteam.com">email</a><br><br />
<p>Louis Briggs is a third year biochemical engineering student who has strong interests in synthetic biology and biomedical sciences. In addition to volunteering for the QGEM team, Louis has worked in biomedical research, specifically in the development of automated clinical systems for cell and tissue therapy.</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/e/e1/Qgem_team_sg.png"><br />
<span class="name">Steve Goldie</span><br />
<a href="mailto:steve.goldie@qgemteam.com">email</a><br><br />
<p>Steve is a Life Sciences major with an interest in Cancer Biology and Genetics. He hopes to eventually go to medical school. He became interested in the QGEM team after being promised copious amounts of lab work. So far the research and planning portion of the project has been his favourite part.</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/a/a3/Qgem_team_glh.png"><br />
<span class="name">Geoff Halliday</span><br />
<a href="mailto:geoff.halliday@qgemteam.com">email</a><br><br />
<p>Geoff is on the fence between biomedical computing and biochemistry. He comes from a computing background, but is very interested in the biological sciences, and synthetic biology in particular. He also thinks he finally got the layout looking okay.</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/5/5a/Qgem_team_kh.png"><br />
<span class="name">Kevin Hong</span><br />
<a href="mailto:kevin.hong@qgemteam.com">email</a><br><br />
<p>Kevin is in his third year of studies in the life sciences. He is passionate about both the fields of synthetic biology and biomimicry.</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><span class="name">Chris Palmer</span><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/e/e3/Qgem_team_cp.png"><br />
<a href="mailto:chrispalmer@qgemteam.com">email</a><br><br />
<p>Chris is a third year Engineering Chemistry student with a strong interest in synthetic biology. In addition to his involvement with the iGEM Team, he is also working with Drs. Ian Chin-Sang and Ken Ko towards bringing a synthetic biology course into existence at Queen's University.</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><span class="name">Thai Phi</span><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/b/bc/Qgem_team_tp.png"><br />
<a href="mailto:thai.phi@qgemteam.com">email</a><br><br />
<p>Thai is a third year biochemical engineering student. He joined the Queen's iGEM team in 2010 due to his interest in the emerging field of synthetic biology.</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><span class="name">Basia Rozinowicz</span><br />
<a href="mailto:basia.r@qgemteam.com">email</a><br><br />
<p>Basia studies biochemistry at Queen's. She naturally became interested in the iGEM as a result of her goal to pursue work in research and development in health biotechnology.</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><span class="name">Mike Schmidt</span><br />
<a href="mailto:mike.schmidt@qgemteam.com">email</a><br><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/7/70/Qgem_team_ms.png"><br />
<p>Mike is a second year undergraduate student, with a concentration in engineering chemistry. Mike is experienced in mathematical modelling, and the physical sciences affiliated with engineering tasks. He came to the iGEM team in 2009 because of his interests in the wide-range of applications in the field of synthetic biology and systematic engineering. Aside from his studies and QGEM he enjoys playing a variety of sports including hockey, soccer and golf.</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><span class="name">Hao Shi</span><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/b/b7/Qgem_team_hs.png"><br />
<a href="mailto:hao.shi@qgemteam.com">email</a><br><br />
<p></html>"''How'' is ''she'' doin'?" Yup, that's how I will introduce myself if we ever meet. I'm just a guy going into 3rd year Life Sciences aiming to enter medical school like any other life-sci. Aside from that ambition, I have a little engineer inside my brain that craves innovative challenges, which is what brought me to iGEM. To top that off, I need to admit my addiction to badminton... it takes up about 20% of my awake-hours each week. Sherlock Holmes would deduce either I sleep a lot or I play a lot of badminton... or I might be over-exaggerating. Summary: Med school + engineer + badminton = Hao Shi = Awesome!! QED =)<html></p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><span class="name">Mary Tao</span><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/6/6e/Qgem_team_mt.png"><br />
<a href="mailto:mary.tao@qgemteam.com">email</a><br><br />
<p>Mary is in her third year of the Life Science and X-ray technology program at Queen's. Her interest in the iGEM team was sparked by her experience in a biotechnology course in high-school, as well as her natural affinity for team work.</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><span class="name">Nelson Yao</span><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/b/b7/Qgem_team_ny.png"><br />
<a href="mailto:nelson.yao@qgemteam.com">email</a><br><br />
<p>Nelson is going into third-year biochemistry. Therefore he contributes knowledge in both molecular biology and chemistry to the team. Nelson joined QGEM 2010 as a volunteer because of his interest in exploring the cool and interesting aspects of synthetic biology and his desire to gain experience in the lab. Also, Nelson has a interest for all kinds of sports including badminton, pingpong, soccer etc, and he is a basketball lunatic.</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><span class="name">Yuli Zhang</span><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/2/27/Qgem_team_yz.png"><br />
<a href="mailto:yuli.zhang@qgemteam.com">email</a><br><br />
<p>Hi. I am a third year life sciences student. I am really interested in molecular genetics and participating in the iGEM competition as a QGEM member has given me the chance to learn more about synthetic biology. The QGEM team worked on engineering <i>C. elegans</i> this past summer. The project was very challenging (if you don’t believe me, try going through all of our gel images) and yet very rewarding (we got a transgenic worm)! The team worked mostly in Dr. Chin-Sang’s lab. When we are running gels we didn’t want to mistakenly take one of the lab’s gel, so we wrote, “You can run, but you can’t hide. iGEM” on tissue paper and laid it under our gel box. What it meant was the DNA can run through the gel by applying an electric field but afterwards, we can image the gel and find the DNA band.</p><br />
<div style="clear: both;"></div><br />
</div></html><br />
<br />
<h1>Advisors</h1><br />
<html><div class="person"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/e/e1/Qgem_prof_ic.png"><br />
<span class="name">Ian Chin-Sang</span><br />
<a href="mailto:chinsang@queensu.ca">email</a> | <a target="_new" href="http://chin-sang.ca">lab</a><br><br />
<br />
<p>Faculty: Arts & Science<br><br />
Department: Biology<br><br />
Position: Associate Professor and CCS/NCIC Research Scientist<br><br />
Research: Molecular Genetics of <i>C. elegans</i> Development<br><br />
<br><br />
<a href="http://130.15.90.245/movies/iGEM%20worms.mov"><i>C. elegans</i> Loves iGEM Movie</a><br />
</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/5/5f/Qgem_prof_pg.png"><br />
<span class="name">Peter Greer</span><br />
<a href="mailto:greerp@queensu.ca">email</a> | <a target="_new" href="http://qcri.queensu.ca/Greer.html">research</a><br><br />
<br />
<p>Faculty: Health Sciences<br><br />
Department: Biochemistry<br><br />
Position: Professor of Biochemistry and Pathology & Molecular Medicine<br><br />
Research: Cancer Signal Transduction</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/b/b4/Qgem_prof_kk.png"><br />
<span class="name">Kenton Ko</span><br />
<a href="mailto:kok@queensu.ca">email</a> | <a target="_new" href="http://post.queensu.ca/~kok/">research</a><br><br />
<br />
<p>Faculty: Arts & Science<br><br />
Department: Biology<br><br />
Position: Professor<br><br />
Research: Protein trafficking and functional proteomics</p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/d/dd/Qgem_prof_nm.png"><br />
<span class="name">Nancy Martin</span><br />
<a href="mailto:nancy.martin@queensu.ca">email</a> | <a target="_new" href="http://microimm.queensu.ca/facultyPages/full_time/martin_n.html">research</a><br><br />
<br />
<p>Faculty: Health Sciences<br><br />
Department: Microbiology and Immunology<br><br />
Position: Associate Professor<br><br />
Research: Sensing and Adaptation to Environmental Changes in <i>Salmonella typhimurium</i></p><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
<div class="person"><br />
<img class="photo" src="https://static.igem.org/mediawiki/2010/2/2d/Qgem_prof_py.png"><br />
<span class="name">Paul Young</span><br />
<a href="mailto:paul.young@queensu.ca">email</a> | <a href="http://130.15.90.59/">homepage</a><br><br />
<br />
<p>Faculty: Arts & Science<br><br />
Department: Biology<br><br />
Position: Professor<br><br />
Research: Cell cycle genetics and molecular biology</p><br />
<div style="clear: both;"></div><br />
</div><br />
</html><br />
<br />
'''[[Team:Queens-Canada/project|Return to WormWorks]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/File:Qgem_team_mt.pngFile:Qgem team mt.png2010-10-28T03:18:51Z<p>Glh: </p>
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<div></div>Glhhttp://2010.igem.org/File:Qgem_team_ms.pngFile:Qgem team ms.png2010-10-28T03:18:35Z<p>Glh: </p>
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<div></div>Glhhttp://2010.igem.org/Team:Queens-Canada/projectTeam:Queens-Canada/project2010-10-28T03:17:32Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks</h1><br />
<br />
The International Genetically-Engineered Machine competition challenges students from universities around the globe to use the vast amount of knowledge that Molecular Biology and Biochemistry have obtained over the past century and put it to use to make the world a better (or simply more interesting) place for us to live in.<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/a/a4/Qgem_content_project_gfp.jpg" style="float: right; margin-left: 10px; width: 40%; max-width: 1024px; box-shadow: 1px 2px 3px #808080;"><br />
</html><br />
<br />
Most iGEM teams have built their projects upon the humble bacterium ''Escherichia coli'', which is well-understood and easy to engineer. No team in past years has engineered a multicellular organism. Since many teams are comprised of students from diverse backgrounds, the more intricate complexities of the biology of higher organisms aren’t always accessible, and this has set a ceiling on project evolution. We set out to make these advancements possible, and bring iGEM into the kingdom Animalia.<br />
<br />
''Caenorhabidits elegans'' is a worm approximately one millimeter in length. It’s a common model organism, easier to work with than most well-studied animals, and feeds on bacteria. Last spring, our team didn’t know much more about it than that. But we learned and studied, and engineered a whole set of foundational BioBricks in the hope that we would be able to offer other teams in future years the tools and information that would let them expand beyond one-cell projects.<br />
<br />
We were able to accomplish this in one short summer, with no BioBricks to work from. We picked up the fundamental worm related lab techniques, did all our own wet work, and met our project goals. We hope that future teams, armed with our fundamental BioBricks and with WormGuide to get them started and help them along the way, will be able to dive into the world of worms and fully utilize synthetic biology in this fascinating and powerful organism.<br />
<br />
An important component of achieving this is passing on the knowledge of background material and techniques for engineering the worm. We spent a significant portion of our summer making sure that the necessary information would be available to teams who need it—and if you're interested in that information, now is a good time to head over to [[Team:Queens-Canada/guide|WormGuide]]. Otherwise, keep reading!<br />
<br />
'''[[Team:Queens-Canada/idea|Continue to Project Overview]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/projectTeam:Queens-Canada/project2010-10-28T03:10:46Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks</h1><br />
<br />
The International Genetically-Engineered Machine competition challenges students from universities around the globe to use the vast amount of knowledge that Molecular Biology and Biochemistry have obtained over the past century and put it to use to make the world a better (or simply more interesting) place for us to live in.<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/a/a4/Qgem_content_project_gfp.jpg" style="float: right; margin-left: 10px; width: 40%; max-width: 1024px; box-shadow: 1px 2px 3px #808080;"><br />
</html><br />
<br />
Most iGEM teams have built their projects upon the humble bacterium ''Escherichia coli'', which is well-understood and easy to engineer. No team in past years has engineered a multicellular organism. Since many teams are comprised of students from diverse backgrounds, the more intricate complexities of the biology of higher organisms aren’t always accessible, and this has set a ceiling on project evolution. We set out to make these advancements possible, and bring iGEM into the kingdom Animalia.<br />
<br />
''Caenorhabidits elegans'' is a worm approximately one millimeter in length. It’s a common model organism, easier to work with than most well-studied animals, and feeds on bacteria. Last spring, our team didn’t know much more about it than that. But we learned and studied, and engineered a whole set of foundational BioBricks in the hope that we would be able to offer other teams in future years the tools and information that would let them expand beyond one-cell projects.<br />
<br />
We were able to accomplish this in one short summer, with no BioBricks to work from. We picked up the fundamental worm related lab techniques, did all our own wet work, and met our project goals. We hope that future teams, armed with our fundamental BioBricks and with WormGuide to get them started and help them along the way, will be able to dive into the world of worms and fully utilize synthetic biology in this fascinating and powerful organism.<br />
<br />
An important component of achieving this is passing on the knowledge of background material and technique for engineering the worm. We spent a significant portion of our summer making sure that the necessary information would be available to teams who need it—and if you're interested in that information, now is a good time to head over to [[Team:Queens-Canada/guide|WormGuide]]. Otherwise, keep reading!<br />
<br />
'''[[Team:Queens-Canada/idea|Continue to Project Overview]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/projectTeam:Queens-Canada/project2010-10-28T03:04:40Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks</h1><br />
<br />
The International Genetically-Engineered Machine competition challenges students from universities around the globe to use the vast amount of knowledge that Molecular Biology and Biochemistry have obtained over the past century and put it to use to make the world a better (or simply more interesting) place for us to live in.<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/a/a4/Qgem_content_project_gfp.jpg" style="float: right; margin-left: 10px; width: 40%; max-width: 1024px; box-shadow: 1px 2px 3px #808080;"><br />
</html><br />
<br />
Most iGEM teams have built their projects upon the humble bacterium ''Escherichia coli'', which is well-understood and easy to engineer. No team in past years has engineered a multicellular organism. Since many teams are comprised of students from diverse backgrounds, the more intricate complexities of the biology of higher organisms aren’t always accessible, and this has set a ceiling on project evolution. We set out to make these advancements possible, and bring iGEM into the kingdom Animalia.<br />
<br />
''Caenorhabidits elegans'' is a worm approximately one millimeter in length. It’s a common model organism, easier to work with than most well-studied animals, and feeds on bacteria. Last spring, our team didn’t know much more about it than that. But we learned and studied, and engineered a whole set of foundational BioBricks in the hope that we would be able to offer other teams in future years the tools and information that would let them expand beyond one-cell projects.<br />
<br />
We were able to accomplish this in one short summer, with no BioBricks to work from. We picked up the fundamental worm related lab techniques, did all our own wet work, and met our project goals. We hope that future teams, armed with our fundamental BioBricks and with WormGuide to get them started and help them along the way, will be able to dive into the world of worms and fully utilize synthetic biology in this fascinating and powerful organism.<br />
<br />
If you want to dive into background material on the worm, now is a good time to head over to [[Team:Queens-Canada/guide|WormGuide]]. Otherwise, keep reading!<br />
<br />
'''[[Team:Queens-Canada/idea|Continue to Project Overview]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/File:Qgem_content_project_gfp.jpgFile:Qgem content project gfp.jpg2010-10-28T03:01:58Z<p>Glh: </p>
<hr />
<div></div>Glhhttp://2010.igem.org/Team:Queens-Canada/projectTeam:Queens-Canada/project2010-10-28T03:00:16Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks</h1><br />
<br />
The International Genetically-Engineered Machine competition challenges students from universities around the globe to use the vast amount of knowledge that Molecular Biology and Biochemistry have obtained over the past century and put it to use to make the world a better (or simply more interesting) place for us to live in.<br />
<br />
Most iGEM teams have built their projects upon the humble bacterium ''Escherichia coli'', which is well-understood and easy to engineer. No team in past years has engineered a multicellular organism. Since many teams are comprised of students from diverse backgrounds, the more intricate complexities of the biology of higher organisms aren’t always accessible, and this has set a ceiling on project evolution. We set out to make these advancements possible, and bring iGEM into the kingdom Animalia.<br />
<br />
''Caenorhabidits elegans'' is a worm approximately one millimeter in length. It’s a common model organism, easier to work with than most well-studied animals, and feeds on bacteria. Last spring, our team didn’t know much more about it than that. But we learned and studied, and engineered a whole set of foundational BioBricks in the hope that we would be able to offer other teams in future years the tools and information that would let them expand beyond one-cell projects.<br />
<br />
We were able to accomplish this in one short summer, with no BioBricks to work from. We picked up the fundamental worm related lab techniques, did all our own wet work, and met our project goals. We hope that future teams, armed with our fundamental BioBricks and with WormGuide to get them started and help them along the way, will be able to dive into the world of worms and fully utilize synthetic biology in this fascinating and powerful organism.<br />
<br />
If you want to dive into background material on the worm, now is a good time to head over to [[Team:Queens-Canada/guide|WormGuide]]. Otherwise, keep reading!<br />
<br />
'''[[Team:Queens-Canada/idea|Continue to Project Overview]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/projectTeam:Queens-Canada/project2010-10-28T02:54:05Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks</h1><br />
<br />
The International Genetically-Engineered Machine competition challenges students from universities around the globe to use the vast amount of knowledge that Molecular Biology and Biochemistry have obtained over the past century and put it to use to make the world a better (or simply more interesting) place for us to live in.<br />
<br />
Most iGEM teams have built their projects upon the humble bacterium ''Escherichia coli'', which is well-understood and easy to engineer. No team in past years has engineered a multicellular organism. Since many teams are comprised of students from diverse backgrounds, the more intricate complexities of the biology of higher organisms aren’t always accessible, and this has set a ceiling on project evolution. We set out to make these advancements possible, and bring iGEM into the kingdom Animalia.<br />
<br />
''Caenorhabidits elegans'' is a worm about a millimeter long. It’s a common model organism, easier to work with than most well-studied animals, and feeds on bacteria. Last spring, our team didn’t know much more about it than that. But we learned and studied, and engineered a whole set of foundational BioBricks in the hope that we would be able to offer other teams in future years the tools and information that would let them expand beyond one-cell projects.<br />
<br />
We were able to accomplish this in one short summer, with no BioBricks to work from. We picked up the fundamental worm related lab techniques, did all our own wet work, and met our project goals. We hope that future teams, armed with our fundamental BioBricks and with WormGuide to get them started and help them along the way, will be able to dive into the world of worms and fully utilize synthetic biology in this fascinating and powerful organism.<br />
<br />
If you want to dive into background material on the worm, now is a good time to head over to [[Team:Queens-Canada/guide|WormGuide]]. Otherwise, keep reading!<br />
<br />
'''[[Team:Queens-Canada/idea|Continue to Project Overview]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/projectTeam:Queens-Canada/project2010-10-28T02:50:32Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks</h1><br />
<br />
The International Genetically-Engineered Machine competition challenges students from universities around the globe to use the vast amount of knowledge that Molecular Biology and Biochemistry have obtained over the past century and put it to use to make the world a better (or simply more interesting) place for us to live in.<br />
<br />
Most iGEM teams have built their projects upon the humble bacterium ''Escherichia coli'', which is well-understood and easy to engineer. No team in past years has engineered a multicellular organism. Since many teams are comprised of students from diverse backgrounds, the more intricate complexities of the biology of higher organisms aren’t always accessible, and this has set a ceiling on project evolution. We set out to make these advancements possible, and bring iGEM into the kingdom Animalia.<br />
<br />
''Caenorhabidits elegans'' is a worm about a millimeter long. It’s a common model organism, easier to work with than most well-studied animals, and feeds on bacteria. Last spring, our team didn’t know much more about it than that. But we learned and studied, and engineered a whole set of foundational BioBricks in the hope that we would be able to offer other teams in future years the tools and information that would let them expand beyond one-cell projects.<br />
<br />
We were able to accomplish this, with no BioBricks to work from, in one short summer. We learned the fundamental techniques to work with worms in the lab, did all our own wet work, and made this project successful. We hope that future teams, armed with some fundamental BioBricks and with WormGuide to get them started and help them along the way, will be able to dive into the world of worms and fully utilize synthetic biology in this fascinating and powerful organism.<br />
<br />
If you want to dive into background material on the worm, now is a good time to head over to [[Team:Queens-Canada/guide|WormGuide]]. Otherwise, keep reading!<br />
<br />
'''[[Team:Queens-Canada/idea|Continue to Project Overview]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/transcriptsTeam:Queens-Canada/transcripts2010-10-28T02:45:41Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<html><div class="section"><h1>Transcripts and Introns</h1></html><br />
<br />
<html><center><img src="http://upload.wikimedia.org/wikipedia/commons/thumb/a/a7/Gene2-plain.svg/708px-Gene2-plain.svg.png" style="max-width: 708px; width: 50%;"><center><br><br />
<a href="http://en.wikipedia.org/wiki/Gene#Functional_structure_of_a_gene">From <i>Wikipedia</i></a></html><br />
<br />
The coding sequence contained in the worm’s DNA is not final. During the creation of the messenger RNA that leaves the nucleus, segments of RNA marked by certain specific sequences are removed. The parts that get removed are called '''introns''', and their surroundings, the parts of the coding sequence that stay, are called '''exons'''. Introns aren’t common in prokaryotes, but they do exist, particularly in the form of '''self-splicing''' introns, which are also found in eukaryotes, but aren’t very common in either case.<br />
<br />
Eukaryotic genes with certain introns in them experience a significantly higher rate of expression than genes without introns; as a result, adding these introns to DNA can often improve the rate of production of imported proteins, and may prove useful to those looking to import BioBricks from the Registry of Standard Biological Parts. They may also be useful as a method for concealing ligation scars, promoters intended for other organisms, or other genetic elements that would benefit from being within the coding sequence, but would run the risk of causing a frameshift mutation or producing an undesirable amino acid sequence.<br />
<br />
Self-splicing introns are possible because normal intron removal is accomplished by a set of RNA molecules that act catalytically (ribozymes). In normal splicing, these are found in the nucleus, in a complex called the '''spliceosome'''. This binds to the exposed pre-messenger RNA by complementary base-pairing and then twists it into the correct shape, snapping off the intron. In order for this to succeed, the middle of the intron must include at least one adenosine. One example of an intron sequence is:<br />
<br />
<blockquote>cag<b><u>gt</u>aagt … <u>a</u> … ttttgttt<u>cag</u></b>g</blockquote><br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Introns</b></p><ul><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Intron">Learn more about introns</a><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/RNA_splicing">Learn more about the splicing process</a></ul><br />
</div></html><br />
<br />
The parts in bold will be removed completely. The unbolded parts of this are not necessary, but appear in a substantial portion of ''C. elegans'' introns, especially the final G. Underlined text is absolutely or almost absolutely necessary; non-underlined text merely helps, although it is very common that the region just upstream from the end of the intron is pyrimidine-rich (lots of C and U/T). Again, this sequence does not require any specific reading frame to function, as intron removal occurs prior to translation. The rather short and simple content of the underlined text (which is the minimum required to define an intron) means that it is sometimes surprisingly easy for a point mutation to trigger a deletion by creating an intron, and this should be considered.<html></div></html><br />
<br />
<html><div class="section"><h2><i>Trans</i>-splicing (and operons)</h2></html><br />
<br />
The nuclear splicing machinery starts to assemble itself at the 5' splice site (the GU... at the start of the intron) and then works its way down, generally to the first 3' splice site (the ...AG at the end) that it can recognize. If a messenger RNA contains an unpaired 3' splice site, however, then a different ribonucleoprotein will catalyze at it instead. Such a site is called an '''outron site'''. When an outron is spliced at, the upstream piece of mRNA is lost, and replaced instead with a leader sequence. In ''C. elegans'', there are two such leaders, SL1 and SL2, which function as both catalytic agents and final components, being consumed in the process. They contain regulatory information and typically replace most of the 5' UTR in the transcript. About half of all genes in ''C. elegans'' use the SL1 leader sequence, 20% use SL2, and only 30% go unspliced.<br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Trans-splicing and Operons</b></p><ul><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_transsplicingoperons/transsplicingoperons.html">WormBook on trans-splicing and operons</a><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_mechregultranslation/mechregultranslation.html">WormBook on mechanism and regulation of translation</a><br />
</ul></div></html><br />
<br />
For the time being, applications of trans-splicing are limited in synthetic biology; it is known that different 5’ leader sequences have different regulatory effects, but not exactly what those effects are. However, there is one particular usage which may prove to be of substantial interest: [[Team:Queens-Canada/promoter|'''operons''']]. These use SL2 to separate their transcripts by placing an intron 3' splice site a small distance (typically about 100 nt) after the polyadenylation signal (AAUAAA). The protein that binds to the poly(A) signal, CstF, appears to recruit SL2 to perform the cut and splice itself in. This mechanism is more efficient if the interim sequence is U-rich.<br />
<br />
<br />
'''[[Team:Queens-Canada/rnai|Continue to RNA Interference and the 3' UTR]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/transcriptsTeam:Queens-Canada/transcripts2010-10-28T02:44:52Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<html><div class="section"><h1>Transcripts and Introns</h1></html><br />
<br />
<html><center><img src="http://upload.wikimedia.org/wikipedia/commons/thumb/a/a7/Gene2-plain.svg/708px-Gene2-plain.svg.png" style="max-width: 708px; width: 100%;"><center><br><br />
<a href="http://en.wikipedia.org/wiki/Gene#Functional_structure_of_a_gene">From <i>Wikipedia</i></a></html><br />
<br />
The coding sequence contained in the worm’s DNA is not final. During the creation of the messenger RNA that leaves the nucleus, segments of RNA marked by certain specific sequences are removed. The parts that get removed are called '''introns''', and their surroundings, the parts of the coding sequence that stay, are called '''exons'''. Introns aren’t common in prokaryotes, but they do exist, particularly in the form of '''self-splicing''' introns, which are also found in eukaryotes, but aren’t very common in either case.<br />
<br />
Eukaryotic genes with certain introns in them experience a significantly higher rate of expression than genes without introns; as a result, adding these introns to DNA can often improve the rate of production of imported proteins, and may prove useful to those looking to import BioBricks from the Registry of Standard Biological Parts. They may also be useful as a method for concealing ligation scars, promoters intended for other organisms, or other genetic elements that would benefit from being within the coding sequence, but would run the risk of causing a frameshift mutation or producing an undesirable amino acid sequence.<br />
<br />
Self-splicing introns are possible because normal intron removal is accomplished by a set of RNA molecules that act catalytically (ribozymes). In normal splicing, these are found in the nucleus, in a complex called the '''spliceosome'''. This binds to the exposed pre-messenger RNA by complementary base-pairing and then twists it into the correct shape, snapping off the intron. In order for this to succeed, the middle of the intron must include at least one adenosine. One example of an intron sequence is:<br />
<br />
<blockquote>cag<b><u>gt</u>aagt … <u>a</u> … ttttgttt<u>cag</u></b>g</blockquote><br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Introns</b></p><ul><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Intron">Learn more about introns</a><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/RNA_splicing">Learn more about the splicing process</a></ul><br />
</div></html><br />
<br />
The parts in bold will be removed completely. The unbolded parts of this are not necessary, but appear in a substantial portion of ''C. elegans'' introns, especially the final G. Underlined text is absolutely or almost absolutely necessary; non-underlined text merely helps, although it is very common that the region just upstream from the end of the intron is pyrimidine-rich (lots of C and U/T). Again, this sequence does not require any specific reading frame to function, as intron removal occurs prior to translation. The rather short and simple content of the underlined text (which is the minimum required to define an intron) means that it is sometimes surprisingly easy for a point mutation to trigger a deletion by creating an intron, and this should be considered.<html></div></html><br />
<br />
<html><div class="section"><h2><i>Trans</i>-splicing (and operons)</h2></html><br />
<br />
The nuclear splicing machinery starts to assemble itself at the 5' splice site (the GU... at the start of the intron) and then works its way down, generally to the first 3' splice site (the ...AG at the end) that it can recognize. If a messenger RNA contains an unpaired 3' splice site, however, then a different ribonucleoprotein will catalyze at it instead. Such a site is called an '''outron site'''. When an outron is spliced at, the upstream piece of mRNA is lost, and replaced instead with a leader sequence. In ''C. elegans'', there are two such leaders, SL1 and SL2, which function as both catalytic agents and final components, being consumed in the process. They contain regulatory information and typically replace most of the 5' UTR in the transcript. About half of all genes in ''C. elegans'' use the SL1 leader sequence, 20% use SL2, and only 30% go unspliced.<br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Trans-splicing and Operons</b></p><ul><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_transsplicingoperons/transsplicingoperons.html">WormBook on trans-splicing and operons</a><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_mechregultranslation/mechregultranslation.html">WormBook on mechanism and regulation of translation</a><br />
</ul></div></html><br />
<br />
For the time being, applications of trans-splicing are limited in synthetic biology; it is known that different 5’ leader sequences have different regulatory effects, but not exactly what those effects are. However, there is one particular usage which may prove to be of substantial interest: [[Team:Queens-Canada/promoter|'''operons''']]. These use SL2 to separate their transcripts by placing an intron 3' splice site a small distance (typically about 100 nt) after the polyadenylation signal (AAUAAA). The protein that binds to the poly(A) signal, CstF, appears to recruit SL2 to perform the cut and splice itself in. This mechanism is more efficient if the interim sequence is U-rich.<br />
<br />
<br />
'''[[Team:Queens-Canada/rnai|Continue to RNA Interference and the 3' UTR]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/transcriptsTeam:Queens-Canada/transcripts2010-10-28T02:42:43Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<html><div class="section"><h1>Transcripts and Introns</h1></html><br />
<br />
<html><div class="asideL" style="width: 40%; max-width: 708px;"><img src="http://upload.wikimedia.org/wikipedia/commons/thumb/a/a7/Gene2-plain.svg/708px-Gene2-plain.svg.png" style="max-width: 708px;"></div></html><br />
<br />
The coding sequence contained in the worm’s DNA is not final. During the creation of the messenger RNA that leaves the nucleus, segments of RNA marked by certain specific sequences are removed. The parts that get removed are called '''introns''', and their surroundings, the parts of the coding sequence that stay, are called '''exons'''. Introns aren’t common in prokaryotes, but they do exist, particularly in the form of '''self-splicing''' introns, which are also found in eukaryotes, but aren’t very common in either case.<br />
<br />
Eukaryotic genes with certain introns in them experience a significantly higher rate of expression than genes without introns; as a result, adding these introns to DNA can often improve the rate of production of imported proteins, and may prove useful to those looking to import BioBricks from the Registry of Standard Biological Parts. They may also be useful as a method for concealing ligation scars, promoters intended for other organisms, or other genetic elements that would benefit from being within the coding sequence, but would run the risk of causing a frameshift mutation or producing an undesirable amino acid sequence.<br />
<br />
Self-splicing introns are possible because normal intron removal is accomplished by a set of RNA molecules that act catalytically (ribozymes). In normal splicing, these are found in the nucleus, in a complex called the '''spliceosome'''. This binds to the exposed pre-messenger RNA by complementary base-pairing and then twists it into the correct shape, snapping off the intron. In order for this to succeed, the middle of the intron must include at least one adenosine. One example of an intron sequence is:<br />
<br />
<blockquote>cag<b><u>gt</u>aagt … <u>a</u> … ttttgttt<u>cag</u></b>g</blockquote><br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Introns</b></p><ul><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Intron">Learn more about introns</a><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/RNA_splicing">Learn more about the splicing process</a></ul><br />
</div></html><br />
<br />
The parts in bold will be removed completely. The unbolded parts of this are not necessary, but appear in a substantial portion of ''C. elegans'' introns, especially the final G. Underlined text is absolutely or almost absolutely necessary; non-underlined text merely helps, although it is very common that the region just upstream from the end of the intron is pyrimidine-rich (lots of C and U/T). Again, this sequence does not require any specific reading frame to function, as intron removal occurs prior to translation. The rather short and simple content of the underlined text (which is the minimum required to define an intron) means that it is sometimes surprisingly easy for a point mutation to trigger a deletion by creating an intron, and this should be considered.<html></div></html><br />
<br />
<html><div class="section"><h2><i>Trans</i>-splicing (and operons)</h2></html><br />
<br />
The nuclear splicing machinery starts to assemble itself at the 5' splice site (the GU... at the start of the intron) and then works its way down, generally to the first 3' splice site (the ...AG at the end) that it can recognize. If a messenger RNA contains an unpaired 3' splice site, however, then a different ribonucleoprotein will catalyze at it instead. Such a site is called an '''outron site'''. When an outron is spliced at, the upstream piece of mRNA is lost, and replaced instead with a leader sequence. In ''C. elegans'', there are two such leaders, SL1 and SL2, which function as both catalytic agents and final components, being consumed in the process. They contain regulatory information and typically replace most of the 5' UTR in the transcript. About half of all genes in ''C. elegans'' use the SL1 leader sequence, 20% use SL2, and only 30% go unspliced.<br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Trans-splicing and Operons</b></p><ul><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_transsplicingoperons/transsplicingoperons.html">WormBook on trans-splicing and operons</a><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_mechregultranslation/mechregultranslation.html">WormBook on mechanism and regulation of translation</a><br />
</ul></div></html><br />
<br />
For the time being, applications of trans-splicing are limited in synthetic biology; it is known that different 5’ leader sequences have different regulatory effects, but not exactly what those effects are. However, there is one particular usage which may prove to be of substantial interest: [[Team:Queens-Canada/promoter|'''operons''']]. These use SL2 to separate their transcripts by placing an intron 3' splice site a small distance (typically about 100 nt) after the polyadenylation signal (AAUAAA). The protein that binds to the poly(A) signal, CstF, appears to recruit SL2 to perform the cut and splice itself in. This mechanism is more efficient if the interim sequence is U-rich.<br />
<br />
<br />
'''[[Team:Queens-Canada/rnai|Continue to RNA Interference and the 3' UTR]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/transcriptsTeam:Queens-Canada/transcripts2010-10-28T02:41:34Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<html><div class="section"><h1>Transcripts and Introns</h1></html><br />
<br />
<html><div class="asideL" style="width: 40%; max-width: 708px;"><img src="http://upload.wikimedia.org/wikipedia/commons/thumb/a/a7/Gene2-plain.svg/708px-Gene2-plain.svg.png" style="width: 40%; max-width: 708px;"></div></html><br />
<br />
The coding sequence contained in the worm’s DNA is not final. During the creation of the messenger RNA that leaves the nucleus, segments of RNA marked by certain specific sequences are removed. The parts that get removed are called '''introns''', and their surroundings, the parts of the coding sequence that stay, are called '''exons'''. Introns aren’t common in prokaryotes, but they do exist, particularly in the form of '''self-splicing''' introns, which are also found in eukaryotes, but aren’t very common in either case.<br />
<br />
Eukaryotic genes with certain introns in them experience a significantly higher rate of expression than genes without introns; as a result, adding these introns to DNA can often improve the rate of production of imported proteins, and may prove useful to those looking to import BioBricks from the Registry of Standard Biological Parts. They may also be useful as a method for concealing ligation scars, promoters intended for other organisms, or other genetic elements that would benefit from being within the coding sequence, but would run the risk of causing a frameshift mutation or producing an undesirable amino acid sequence.<br />
<br />
Self-splicing introns are possible because normal intron removal is accomplished by a set of RNA molecules that act catalytically (ribozymes). In normal splicing, these are found in the nucleus, in a complex called the '''spliceosome'''. This binds to the exposed pre-messenger RNA by complementary base-pairing and then twists it into the correct shape, snapping off the intron. In order for this to succeed, the middle of the intron must include at least one adenosine. One example of an intron sequence is:<br />
<br />
<blockquote>cag<b><u>gt</u>aagt … <u>a</u> … ttttgttt<u>cag</u></b>g</blockquote><br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Introns</b></p><ul><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Intron">Learn more about introns</a><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/RNA_splicing">Learn more about the splicing process</a></ul><br />
</div></html><br />
<br />
The parts in bold will be removed completely. The unbolded parts of this are not necessary, but appear in a substantial portion of ''C. elegans'' introns, especially the final G. Underlined text is absolutely or almost absolutely necessary; non-underlined text merely helps, although it is very common that the region just upstream from the end of the intron is pyrimidine-rich (lots of C and U/T). Again, this sequence does not require any specific reading frame to function, as intron removal occurs prior to translation. The rather short and simple content of the underlined text (which is the minimum required to define an intron) means that it is sometimes surprisingly easy for a point mutation to trigger a deletion by creating an intron, and this should be considered.<html></div></html><br />
<br />
<html><div class="section"><h2><i>Trans</i>-splicing (and operons)</h2></html><br />
<br />
The nuclear splicing machinery starts to assemble itself at the 5' splice site (the GU... at the start of the intron) and then works its way down, generally to the first 3' splice site (the ...AG at the end) that it can recognize. If a messenger RNA contains an unpaired 3' splice site, however, then a different ribonucleoprotein will catalyze at it instead. Such a site is called an '''outron site'''. When an outron is spliced at, the upstream piece of mRNA is lost, and replaced instead with a leader sequence. In ''C. elegans'', there are two such leaders, SL1 and SL2, which function as both catalytic agents and final components, being consumed in the process. They contain regulatory information and typically replace most of the 5' UTR in the transcript. About half of all genes in ''C. elegans'' use the SL1 leader sequence, 20% use SL2, and only 30% go unspliced.<br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Trans-splicing and Operons</b></p><ul><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_transsplicingoperons/transsplicingoperons.html">WormBook on trans-splicing and operons</a><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_mechregultranslation/mechregultranslation.html">WormBook on mechanism and regulation of translation</a><br />
</ul></div></html><br />
<br />
For the time being, applications of trans-splicing are limited in synthetic biology; it is known that different 5’ leader sequences have different regulatory effects, but not exactly what those effects are. However, there is one particular usage which may prove to be of substantial interest: [[Team:Queens-Canada/promoter|'''operons''']]. These use SL2 to separate their transcripts by placing an intron 3' splice site a small distance (typically about 100 nt) after the polyadenylation signal (AAUAAA). The protein that binds to the poly(A) signal, CstF, appears to recruit SL2 to perform the cut and splice itself in. This mechanism is more efficient if the interim sequence is U-rich.<br />
<br />
<br />
'''[[Team:Queens-Canada/rnai|Continue to RNA Interference and the 3' UTR]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/transcriptsTeam:Queens-Canada/transcripts2010-10-28T02:40:59Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
<br />
<html><div class="section"><h1>Transcripts and Introns</h1></html><br />
<br />
<html><div class="asideR"><img src="http://upload.wikimedia.org/wikipedia/commons/thumb/a/a7/Gene2-plain.svg/708px-Gene2-plain.svg.png" style="width: 40%; max-width: 708px;"></div></html><br />
<br />
The coding sequence contained in the worm’s DNA is not final. During the creation of the messenger RNA that leaves the nucleus, segments of RNA marked by certain specific sequences are removed. The parts that get removed are called '''introns''', and their surroundings, the parts of the coding sequence that stay, are called '''exons'''. Introns aren’t common in prokaryotes, but they do exist, particularly in the form of '''self-splicing''' introns, which are also found in eukaryotes, but aren’t very common in either case.<br />
<br />
Eukaryotic genes with certain introns in them experience a significantly higher rate of expression than genes without introns; as a result, adding these introns to DNA can often improve the rate of production of imported proteins, and may prove useful to those looking to import BioBricks from the Registry of Standard Biological Parts. They may also be useful as a method for concealing ligation scars, promoters intended for other organisms, or other genetic elements that would benefit from being within the coding sequence, but would run the risk of causing a frameshift mutation or producing an undesirable amino acid sequence.<br />
<br />
Self-splicing introns are possible because normal intron removal is accomplished by a set of RNA molecules that act catalytically (ribozymes). In normal splicing, these are found in the nucleus, in a complex called the '''spliceosome'''. This binds to the exposed pre-messenger RNA by complementary base-pairing and then twists it into the correct shape, snapping off the intron. In order for this to succeed, the middle of the intron must include at least one adenosine. One example of an intron sequence is:<br />
<br />
<blockquote>cag<b><u>gt</u>aagt … <u>a</u> … ttttgttt<u>cag</u></b>g</blockquote><br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Introns</b></p><ul><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Intron">Learn more about introns</a><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/RNA_splicing">Learn more about the splicing process</a></ul><br />
</div></html><br />
<br />
The parts in bold will be removed completely. The unbolded parts of this are not necessary, but appear in a substantial portion of ''C. elegans'' introns, especially the final G. Underlined text is absolutely or almost absolutely necessary; non-underlined text merely helps, although it is very common that the region just upstream from the end of the intron is pyrimidine-rich (lots of C and U/T). Again, this sequence does not require any specific reading frame to function, as intron removal occurs prior to translation. The rather short and simple content of the underlined text (which is the minimum required to define an intron) means that it is sometimes surprisingly easy for a point mutation to trigger a deletion by creating an intron, and this should be considered.<html></div></html><br />
<br />
<html><div class="section"><h2><i>Trans</i>-splicing (and operons)</h2></html><br />
<br />
The nuclear splicing machinery starts to assemble itself at the 5' splice site (the GU... at the start of the intron) and then works its way down, generally to the first 3' splice site (the ...AG at the end) that it can recognize. If a messenger RNA contains an unpaired 3' splice site, however, then a different ribonucleoprotein will catalyze at it instead. Such a site is called an '''outron site'''. When an outron is spliced at, the upstream piece of mRNA is lost, and replaced instead with a leader sequence. In ''C. elegans'', there are two such leaders, SL1 and SL2, which function as both catalytic agents and final components, being consumed in the process. They contain regulatory information and typically replace most of the 5' UTR in the transcript. About half of all genes in ''C. elegans'' use the SL1 leader sequence, 20% use SL2, and only 30% go unspliced.<br />
<br />
<html><div class="asideR style="max-width: 40%"><p><b>More on Trans-splicing and Operons</b></p><ul><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_transsplicingoperons/transsplicingoperons.html">WormBook on trans-splicing and operons</a><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_mechregultranslation/mechregultranslation.html">WormBook on mechanism and regulation of translation</a><br />
</ul></div></html><br />
<br />
For the time being, applications of trans-splicing are limited in synthetic biology; it is known that different 5’ leader sequences have different regulatory effects, but not exactly what those effects are. However, there is one particular usage which may prove to be of substantial interest: [[Team:Queens-Canada/promoter|'''operons''']]. These use SL2 to separate their transcripts by placing an intron 3' splice site a small distance (typically about 100 nt) after the polyadenylation signal (AAUAAA). The protein that binds to the poly(A) signal, CstF, appears to recruit SL2 to perform the cut and splice itself in. This mechanism is more efficient if the interim sequence is U-rich.<br />
<br />
<br />
'''[[Team:Queens-Canada/rnai|Continue to RNA Interference and the 3' UTR]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/projectTeam:Queens-Canada/project2010-10-28T02:37:49Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks</h1><br />
<br />
The International Genetically-Engineered Machine competition challenges students from universities around the globe to use the vast amount of knowledge that Molecular Biology and Biochemistry have obtained over the past century and put it to use to make the world a better (or simply more interesting) place for us to live in.<br />
<br />
Most iGEM teams have built their projects upon the humble bacterium ''Escherichia coli'', which is well-understood and easy to engineer. No team in past years has engineered a multicellular organism. Since many teams are comprised of students from diverse backgrounds, the more intricate complexities of the biology of higher organisms aren’t always accessible, and this has set a ceiling on project evolution. We set out to make these advancements possible, and bring iGEM into the kingdom Animalia.<br />
<br />
''Caenorhabidits elegans'' is a worm about a millimeter long. It’s a common model organism, easier to work with than most well-studied animals, and feeds on bacteria. Last spring, our team didn’t know much more about it than that. But we learned and studied, and engineered a whole set of foundational BioBricks in the hope that we would be able to offer other teams in future years the tools and information that would let them expand beyond one-cell projects. W<br />
<br />
If you want to dive into background material on the worm, now is a good time to head over to [[Team:Queens-Canada/guide|WormGuide]]. Otherwise, keep reading!<br />
<br />
'''[[Team:Queens-Canada/idea|Continue to Project Overview]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/guideTeam:Queens-Canada/guide2010-10-28T02:25:24Z<p>Glh: </p>
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<br />
<html><div class="section"><h1>WormGuide</h1></div></html><br />
<br />
<html><img src="https://static.igem.org/mediawiki/2010/6/60/Qgem_content_guide_wormpile.jpg" style="float: left; width: 40%; max-width: 350px; box-shadow: 1px 2px 3px #808080; margin-right: 10px;"></html><br />
<br />
Without exception, all of the work at <html><a target="_new" href="/">iGEM</a></html> in previous years has been restricted to single-celled organisms, mostly bacteria. This has naturally imposed limitations on the scope and application of iGEM projects. We decided to introduce ''Caenorhabditis elegans'', an easily-engineered nematode worm, as a new chassis for future teams to use as the platform for their projects. This opens up a new tier of possibilities for projects in the future, as iGEM enters the multicellular world.<br />
<br />
Before an iGEM team can begin work on a project using this new chassis, a lot of learning is necessary. WormGuide is a comprehensive educational and reference work that we have designed to allow future iGEM teams to quickly and easily learn about ''C. elegans'', and whether a worm project is right for them. We hope that it will help people learn the background knowledge necessary for the use of this organism for work in synthetic biology, while simultaneously helping them understand what opportunities the new chassis creates.<br />
<br />
<html><div class="section"><h2>Contents</h2></html><br />
<br />
''You can also browse WormGuide by using the graphical navigation at the top.''<br />
<br />
* Context<br />
** '''[[Team:Queens-Canada/intro|Introduction]]''': A little bit more information on what these worms are.<br />
** '''[[Team:Queens-Canada/history|History]]''': A brief history of ''C. elegans'' in biology and why it is so widely studied.<br />
** '''[[Team:Queens-Canada/resources|Online Resources]]''': Some of the pages you'll be using as a synthetic worm biologist.<br />
* Physiology and Anatomy<br />
** '''[[Team:Queens-Canada/pseudocoelom|Pseudocoelom]]''': The "blood" of the worm which bathes all tissues and allows for gas, nutrient and hormone transfer between cells, as well as maintaining osmotic and internal pressure.<br />
** '''[[Team:Queens-Canada/digestive|Digestive]]''': The pharynx and intestine.<br />
** '''[[Team:Queens-Canada/nervous|Nervous System]]''': The worm's sensory apparatus, the worm's muscles, and how they all fit together.<br />
** '''[[Team:Queens-Canada/skin|Exterior]]''': The cuticle and epicuticle that surround the worm.<br />
** '''[[Team:Queens-Canada/reproductive|Reproductive]]''': The reproductive anatomy and the worm's lifecycle.<br />
* Genetics<br />
** '''[[Team:Queens-Canada/promoter|Promoter and 5' UTR]]''': Everything upstream of the protein-coding sequence.<br />
** '''[[Team:Queens-Canada/transcripts|Introns and Transcripts]]''': How to make and use introns and operons in ''C. elegans''.<br />
** '''[[Team:Queens-Canada/rnai|RNAi and 3' UTR]]''': Not all forms of regulation occur before transcription!<br />
** '''[[Team:Queens-Canada/strains|Strains and Mutants]]''': How to get worms from the CGC and a selection of mutant strains that might prove useful to various kinds of projects.<br />
* Protocols<br />
** '''[[Team:Queens-Canada/transformation|Transformation]]''': How to get DNA into the worm.<br />
** '''[[Team:Queens-Canada/care|Care and Keeping]]''': How to keep the worms alive and work with them.<br />
** '''[[Team:Queens-Canada/protocols|Miscellaneous]]''': The non-worm protocols that we used in WormWorks.<br />
<br />
<html></div></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/guideTeam:Queens-Canada/guide2010-10-28T02:24:21Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<html><div class="section"><h1>WormGuide</h1></div></html><br />
<br />
Without exception, all of the work at <html><a target="_new" href="/">iGEM</a></html> in previous years has been restricted to single-celled organisms, mostly bacteria. This has naturally imposed limitations on the scope and application of iGEM projects. We decided to introduce ''Caenorhabditis elegans'', an easily-engineered nematode worm, as a new chassis for future teams to use as the platform for their projects. This opens up a new tier of possibilities for projects in the future, as iGEM enters the multicellular world.<br />
<br />
<html><img src="https://static.igem.org/mediawiki/2010/6/60/Qgem_content_guide_wormpile.jpg" style="float: left; width: 40%; max-width: 350px; box-shadow: 1px 2px 3px #808080; margin-right: 10px;"></html><br />
<br />
Before an iGEM team can begin work on a project using this new chassis, a lot of learning is necessary. WormGuide is a comprehensive educational and reference work that we have designed to allow future iGEM teams to quickly and easily learn about ''C. elegans'', and whether a worm project is right for them. We hope that it will help people learn the background knowledge necessary for the use of this organism for work in synthetic biology, while simultaneously helping them understand what opportunities the new chassis creates.<br />
<br />
<html><div class="section"><h2>Contents</h2></html><br />
<br />
''You can also browse WormGuide by using the graphical navigation at the top.''<br />
<br />
* Context<br />
** '''[[Team:Queens-Canada/intro|Introduction]]''': A little bit more information on what these worms are.<br />
** '''[[Team:Queens-Canada/history|History]]''': A brief history of ''C. elegans'' in biology and why it is so widely studied.<br />
** '''[[Team:Queens-Canada/resources|Online Resources]]''': Some of the pages you'll be using as a synthetic worm biologist.<br />
* Physiology and Anatomy<br />
** '''[[Team:Queens-Canada/pseudocoelom|Pseudocoelom]]''': The "blood" of the worm which bathes all tissues and allows for gas, nutrient and hormone transfer between cells, as well as maintaining osmotic and internal pressure.<br />
** '''[[Team:Queens-Canada/digestive|Digestive]]''': The pharynx and intestine.<br />
** '''[[Team:Queens-Canada/nervous|Nervous System]]''': The worm's sensory apparatus, the worm's muscles, and how they all fit together.<br />
** '''[[Team:Queens-Canada/skin|Exterior]]''': The cuticle and epicuticle that surround the worm.<br />
** '''[[Team:Queens-Canada/reproductive|Reproductive]]''': The reproductive anatomy and the worm's lifecycle.<br />
* Genetics<br />
** '''[[Team:Queens-Canada/promoter|Promoter and 5' UTR]]''': Everything upstream of the protein-coding sequence.<br />
** '''[[Team:Queens-Canada/transcripts|Introns and Transcripts]]''': How to make and use introns and operons in ''C. elegans''.<br />
** '''[[Team:Queens-Canada/rnai|RNAi and 3' UTR]]''': Not all forms of regulation occur before transcription!<br />
** '''[[Team:Queens-Canada/strains|Strains and Mutants]]''': How to get worms from the CGC and a selection of mutant strains that might prove useful to various kinds of projects.<br />
* Protocols<br />
** '''[[Team:Queens-Canada/transformation|Transformation]]''': How to get DNA into the worm.<br />
** '''[[Team:Queens-Canada/care|Care and Keeping]]''': How to keep the worms alive and work with them.<br />
** '''[[Team:Queens-Canada/protocols|Miscellaneous]]''': The non-worm protocols that we used in WormWorks.<br />
<br />
<html></div></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/File:Qgem_content_guide_wormpile.jpgFile:Qgem content guide wormpile.jpg2010-10-28T02:23:00Z<p>Glh: </p>
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<div></div>Glhhttp://2010.igem.org/Team:Queens-Canada/introTeam:Queens-Canada/intro2010-10-28T02:19:13Z<p>Glh: </p>
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<br />
<h1>Introduction</h1><br />
<br />
<html><img src="https://static.igem.org/mediawiki/2010/d/d0/Qgem_content_intro_glowy.jpg" style="float: right; width: 40%; max-width: 404px; box-shadow: 1px 2px 3px #808080; margin-left: 10px;"></html><br />
<br />
''Caenorhabditis elegans'' is a nematode worm, which is about a milimetre in length and free-living. (Unlike many other nematodes, ''C. elegans'' is not parasitic.) It has been used by the scientific community as a model organism since 1965. It fills this role well, as a result of the ease with which it can be bred and stored; its fast generation time; its simple, transparent body, which consists of about 1000 cells that do not change once the worm has matured; its basic neural and sensory system; and the fact that the vast majority of individuals are self-fertilizing hermaphrodites, which allows a genetically homogeneous population to be easily maintained.<br />
<br />
The worm is also extremely simple to work with in the lab, as populations can be grown on agar plates or in liquid media, need only a lawn of ''E. coli'' for sustenance, and can even be stored for decades if frozen at –80°C or in liquid nitrogen. The full worm lifecycle, from spawning an F1 to spawning an F2, takes about a week, but worms are mature enough after 3 days for their expression patterns to be almost fully adult; thus the success or failure of transgenic constructs can be assessed at that time.<br />
<br />
The transparent body of ''C. elegans'' allows fluorescent proteins to be used as markers that can easily be detected in any tissues, while keeping the worm completely intact. The worm’s simple nervous system allows it to react to basic inputs in an appropriate manner. For example, mechanoreceptor neurons allow the organism to realize when it bumps into something, and then to back up and turn. ''C. elegans'' is also capable of thermotaxis (towards a preferred temperature range), phototaxis (away from light), and chemotaxis (towards evidence of food, away from evidence of hazards, and towards its favourite pH). The organism's ability react to stimuli can be easily manipulated through the introduction of new elements into the sensory neurons (individual neurons can be selectively targeted using the appropriate promoter, as described [[Team:Queens-Canada/nervous|in our article on the nervous system]].) This represents a wealth of opportunities in synthetic biology, as what ''C. elegans'' perceives itself to be experiencing can be manipulated to achieve a desired behaviour. A genetically distinct hermaphrodite can be cultured in isolation to produce a genetically identical population that can be maintained with ease. This allows a line of transformed worms to be easily kept.<br />
<br />
There are a number of further reasons as to why ''C. elegans'' is especially useful in the field of synthetic biology. It is relatively easy to transform (in addition to the week-long generation cycle, microinjecting the gonad with a plasmid is sufficient), has a fixed number of cells at maturity (959 somatic nuclei for the hermaphrodite and 1031 for the male), can have its genes selectively silenced through RNAi (which was discovered in ''C. elegans''), and lives naturally in temperate soil (and so thrives at laboratory temperatures).<br />
<br />
Developmental biologists have been attracted to ''C. elegans'', in addition to all of the reasons above, because of a number of striking analogues that can be found between the nematode and other model organisms. Riddle et al. in <html><a href="http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ce2&part=A24" target="_new">1.I of <i>C. elegans II</i></a></html> put it this way:<br />
<br />
<blockquote>“Whether by chance or by design, basic biomedical research in the past 30 years has concentrated on a relatively small number of model systems (primarily prokaryotic cells, yeast, protozoans, ''C. elegans'', ''Drosophila'', ''Xenopus'', ''Mus'', primates, and mammalian cells in culture). Although these are quite different from each other, an astounding degree of connectivity between them has been revealed in the past decade. The emerging parallels between the development of the body plan in nematodes, flies, and mice <html>(<a href="http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ce2&part=A680" target="_new">Ruvkun</a>, this volume), and the fact that similar proteins are used for programmed cell death in both nematodes and humans (<a href="http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ce2&part=A485" target="_new">Hengartner</a>, this volume)</html>, provide two examples.”</blockquote><br />
<br />
Most importantly, however, there is a high degree of portability of genes between different complex animals, including the worm. Transgenes containing G-protein coupled receptors from humans and mice have been introduced into ''C. elegans'' before, and remarkably these receptors were also able to interact with the native G-protein subunits and function in the intended fashion, despite hundreds of millions of years of divergent evolution. This suggests that much of the work we will do in this little nematode will still be relevant if and when synthetic biology as a whole shifts its focus to larger animals.<br />
<br />
'''[[Team:Queens-Canada/history|Continue to History in Science]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/File:Qgem_content_intro_glowy.jpgFile:Qgem content intro glowy.jpg2010-10-28T02:13:17Z<p>Glh: </p>
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<div></div>Glhhttp://2010.igem.org/Team:Queens-Canada/partsTeam:Queens-Canada/parts2010-10-28T00:24:29Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks Parts List</h1><br />
<br />
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.<br />
<br />
<html><div class="section"><h2>Regulatory</h2></html><br />
<br />
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.<br />
<br />
<h3>Constitutive</h3><br />
* '''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.<br />
* '''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.<br />
* '''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>.<br />
<br />
<h3>Inducible</h3><br />
<br />
* '''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.<br />
<br />
<h3>Tissue-Specific</h3><br />
<br />
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]].<br />
<br />
* '''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>.<br />
* '''pOdr-1''': This targets the AWC sensory neuron.<br />
* '''pStr-1''': This targets the AWB sensory neuron.<br />
* '''pOsm-10''': This targets the ASH and ASI sensory neurons, as well as the PHA and PHB phasmid neurons.<br />
* '''pFlp-1''': This targets the AVA sensory neuron.<br />
* '''pSra-10''': This targets the AVB sensory neuron.<br />
* '''pStr-220''': This targets the AWC sensory neuron.<br />
<br />
<html></div><div class="section"><h2>Reporters</h2></html><br />
<br />
* '''eGFP''': excitation: 395 nm; emission: 509 nm.<br />
* '''eCFP''': excitation: 433 nm; emission: 475 nm.<br />
* '''eYFP''': excitation: ~485 nm; emission: ~700 nm.<br />
* '''mCherry''': excition: ~580 nm; emission: ~620 nm.<br />
<br />
<html></div><div class="section"><h2>Optogenetics Proteins</h2></html><br />
<br />
* '''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.<br />
* '''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.<br />
* ''Fusions'': ChR2::eYFP and NpHR::eCFP are both also provided. These are useful for ensuring that the channels localized successfully to their target.<br />
<br />
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.<br />
<br />
<html></div><div class="section"><h2>Constructs</h2></html><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/72/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"><br />
</html><br />
<br />
Unlike the fusions listed under Optogenetics Proteins, these were assembled through BioBrick digestion/ligation.<br />
<br />
* '''pStr-220::eCFP::unc-54 3' UTR'''<br />
* '''pOdr-10::eCFP::unc-54 3' UTR'''<br />
* '''pGpd-2::eCFP::unc-54 3' UTR'''<br />
* '''pSip1::eCFP::unc-54 3' UTR'''<br />
* '''pFlp1::eCFP:unc-54 3' UTR'''<br />
* '''ChR2::eYFP::unc-54 3' UTR'''<br />
* '''pHsp3::eCFP::unc-54 3' UTR'''<br />
* '''pOsm-10::eCFP::unc-54 3' UTR'''<br />
* '''mCherry::unc-54 3' UTR'''<br />
* '''eYFP::unc-54 3' UTR'''<br />
* '''pOdr-1::eCFP::unc-54 3' UTR'''<br />
* '''pMec-7::eCFP::unc-54 3' UTR'''<br />
<br />
<html></div><div class="section"><h2>Getting the Parts</h2></html><br />
<br />
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.<br />
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{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/transformationTeam:Queens-Canada/transformation2010-10-27T23:40:44Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
<br />
<html><div class="section"><h1>Microinjection</h1></html><br />
<br />
Microinjection takes about a week to learn how to do, and a few months to get highly proficient at. Once experienced, however, the actual act of injection only takes a few minutes. Most universities have at least one microinjection table, and we recommend that teams not comfortable with devoting the resources to training one of their members to microinject ask for assistance from an experienced technician. The actual process involves injecting DNA into the mother’s ovary using a very fine needle under a high-powered microscope.<br />
<br />
Below is our standard protocol for performing microinjections as used by Hao Shi. He spent most of the summer practicing and was successful when we started our real injections in August. Learning the microinjection process is best done with the assistance of a live instructor, but these resources may also help you:<br />
<br />
* <html><a href="http://130.15.90.245/microinjection.htm" target="_new">Chin-Sang Lab Microinjection Protocol</a></html><br />
* <html><a href="http://wormbook.org/chapters/www_transformationmicroinjection/transformationmicroinjection.html" target="_new">Microinjection Information from WormMethods</a></html><br />
<br />
The WormMethods link also includes information on techniques for integrating DNA into chromosomes. Since microinjection isn’t perfect, these may still be of use.<br />
<br />
<html></div><div class="section"><h2>Materials</h2></html><br />
<br />
<html><div class="asideR" style="max-width: 60%"><p><b>Why a plasmid?</b></p><br />
<p><i>C. elegans</i> is unusual in that its chromosome duplication is <b>holocentric</b>: the proteins necessary to split the duplicated DNA strands don’t focus on one specific, defined spot, but distribute evenly over the length of the molecule. As a result, we can use just about any DNA format we want to transform worms with, including bacterial plasmids and PCR products. This isn’t perfect; worms have been shown to have up to a 2% chance of loss of artificially-introduced high-copy DNA, compared to a maximum reported rate of 0.5% for chromosomal loss, suggesting that the structure that forms isn’t quite as stable as a natural chromosome. Whether this is structural or if some sequence on the chromosomes assists efficient replication isn’t clear.</p><br />
</div></html><br />
<br />
* Linear gene construct or plasmid<br />
* Salmon sperm DNA (filler DNA)<br />
* Inverted microscope with microinjection stage<br />
* Micropipette puller<br />
* Microcapillaries<br />
* M9 microinjection oil<br />
* Injection pads<br />
* Microscope slide with glass cover slip<br />
<br />
<html></div><div class="section"><h2>Injection Pad Preparation</h2></html><br />
<br />
# Mix '''0.2 g''' of agarose and '''10 mL''' of ddH<sub>2</sub>O and microwave for '''30 s'''.<br />
# Place 2–4 drops of 2% agarose onto the center of a thin glass slide.<br />
# Flatten the drop with another slide placed perpendicularly on top.<br />
# Allow slides to dry for '''5–10 minutes''' and separate the two glass slides.<br />
# Allow slides to dry over night before use.<br />
<br />
<html></div><div class="section"><h2>Needle Preparation</h2></html><br />
<br />
# Place the microcapillary tube into top slot of micropipette puller and tighten the top knob. # The capillary should be protruding approximately 1.5-2.0 inch from the top ridge. <br />
# Slide up the bottom unit and clamp onto microcapillary with bottom knob.<br />
# Turn on machine.<br />
# Fix machine settings to '''Temperature = 740''' and '''Pull = 940'''.<br />
# Close cover and press “start” button.<br />
# The microcapillary tube will be pulled into two needles after a few seconds.<br />
# Remove needle in top clamp and place horizontally onto a piece of clay.<br />
<br />
<html></div><div class="section"><h2>Sample Preparation</h2></html><br />
<br />
# Prepare sample by mixing '''30 µg/mL''' of gene construct, '''30 µg/mL''' of reporter, and '''40 µg/mL''' of filler DNA. Alternatively, if reporter is already included in the construct, then add '''30 µg/mL''' of sample and '''70 µg/mL''' of filler DNA.<br />
# Centrifuge mixture at 13,000 rpm for 10 min to pellet the impurities.<br />
# Pipette '''1.5 µL''' of the supernatant into the un-pulled end of the microcapillary.<br />
# Allow '''3-5 min''' for the sample to move into the needle tip.<br />
<br />
<html></div><div class="section"><h2>Breaking the Needle</h2></html><br />
<br />
# Mount needle onto microinjection apparatus of microscope.<br />
# Prepare a needle-breaking slide by adding a few drops of M9 microinjection oil onto a thin glass slide and covering that with a small glass cover slide. Some oil should squeeze out from the sides of the cover slide.<br />
# Place slide with glass cover slip on stage.<br />
# Locate the edge of cover slip closest to the needle and bring it into focus.<br />
# Use the XYZ axis movement control knobs to bring needle tip into view and in focus.<br />
# Gently touch the edge of the cover slip to the tip of the needle '''by moving the stage, NOT the needle!''' This should break the needle.<br />
# Tap the foot pedal for releasing needle contents to check if needle tip is broken properly. The droplet of water should be approximately 10 times the width of the needle tip.<br />
<br />
<html></div><div class="section"><h2>Injection</h2></html><br />
<br />
# Apply a small drop of M9 onto the agar region of the injection pad.<br />
# Flame a pick and dip it in the drop of M9.<br />
# Pick a young adult N2 worm from a plate and transfer it to the M9 on the injection pad.<br />
# Very gently press the worm onto the agar with the pick to prevent it from moving.<br />
# Place the injection pad onto stage. Locate the worm and bring it into focus.<br />
# Bring the needle into focus in the same field as the worm.<br />
# Orient the slide so that the site of injection (the gonads) makes a 45° angle with the length of the needle.<br />
# Use the fine adjustment knob to focus on two rows of cells separated by a clear area beside the row of oocytes. This area represents the gonad of the worm. There are two of such areas (“arms”) in each worm, one in the anterior and one in the posterior.<br />
# Use the Z axis fine control to bring the '''tip''' of the needle into focus. The two rows of cells in one arm of the gonads and the needle tip should '''both''' now be in focus.<br />
# Slowly '''bring the worm towards the needle''' ('''NOT the other way around''') and puncture the side of the worm so that the tip of the needle ends up in the space between the two rows of cells mentioned above.<br />
# Tap the foot pedal to inject DNA into the worm’s gonad. It is important to not bloat the worm.<br />
# Slowly remove the worm from the needle and lift needle away from the slide.<br />
# Repeat steps 5-12 for the other arm of the gonads if possible.<br />
# Flame a pick and transfer the injected worm into a new, labelled plate.<br />
# Store injected worms at 18° C for 3 days and check for F1 offspring displaying the transgenic phenotype. These worms should be transferred onto individual plates for further analysis.<html></div></html><br />
<br />
'''[[Team:Queens-Canada/care|Continue to Care and Keeping]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/1_September_2010Team:Queens-Canada/1 September 20102010-10-27T23:11:19Z<p>Glh: New page: {{:Team:Queens-Canada/head}} <h2>1 September 2010</h2> <p>We imaged the results of a successful microinjection of our pMec-7::eCFP::unc-54 3' UTR construct:</p> <html><center><img src="...</p>
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<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h2>1 September 2010</h2><br />
<br />
<p>We imaged the results of a successful microinjection of our pMec-7::eCFP::unc-54 3' UTR construct:</p><br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/7/72/Qgem_content_mec7_ecfp.jpg" style="max-width: 960px; width: 100%; box-shadow: 1px 2px 3px #808080;"></center></html><br />
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<p>It can be found in the parts registry <html><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K309032" target="_new">here</a>.</html><br />
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{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/partsTeam:Queens-Canada/parts2010-10-27T23:06:46Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks Parts List</h1><br />
<br />
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.<br />
<br />
<html><div class="section"><h2>Regulatory</h2></html><br />
<br />
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.<br />
<br />
<h3>Constitutive</h3><br />
* '''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.<br />
* '''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.<br />
* '''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>.<br />
<br />
<h3>Inducible</h3><br />
<br />
* '''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.<br />
<br />
<h3>Tissue-Specific</h3><br />
<br />
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]].<br />
<br />
* '''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>.<br />
* '''pOdr-1''': This targets the AWC sensory neuron.<br />
* '''pStr-1''': This targets the AWB sensory neuron.<br />
* '''pOsm-10''': This targets the ASH and ASI sensory neurons, as well as the PHA and PHB phasmid neurons.<br />
* '''pFlp-1''': This targets the AVA sensory neuron.<br />
* '''pSra-10''': This targets the AVB sensory neuron.<br />
* '''pStr-220''': This targets the AWC sensory neuron.<br />
<br />
<html></div><div class="section"><h2>Reporters</h2></html><br />
<br />
* '''eGFP''': excitation: 395 nm; emission: 509 nm.<br />
* '''eCFP''': excitation: 433 nm; emission: 475 nm.<br />
* '''eYFP''': excitation: ~485 nm; emission: ~700 nm.<br />
* '''mCherry''': excition: ~580 nm; emission: ~620 nm.<br />
<br />
<html></div><div class="section"><h2>Optogenetics Proteins</h2></html><br />
<br />
* '''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.<br />
* '''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.<br />
* ''Fusions'': ChR2::eYFP and NpHR::eCFP are both also provided. These are useful for ensuring that the channels localized successfully to their target.<br />
<br />
<html></div><div class="section"><h2>Constructs</h2></html><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/7/72/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"><br />
</html><br />
<br />
Unlike the fusions listed under Optogenetics Proteins, these were assembled through BioBrick digestion/ligation.<br />
<br />
* '''pStr-220::eCFP::unc-54 3' UTR'''<br />
* '''pOdr-10::eCFP::unc-54 3' UTR'''<br />
* '''pGpd-2::eCFP::unc-54 3' UTR'''<br />
* '''pSip1::eCFP::unc-54 3' UTR'''<br />
* '''pFlp1::eCFP:unc-54 3' UTR'''<br />
* '''ChR2::eYFP::unc-54 3' UTR'''<br />
* '''pHsp3::eCFP::unc-54 3' UTR'''<br />
* '''pOsm-10::eCFP::unc-54 3' UTR'''<br />
* '''mCherry::unc-54 3' UTR'''<br />
* '''eYFP::unc-54 3' UTR'''<br />
* '''pOdr-1::eCFP::unc-54 3' UTR'''<br />
* '''pMec-7::eCFP::unc-54 3' UTR'''<br />
<br />
<html></div><div class="section"><h2>Getting the Parts</h2></html><br />
<br />
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.<br />
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{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/File:Qgem_content_mec7_ecfp.jpgFile:Qgem content mec7 ecfp.jpg2010-10-27T23:05:40Z<p>Glh: </p>
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<div></div>Glhhttp://2010.igem.org/Team:Queens-Canada/introTeam:Queens-Canada/intro2010-10-27T22:58:47Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>Introduction</h1><br />
<br />
''Caenorhabditis elegans'' is a nematode worm, which is about a milimetre in length and free-living. (Unlike many other nematodes, ''C. elegans'' is not parasitic.) It has been used by the scientific community as a model organism since 1965. It fills this role well, as a result of the ease with which it can be bred and stored; its fast generation time; its simple, transparent body, which consists of about 1000 cells that do not change once the worm has matured; its basic neural and sensory system; and the fact that the vast majority of individuals are self-fertilizing hermaphrodites, which allows a genetically homogeneous population to be easily maintained.<br />
<br />
The worm is also extremely simple to work with in the lab, as populations can be grown on agar plates or in liquid media, need only a lawn of ''E. coli'' for sustenance, and can even be stored for decades if frozen at –80°C or in liquid nitrogen. The full worm lifecycle, from spawning an F1 to spawning an F2, takes about a week, but worms are mature enough after 3 days for their expression patterns to be almost fully adult; thus the success or failure of transgenic constructs can be assessed at that time.<br />
<br />
The transparent body of ''C. elegans'' allows fluorescent proteins to be used as markers that can easily be detected in any tissues, while keeping the worm completely intact. The worm’s simple nervous system allows it to react to basic inputs in an appropriate manner. For example, mechanoreceptor neurons allow the organism to realize when it bumps into something, and then to back up and turn. ''C. elegans'' is also capable of thermotaxis (towards a preferred temperature range), phototaxis (away from light), and chemotaxis (towards evidence of food, away from evidence of hazards, and towards its favourite pH). The organism's ability react to stimuli can be easily manipulated through the introduction of new elements into the sensory neurons (individual neurons can be selectively targeted using the appropriate promoter, as described [[Team:Queens-Canada/nervous|in our article on the nervous system]].) This represents a wealth of opportunities in synthetic biology, as what ''C. elegans'' perceives itself to be experiencing can be manipulated to achieve a desired behaviour. A genetically distinct hermaphrodite can be cultured in isolation to produce a genetically identical population that can be maintained with ease. This allows a line of transformed worms to be easily kept.<br />
<br />
There are a number of further reasons as to why ''C. elegans'' is especially useful in the field of synthetic biology. It is relatively easy to transform (in addition to the week-long generation cycle, microinjecting the gonad with a plasmid is sufficient), has a fixed number of cells at maturity (959 somatic nuclei for the hermaphrodite and 1031 for the male), can have its genes selectively silenced through RNAi (which was discovered in ''C. elegans''), and lives naturally in temperate soil (and so thrives at laboratory temperatures).<br />
<br />
Developmental biologists have been attracted to ''C. elegans'', in addition to all of the reasons above, because of a number of striking analogues that can be found between the nematode and other model organisms. Riddle et al. in <html><a href="http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ce2&part=A24" target="_new">1.I of <i>C. elegans II</i></a></html> put it this way:<br />
<br />
<blockquote>“Whether by chance or by design, basic biomedical research in the past 30 years has concentrated on a relatively small number of model systems (primarily prokaryotic cells, yeast, protozoans, ''C. elegans'', ''Drosophila'', ''Xenopus'', ''Mus'', primates, and mammalian cells in culture). Although these are quite different from each other, an astounding degree of connectivity between them has been revealed in the past decade. The emerging parallels between the development of the body plan in nematodes, flies, and mice <html>(<a href="http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ce2&part=A680" target="_new">Ruvkun</a>, this volume), and the fact that similar proteins are used for programmed cell death in both nematodes and humans (<a href="http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ce2&part=A485" target="_new">Hengartner</a>, this volume)</html>, provide two examples.”</blockquote><br />
<br />
Most importantly, however, there is a high degree of portability of genes between different complex animals, including the worm. Transgenes containing G-protein coupled receptors from humans and mice have been introduced into ''C. elegans'' before, and remarkably these receptors were also able to interact with the native G-protein subunits and function in the intended fashion, despite hundreds of millions of years of divergent evolution. This suggests that much of the work we will do in this little nematode will still be relevant if and when synthetic biology as a whole shifts its focus to larger animals.<br />
<br />
'''[[Team:Queens-Canada/history|Continue to History in Science]]'''<br />
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{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/reproductiveTeam:Queens-Canada/reproductive2010-10-27T22:02:44Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>The Reproductive System</h1><br />
<br />
The majority of ''C. elegans'' nematodes develop as hermaphrodites, and contain both male and female reproductive organs and fertilize their own eggs in order to reproduce. The remaining ''C. elegans'' males develop unique structures which they then use to fertilize the eggs of hermaphroditic organisms.<br />
<br />
<html><div class="section"><h2>Hermaphrodite System</h2></html><br />
<br />
The hermaphroditic reproductive system is structured to produce both sperm and eggs. The sperm fertilize the eggs internally, and additional anatomy works to lay the developing embryos.<br />
<br />
The system is organized bilaterally into two gonadal arms. Each arm contains one copy of the gamete development and fertilization apparati and the two arms join in the middle to form one egg-laying apparatus.<br />
<br />
<html><center><img src="http://wormatlas.org/hermaphrodite/reproductive/Images/ReproFIG1lr.jpg" style="width: 100%; max-width: 800px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/hermaphrodite/reproductive/Images/reprofig1leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
<b>Distal tip cell (DTC)</b>: A single large cell at the end of the gonadal arm. It is the first cell present in the developing reproductive system. It helps to direct the development of the system and the gametes contained therein.<br />
<br />
<b>Gonadal sheath</b>: A single layer of cells covering the germ line cells in the gonadal arm. The sheath consists of five pairs of identical cells each with different roles in development. The cells envelop germ line cells and aid in spermatogenesis and oogenesis. As the germ line cells travel from the DTC to the spermatheca through the gonadal sheath they mature and fully develop.<br />
<br />
<b>Spermatheca</b>: A twenty-four cell structure distal to the gonadal sheath. This is the fertilization apparatus of the worm. Mature sperm colonize the structure during development and await the entrance of oocytes. Oocytes enter one at a time and are fertilized. The spermatheca contains a valve on either end in order to control the entry and exit of oocytes into the structure. The distal valve is called the spermathecal-uterine valve (sp-ut valve)<br />
<br />
<b>Egg-laying apparatus</b> (uterus, vulva, VC, HSN neurons): A structure distal to the spermathecae, which contains and nourishes developing embryos and then lays the embryos. The apparatus is made of the uterus, uterine muscles, the vulva, vulval muscles, and a neuropil made up of neurons that direct egg laying. The vulva is the reproductive tract’s opening to the external environment.<html></div></html><br />
<br />
<html><div class="section"><h2>Male System</h2></html><br />
<br />
The male reproductive system is structured to produce mature sperm which are then used for the fertilization of hermaphroditic eggs.<br />
<br />
Male worms contain a single germ line apparatus that develops germ line cells and produces spermatids, a somatic gonad that stores and transports mature sperm and accessory liquids, and a proctodeum which is a modified rectum that contains the outflow of the alimentary canal and the reproductive system. The proctodeum also contains the structures necessary for copulation.<br />
<br />
<html><center><img src="http://wormatlas.org/male/reproductive/images/MaleReproFIG1.jpg" style="width: 100%; max-width: 733px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/male/reproductive/Reprointroframeset.html">From <i>WormAtlas</i></a></center></html><br />
<br />
<b>Distal tip cells</b> (DTC): Two large cells on the distal end of the germ line apparatus. They help to regulate the mitotic and meiotic division of germ line cells.<br />
<br />
<b>Germ cell apparatus</b>: A region of the male reproductive tract where stem cells undergo mitosis and meoisis as the migrate proximally towards the somatic gonad. The worm’s spermatids form in this region. They will remain spermatids until they enter the hermaphrodite’s uterus and become spermatazoa.<br />
<br />
<b>Seminal vesicle</b>: A structure distal to the germ cell apparatus that consists of an inner tube of twenty secretory cells surrounded by the cytoplasmic processes of three larger cells. The structure stores spermatids before they are ejaculated.<br />
<br />
<b>Vas deferens</b>: A long secretory tube made up of thirty cells that conduct spermatids from the seminal vesicle to the cloaca for ejaculation. Cells of three distinct morphologies are present.<br />
<br />
<html><div class="asideL" style="max-width: 40%"><br />
<p><b>More on Worm Reproduction</b></p><br />
<ul><br />
<li><a target="_new" href="http://wormatlas.org/hermaphrodite/reproductive/Reproframeset.html">WormAtlas on the hermaphrodite system</a><br />
<li><a target="_new" href="http://wormatlas.org/male/reproductive/Reprointroframeset.html">WormAtlas on the male system</a><br />
<li><a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed/20212008">Outcrossing and the Maintenance of Males within C. elegans Populations</a><br />
<li><a target="_new" href="http://wormbook.org/chapters/www_malematingbehavior/malematingbehavior.html">Male mating behavior</a><br />
</ul><br />
</div></html><br />
<br />
<b>Cloaca</b> (spicules, opening): An epithelial structure that joins the openings of the alimentary canal and the vas deferens to the exterior world at the end of the worm. The cloacal opening is the actual opening of the epithelial structure to the environment. Copulatory spicules are housed within the cloaca. These structures protrude from the worm, probe for the hermaphrodite vulva, and attach to the hermaphrodite during copulation.<html></div></html><br />
<br />
<h2>Reproduction and the Worm Lifecycle</h2><br />
<br />
<html><center><img src="http://130.15.90.245/images/C.%20elegans%20embryogenesis.jpg" style="width: 100%; max-width: 821px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://130.15.90.245/photos.htm">From the Chin-Sang Lab</a></center></html><br />
<br />
<html><div class="section"><h3>Embryonic Development</h3></html><br />
<br />
''C. elegans'' takes about thirteen hours to develop from zygote to larva. During this time, the first five and a half hours contain only division into more undifferentiated cells within the egg—the process of forming organs (morphogenesis) doesn’t begin until after this. Organs develop for the next six and a half to eight and a half hours, at which point the worm’s development halts unless food is available. Laying by the mother typically occurs around two and a half hours into development; hatching at around ten. The mature embryo has a well-developed nervous system and is capable of finding food on its own. At the end of embryonic development, the worm has a little over half of its total cells.<html></div></html><br />
<br />
<html><div class="section"><h3>Larval Development</h3></html><br />
<br />
Once food has been found, development proceeds, and cell division resumes three hours after hatching under ideal conditions, although arrested larvae can survive 6–10 days without food. The worm progresses through four distinctive stages, termed L1–L4, which are separated from each other by molting events, in which the worm sheds its cuticle. Collectively, larval development takes forty-five to fifty hours, and ends with the laying of eggs in hermaphrodites shortly after the fifth molt. The reproductive system, somewhat neglected during embryonic development, grows substantially during the larval phase.<html></div></html><br />
<br />
<html><a name="dauer"></a><div class="section"><h3>Dauer</h3></html><br />
<br />
If the worm determines that its environment is particularly food-poor, overcrowded, or of an unfavorable temperature, it will enter a form of hibernation called the dauer state. (There is a preface state to dauer, called L2d, in which it is still possible to revert into an L3 larva.) The worm becomes thin, develops a thick, protective cuticle, closes some of its orifices, and waits until better conditions present themselves. A worm in the dauer state does not experience a diminished adult lifespan, and will reach leave dauer within an hour of locating food, reaching L4 some nine hours later.<html></div></html><br />
<br />
<html><div class="section"><h3>Adulthood</h3></html><br />
<br />
The adult hermaphrodite lives for 13 to 19 days, including 3–4 days of egg laying. The hermaphrodite only has a supply of about 300 spermatozoa, developed during L4, but can produce up to 1400 progeny over the adult lifespan if a male is available to fertilize it. Τhe hermaphrodite has 959 somatic nuclei, 302 of which are neurons and 95 are body wall muscle cells, which are multinucleated. The male has 1031 somatic nuclei. 381 of these are neurons.<html></div></html><br />
<br />
'''[[Team:Queens-Canada/guide|Return to the Guide Hub]]'''<br />
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{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/reproductiveTeam:Queens-Canada/reproductive2010-10-27T21:59:59Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>The Reproductive System</h1><br />
<br />
The majority of ''C. elegans'' nematodes develop as hermaphrodites, and contain both male and female reproductive organs and fertilize their own eggs in order to reproduce. The remaining ''C. elegans'' males develop unique structures which they then use to fertilize the eggs of hermaphroditic organisms.<br />
<br />
<html><div class="section"><h2>Hermaphrodite System</h2></html><br />
<br />
The hermaphroditic reproductive system is structured to produce both sperm and eggs. The sperm fertilize the eggs internally, and additional anatomy works to lay the developing embryos.<br />
<br />
The system is organized bilaterally into two gonadal arms. Each arm contains one copy of the gamete development and fertilization apparati and the two arms join in the middle to form one egg-laying apparatus.<br />
<br />
<html><center><img src="http://wormatlas.org/hermaphrodite/reproductive/Images/ReproFIG1lr.jpg" style="width: 100%; max-width: 800px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/hermaphrodite/reproductive/Images/reprofig1leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
<b>Distal tip cell (DTC)</b>: A single large cell at the end of the gonadal arm. It is the first cell present in the developing reproductive system. It helps to direct the development of the system and the gametes contained therein.<br />
<br />
<b>Gonadal sheath</b>: A single layer of cells covering the germ line cells in the gonadal arm. The sheath consists of five pairs of identical cells each with different roles in development. The cells envelop germ line cells and aid in spermatogenesis and oogenesis. As the germ line cells travel from the DTC to the spermatheca through the gonadal sheath they mature and fully develop.<br />
<br />
<b>Spermatheca</b>: A twenty-four cell structure distal to the gonadal sheath. This is the fertilization apparatus of the worm. Mature sperm colonize the structure during development and await the entrance of oocytes. Oocytes enter one at a time and are fertilized. The spermatheca contains a valve on either end in order to control the entry and exit of oocytes into the structure. The distal valve is called the spermathecal-uterine valve (sp-ut valve)<br />
<br />
<b>Egg-laying apparatus</b> (uterus, vulva, VC, HSN neurons): A structure distal to the spermathecae, which contains and nourishes developing embryos and then lays the embryos. The apparatus is made of the uterus, uterine muscles, the vulva, vulval muscles, and a neuropil made up of neurons that direct egg laying. The vulva is the reproductive tract’s opening to the external environment.<html></div></html><br />
<br />
<html><div class="section"><h2>Male System</h2></html><br />
<br />
The male reproductive system is structured to produce mature sperm which are then used for the fertilization of hermaphroditic eggs.<br />
<br />
Male worms contain a single germ line apparatus that develops germ line cells and produces spermatids, a somatic gonad that stores and transports mature sperm and accessory liquids, and a proctodeum which is a modified rectum that contains the outflow of the alimentary canal and the reproductive system. The proctodeum also contains the structures necessary for copulation.<br />
<br />
<html><center><img src="http://wormatlas.org/male/reproductive/images/MaleReproFIG1.jpg" style="width: 100%; max-width: 733px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/male/reproductive/Reprointroframeset.html">From <i>WormAtlas</i></a></center></html><br />
<br />
<b>Distal tip cells</b> (DTC): Two large cells on the distal end of the germ line apparatus. They help to regulate the mitotic and meiotic division of germ line cells.<br />
<br />
<b>Germ cell apparatus</b>: A region of the male reproductive tract where stem cells undergo mitosis and meoisis as the migrate proximally towards the somatic gonad. The worm’s spermatids form in this region. They will remain spermatids until they enter the hermaphrodite’s uterus and become spermatazoa.<br />
<br />
<b>Seminal vesicle</b>: A structure distal to the germ cell apparatus that consists of an inner tube of twenty secretory cells surrounded by the cytoplasmic processes of three larger cells. The structure stores spermatids before they are ejaculated.<br />
<br />
<b>Vas deferens</b>: A long secretory tube made up of thirty cells that conduct spermatids from the seminal vesicle to the cloaca for ejaculation. Cells of three distinct morphologies are present.<br />
<br />
<html><div class="asideL" style="max-width: 40%"><br />
<p><b>More on Worm Reproduction</b></p><br />
<ul><br />
<li><a target="_new" href="http://wormatlas.org/hermaphrodite/reproductive/Reproframeset.html">WormAtlas on the hermaphrodite system</a><br />
<li><a target="_new" href="http://wormatlas.org/male/reproductive/Reprointroframeset.html">WormAtlas on the male system</a><br />
<li><a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed/20212008">Outcrossing and the Maintenance of Males within C. elegans Populations</a><br />
<li><a target="_new" href="http://wormbook.org/chapters/www_malematingbehavior/malematingbehavior.html">Male mating behavior</a><br />
</ul><br />
</div></html><br />
<br />
<b>Cloaca</b> (spicules, opening): An epithelial structure that joins the openings of the alimentary canal and the vas deferens to the exterior world at the end of the worm. The cloacal opening is the actual opening of the epithelial structure to the environment. Copulatory spicules are housed within the cloaca. These structures protrude from the worm, probe for the hermaphrodite vulva, and attach to the hermaphrodite during copulation.<html></div></html><br />
<br />
<h2>Reproduction and the Worm Lifecycle</h2><br />
<br />
<html><div class="section"><h3>Embryonic Development</h3></html><br />
<br />
''C. elegans'' takes about thirteen hours to develop from zygote to larva. During this time, the first five and a half hours contain only division into more undifferentiated cells within the egg—the process of forming organs (morphogenesis) doesn’t begin until after this. Organs develop for the next six and a half to eight and a half hours, at which point the worm’s development halts unless food is available. Laying by the mother typically occurs around two and a half hours into development; hatching at around ten. The mature embryo has a well-developed nervous system and is capable of finding food on its own. At the end of embryonic development, the worm has a little over half of its total cells.<html></div></html><br />
<br />
<html><div class="section"><h3>Larval Development</h3></html><br />
<br />
Once food has been found, development proceeds, and cell division resumes three hours after hatching under ideal conditions, although arrested larvae can survive 6–10 days without food. The worm progresses through four distinctive stages, termed L1–L4, which are separated from each other by molting events, in which the worm sheds its cuticle. Collectively, larval development takes forty-five to fifty hours, and ends with the laying of eggs in hermaphrodites shortly after the fifth molt. The reproductive system, somewhat neglected during embryonic development, grows substantially during the larval phase.<html></div></html><br />
<br />
<html><a name="dauer"></a><div class="section"><h3>Dauer</h3></html><br />
<br />
If the worm determines that its environment is particularly food-poor, overcrowded, or of an unfavorable temperature, it will enter a form of hibernation called the dauer state. (There is a preface state to dauer, called L2d, in which it is still possible to revert into an L3 larva.) The worm becomes thin, develops a thick, protective cuticle, closes some of its orifices, and waits until better conditions present themselves. A worm in the dauer state does not experience a diminished adult lifespan, and will reach leave dauer within an hour of locating food, reaching L4 some nine hours later.<html></div></html><br />
<br />
<html><div class="section"><h3>Adulthood</h3></html><br />
<br />
The adult hermaphrodite lives for 13 to 19 days, including 3–4 days of egg laying. The hermaphrodite only has a supply of about 300 spermatozoa, developed during L4, but can produce up to 1400 progeny over the adult lifespan if a male is available to fertilize it. Τhe hermaphrodite has 959 somatic nuclei, 302 of which are neurons and 95 are body wall muscle cells, which are multinucleated. The male has 1031 somatic nuclei. 381 of these are neurons.<html></div></html><br />
<br />
'''[[Team:Queens-Canada/guide|Return to the Guide Hub]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/skinTeam:Queens-Canada/skin2010-10-27T21:56:02Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>The Worm's Exterior</h1><br />
<br />
<html><center><img src="http://wormatlas.org/hermaphrodite/cuticle/Images/CutFIG1lr.jpg" style="width: 100%; max-width: 650px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/hermaphrodite/cuticle/Images/cutfig1leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
In ''E. coli'', synthetic biology has free reign to insert channels, receptors, and other proteins into the external membrane of the bacterium. The story in ''C. elegans'' is somewhat more complicated, as the nematode’s soft outer cells, the '''hypodermis''', are protected by a layer of collagens and other proteins, called the '''cuticle'''.<br />
<br />
Anatomically, the cuticle of ''C. elegans'' is divided into dorsal and ventral regions. In some developmental stages, including the adult worm, these regions are separated by longitudinal ridges called '''alae'''. The alae overlay the seam cells on the lateral side of the worm’s body, have different protein composition, and are distinct in appearance under an electron microscope. The cuticular structure is produced outside-first by cells underneath the cuticle, which insert additional layers of collagens during each developmental stage.<br />
<br />
The cuticle itself is always covered with a layer of lipids and proteins called the '''epicuticle''', some of which are glycosylated. These sugar moieties are diverse, and so complicate colonization by bacteria who thus need a significant number of different surface proteins to bind effectively and specifically, although there are some mutants (see section below) that disrupt collagen structure and may create a topology more friendly to retaining engineered bacteria, and some bacteria that bind despite this. Other known mutants (see section below) can affect the presence of various glycolipids and proteins in the epicuticle more directly. Similar glycosylation occurs inside the intestine, and is sometimes targeted by colonizing bacteria as well (see the article on [[Team:Queens-Canada/digestive|the digestive system]]). The cuticle also bears holes and swellings where some neuronal cilia are exposed to the environment, and has a slightly negative electric charge that helps repel bacteria. The composition of the epicuticle changes during the [[Team:Queens-Canada/reproductive#dauer|dauer hibernation state]] and late larval development, but at adulthood returns to its earlier developmental form, suggesting it is well-maintained.<br />
<br />
There are a number of bacteria known to be able to bind to ''C. elegans'' and other nematodes directly, however the majority are quite toxic to the worm and not very useful. A noteworthy exception to this <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed/11137017">is <i>Microbacterium nematophilum</i></a></html>, which attaches to the anal region of the cuticle as a method of hiding from its nematode predator, but is not particularly lethal to ''C. elegans''. (''M. nematophilum'' is, however, lethal to other nematodes in the ''Caenorhabditis'' genus.) However, the ''M. nematophilum'' genome is not well-studied and has not been sequenced. A lectin-binding assay may suggest a means to enable binding to the worm’s glycosylation.<br />
<br />
Binding bacteria to the exterior of the nematode presents an exciting opportunity for bringing the benefits of nematodes to current synthetic biology work without requiring current prokaryotic solutions to be translated into eukaryotic genes. Such '''episymbiosis''' could permit a layer of “backwards compatibility” that would allow older projects to exploit the benefits of the worm's superior chemotaxis and [[Team:Queens-Canada/nervous|powerful, precise sensory abilities]] for navigation without altering effector circuitry.<br />
<br />
Embedding engineered proteins in the cuticle may also be possible. <html><a target="_new" href="http://www.wormbase.org/db/searches/basic?class=AnyGene&query=cut">The <i>cut</i> family of genes</a></html> codes for the cuticulins, a set of proteins that form structural polymers and complement the collagens. Importantly, they are well-understood as localized to specific regions of the cuticle, and not nearly as numerous, although their export mechanism is not yet well-understood. Removing the cuticle, however, is not practical, as it necessary to maintain hydrostatic pressure inside the worm. Punctures to it cause the worm to burst.<br />
<br />
<html><a name="cuticle"></a></html><h2>Cuticle Mutants</h2><br />
<br />
There are more than 170 collagen proteins that contribute to cuticular extracellular matrix. The majority of those that have been tested do not result in any distinctive phenotype, but <html><a target="_new" href="http://www.wormbase.org/db/searches/basic?class=AnyGene&query=bus">members of the <i>bli</i> gene family</a></html> such as <html><a target="_new" href="http://www.wormbase.org/db/gene/gene?name=WBGene00000251;class=Gene"><i>bli-1</i></a></html> and <html><a target="_new" href="http://www.wormbase.org/db/gene/gene?name=WBGene00000252;class=Gene"><i>bli-2</i></a></html> produce a ‘blistered’ appearance of particular interest without compromising normal development. (A number of cuticle mutants produce abnormal worm movement or shape; these have been listed [[Team:Queens-Canada/strains#cuticle|under cuticular mutants as well]].)<br />
<br />
<html><a name="epicuticle"></a></html><h2>Epicuticle Mutants</h2><br />
<br />
The <html><a target="_new" href="http://www.wormbase.org/db/searches/basic?class=AnyGene&query=srf"><i>srf</i></a></html> family of genes affects the presence and composition of the glycosylated proteins and lipids that surround the epicuticle. ''srf-1'' is known to be necessary for the creation of an antigen that greatly reduces ''M. nematophilum''’s ability to bind when it is absent. The <html><a target="_new" href="http://www.wormbase.org/db/searches/basic?class=AnyGene&query=bus"><i>bus</i></a></html> family is similar, and contains many more genes that ''M. nematophilum'' requires in order to colonize ''C. elegans''. It is probable that one of these can be exploited or over-expressed in order to make a more reliable binding mechanism. In pathogenic nematodes, these carbohydrates are also required for effective immune system evasion. (Medical projects utilizing ''C. elegans'' are, however, ill-advised.)<br />
<br />
<html><div class="aside"><p><b>Cuticular Binding</b></p><br />
<ul><br />
<li><a href="http://www.ncbi.nlm.nih.gov/pubmed/3692138?dopt=abstract" target="_new">Genetic analysis of adult-specific surface antigenic differences between varieties of the nematode <i>Caenorhabditis elegans</i>.</a><br />
<li><a href="http://www.ncbi.nlm.nih.gov/pubmed/11137017" target="_new">A novel bacterial pathogen, Microbacterium nematophilum, induces morphological change in the nematode C. elegans.<br />
<li><a href="http://www.ncbi.nlm.nih.gov/pubmed/15123614" target="_new">Loss of <i>srf-3</i>-encoded nucleotide sugar transporter activity in Caenorhabditis elegans alters surface antigenicity and prevents bacterial adherence.</a><br />
<li><a href="http://en.wikipedia.org/wiki/Lectin" target="_new">Wikipedia on lectins</a><br />
<li><a href="http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ce2&part=A1025" target="_new"><i>C. elegans II</i> on the worm’s exterior surface</a><br />
</ul></div></html><br />
<br />
'''[[Team:Queens-Canada/reproductive|Continue to the Reproductive System]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/nervousTeam:Queens-Canada/nervous2010-10-27T21:54:46Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>The Nervous System</h1><br />
<br />
<html><div class="section"><h2>The Amphid</h2></html><br />
<br />
<html><center><img src="http://wormatlas.org/hermaphrodite/nervous/Images/NeuroFIG14lr.jpg" style="width: 100%; max-width: 700px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/hermaphrodite/nervous/Images/neurofig14leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
The amphid of ''C. elegans'' is equivalent (and homologous) to the nose of a human. It is used for sensing volatile and water soluble chemicals, and is found in the anterior region of the worm, at the base of the lips. Consisting of 12 sensory neurons, some of which are ciliated, the amphid is the primary sensory organ of ''C. elegans''.<br />
<br />
There are only two responses that ''C. elegans'' has to olfactory stimuli: attraction and repulsion. These are governed by separate sensory neurons, which are hard-wired upstream to produce those responses, and use largely interchangeable sensors in the form of receptor proteins. The majority of these receptors are GPCRs and membrane-bound guanylyl cyclases. <html><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550261/" target="_new">It has been shown</a></html> that adding new GPCRs can create either reaction depending on where the GPCR is expressed, and that at least some GPCRs from humans and mice will work in ''C. elegans'' without modification.<br />
<br />
<html><div class="asideR">For more information on GPCRs, see <a href="#gpcr_table">below</a>.</div></html><br />
<br />
There are a large number of GPCRs and receptor GCs that occur natively in ''C. elegans''; over one thousand GPCR candidates have been identified through genomic analysis. However, their functions and stimuli are not well understood.<br />
<br />
The primary appeal of engineering the amphid is to add new receptors so that the worm is either attracted to, or repulsed by, some sensory input. The worm has very powerful chemotaxis, and could effectively replace bacteria in most biosensor applications. Below is a partial table from WormBook listing the neurons of the amphid, their functions, and some of the receptors that occur in each. The gpa family encodes not receptors, but G-protein α subunits, which are required for signal transduction from GPCRs and are the major influential factor in whether or not a specific GPCR can be used in a given neuron without modification.<br />
<br />
<html><div class="aside" style="width: auto; display: inline-block"></html><br />
{|<br />
|Neuron<br />
|Function<br />
|Receptors; G proteins<br />
|-<br />
|ASE<br />
|Water-soluble chemotaxis<br />
|Receptor guanylate cyclases?; gpa-3<br />
|-<br />
|AWC<br />
|Volatile chemotaxis, Lifespan, Navigation<br />
|GPCRs (str-2); odr-3 (major), gpa-3, gpa-2, gpa-5, gpa-13<br />
|-<br />
|AWA<br />
|Volatile chemotaxis, Lifespan (minor)<br />
|GPCRs (odr-10); odr-3 (major), gpa-3, gpa-5; gpa-13; gpa-6<br />
|-<br />
|AWB<br />
|Volatile avoidance<br />
|GPCRs; odr-3<br />
|-<br />
|ASH<br />
|Nociception: Osmotic avoidance, Nose touch avoidance, Chemical avoidance, Social feeding<br />
|GPCRs; odr-3 (major), gpa-3 (major), gpa-11, gpa-1, gpa-13, gpa-14, gpa-15<br />
|-<br />
|ASI<br />
|Dauer formation, Chemotaxis(minor), Navigation<br />
|GPCRs; gpa-1, gpa-3, gpa-4, gpa-5, gpa-6, gpa-10, gpa-14<br />
|-<br />
|ADF<br />
|Dauer formation, Chemotaxis (minor)<br />
|GPCRs; odr-3, gpa-3, gpa-10, gpa-13<br />
|-<br />
|ASG<br />
|Dauer formation (minor), Lifespan, Chemotaxis (minor)<br />
|GPCRs; gpa-3<br />
|-<br />
|ASJ<br />
|Dauer formation and recovery, Chemotaxis (minor), Lifespan<br />
|GPCRs; gpa-1, gpa-3, gpa-9, gpa-10, gpa-14<br />
|-<br />
|ASK<br />
|Avoidance (minor), Chemotaxis (minor), Lifespan, Navigation<br />
|GPCRs; gpa-2, gpa-3, gpa-14, gpa-15<br />
|-<br />
|ADL<br />
|Avoidance (minor), Social feeding<br />
|GPCRs; gpa-1, gpa-3, gpa-11, gpa-15<br />
|-<br />
|URX, AQR, PQR<br />
|Oxygen/aerotaxis, Social feeding<br />
|Soluble guanylate cyclases (gcy-35, gcy-36); gpa-8<br />
|-<br />
|PHA, PHB<br />
|Avoidance (antagonistic)<br />
|GPCRs; gpa-1, gpa-2, gpa-3, gpa-6, gpa-9, gpa-13, gpa-14, gpa-15<br />
|}<br />
<html></div></html><br />
<br />
The major object of desire when working with sensory neurons is the availability of a specific promoter that can target each individually. We have endeavored to provide a number of these; see [[Team:Queens-Canada/parts|our parts list]]. <html><a href="http://wormbook.org/chapters/www_chemosensation/chemosensation.html" target="_new">More detail on chemosensation in <i>C. elegans</i></a></html> is available from WormBook.<html></div></html><br />
<br />
<html><div class="section"><h2>Other Forms of Sensation</h2></html><br />
<br />
''C. elegans'' is also natively able to detect mechanical force exerted on its body as well as heat and cold. The potential applications for engineering these traits is minimal, although it is worth noting that ''C. elegans'' naturally moves towards temperatures at which it has known feeding conditions to be favorable, and away from those at which it was not able to feed. There are, however, mutants that habitually seek out cold (''cryophilic'') and heat (''thermophilic'') without regard for this process. These may be of use, and can be found in our article on getting strains.<html></div></html><br />
<br />
<html><div class="section"><h2>The Neuron</h2></html><br />
<br />
<html><center><img src="http://wormatlas.org/hermaphrodite/nervous/Images/NeuroFIG3lr.jpg" style="width: 100%; max-width: 800px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/hermaphrodite/nervous/Images/neurofig3leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
The neuron is a highly-specialized cell that provides the worm (and many other animals) with the ability to send signals more rapidly than chemical diffusion, by using localized ion gradients to generate an electrical current down the length of their exterior. There are 302 neurons in the adult hermaphroditic ''C. elegans'', which may seem like a tiny number compared to the hundred billion neurons that humans have in the brain alone, but in fact we have not yet fully deciphered their behavior; how the temperature-remembering behavior mentioned above works is not yet known.<br />
<br />
In order to be functional, a neuron must consist of one or more ‘inputs’, a cell body which contains the nucleus and also decides whether or not to take action based on a given pattern of these inputs, and an ‘output’, along which the final signal is sent. If a neuron is capable of receiving input from another neuron, the input portion is a '''dendrite''', so-named because introns in higher organisms often have many, many inputs and these portions of their structure resemble trees. The output portion is called an '''axon''', and is generally non-branching until its terminal region.<br />
<br />
Neurons meet at structures called '''synapses''', which are pockets of intercellular fluid into which special signalling molecules called '''neurotransmitters''' are released from the axon following a raise in calcium levels, received by surface receptors on the next dendrite, and then recycled. These receptors are often GPCRs or guanylate cyclases, making the evolution of sensory cells like the amphid a straight-forward affair. Reception by GPCRs is enough to start a '''post-synaptic potential''' (either ''excitatory'' or ''inhibitory'') which will be integrated at the cell body with input from any other dendrites to determine whether or not an '''action potential''' is appropriate.<br />
<br />
<html><div class="asideR" style="max-width: 40%"><br />
<b>Gap Junctions</b>: Not all neurons actually connect via traditional chemical synapses; a minority of connections use the faster gap junction method, where the electrical gradient from one neuron is able to flow directly into the next one. These are also known as electrical synapses. Different speeds of neuron function are important to ensure complex timing of processes.</div></html><br />
<br />
The details of how potentials and synapses work is a favorite subject of physiology and introductory biology courses, and most students are likely already familiar with their inner workings. The links below hopefully contain enough information to subsidize the mending of any discrepancies:<br />
<br />
<html><div class="asideL"><br />
<p><b>The Nervous System in C. elegans</b></p><ul><br />
<li><a target="_new" href="http://wormbook.org/chapters/www_synapticfunction/synapticfunction.html">WormBook: Synaptic function in C. elegans</a><br />
<li><a target="_new" href="http://ims.dse.ibaraki.ac.jp/ccep/">The C. elegans Neural Network</a><br />
<li><a target="_new" href="http://wormweb.org/neuralnet#c=BAG&m=1">WormWeb: C. elegans Interactive Neural Network</a></ul><br />
<br />
<p><b>Neurons (General Reading)</b></p><ul><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Action_potential">Wikipedia: Action Potential</a><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Excitatory_postsynaptic_potential">Wikipedia: Excitatory Post-Synaptic Potential</a><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Inhibitory_postsynaptic_potential">Wikipedia: Inhibitory Postsynaptic Potential</a></ul><br />
</div></html><br />
<br />
The nervous system is one of the worm’s best-studied subsystems, and may potentially be engineerable to a much greater extent than other systems. <html><a target="_new" href="http://wormbook.org/chapters/www_specnervsys/specnervsys.html">“Specification of the nervous system”</a></html> in WormBook documents a considerable amount of material regarding the lineage of the worm’s neurons, and may help with targeting neurons other than those for which we have already prepared promoters.<br />
<br />
One simple way of engineering the worm’s nervous system is through '''optogenetics''', which involves placing light-gated ion channels in the membranes of nerve cells and then stimulating them to induce (or repress) action potentials in a controllable manner. We have endeavored to do this with our initial toolkit; the resultant channels and neuron-targeting promoters can be found on [[Team:Queens-Canada/parts|our parts list]].<html></div></html><br />
<br />
<html><div class="section"><h2>Locomotion: The Consequences of Sensation</h2></html><br />
<br />
Movement of the worm is controlled by interactions between the nervous and muscular systems. About 75 of the worm’s neurons innervate body wall muscles that control movement. The motor neurons of ''C. elegans'' are classified into groups by their anatomical position and by the neurotransmitters they release:<br />
<br />
<html><div class="aside" style="width: auto; display: inline-block"></html><br />
{|<br />
|<br />
|Ventral<br />
|Dorsal<br />
|-<br />
|Acetylcholine<br />
(excitatory neuron)<br />
|VA (backward movement)<br />
VB (forward movement)<br />
|DA (backward movement)<br />
DB (backward movement)<br />
AS<br />
|-<br />
|GABA (inhibitory neuron)<br />
|VD<br />
|DD<br />
|-<br />
|Other<br />
|VC (egg-laying behaviour)<br />
|<br />
|}<br />
<html></div></html><br />
<br />
The neurotransmitters from these motor neurons bind to very specific receptors on the muscle cell’s outer membrane (called the '''sarcolemma'''), which accordingly produces localized depolarization or repolarization at the area of binding. When sufficient depolarization occurs, an action potential is triggered, just like in interneurons, and this travels down the sarcolemma.<br />
<br />
<h3>Muscle Function</h3><br />
<br />
Action potentials produced in the sarcolemma eventually travel into the t-tubules, which are structures inside of the muscle cell that exist solely for the purpose of communicating these signals. The t-tubules connect directly to the muscle cell’s endoplasmic reticulum, known as the '''sarcoplasmic reticulum''', where the action potentials alter the conformation of dihydropyridine receptors (DHPR) and Ryanodine receptors (RyR) located thereon. These proteins then work together as voltage-sensitive calcium channels, their conformational change allowing calcium ions to be released from the SR into the muscle fibers.<br />
<br />
Muscle fibers consist of a chain of myosin proteins surrounded by six chains of actin proteins. '''Myosin''' is a motor protein that changes shape in response to the state of its binding sites and is capable of crawling up an '''actin filament''' (in this case, a molecular rope) if conditions are favorable. Six actin filaments are necessary because myosin heads align 60° to each other when stacked. There are two sets of actin filaments anchored to opposite walls of the muscle cell, accompanied by a myosin fiber that spans them, and switches directions in the middle (called the M-line). When the muscle contracts, the myosin chain pulls both sides of the muscle cell membrane towards the middle.<br />
<br />
<html><div class="asideL" style="max-width: 40%">The muscle fiber unit outlined here is actually repeated up to ten times in the adult down the length of a body-wall muscle cell.</div></html><br />
<br />
The myosin and actin filaments are not unaccompanied, however. The head domain of myosin is normally blocked from advancing up the actin filaments by '''tropomyosin''', a long molecule that wraps around the myosin filaments and covers their active sites. Tropomyosin is removed by '''troponin''' when calcium ions are available; thus, when the sarcoplasmic reticulum releases the necessary calcium ions, muscle contraction occurs. ATP is responsible for providing the energy to rhythmically change the conformation of the myosin heads. Returning to a blocked state causes the myosin fibres to lose their grip, and the actin fibres are pulled away as the cell returns to its natural elastic state.<br />
<br />
<html><div class="asideL" style="max-width: 40%"><a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed/18616971">This review article</a> is an exhaustive overview of invertebrate muscle fiber research. ''C. elegans II'' <a target="_new" href="http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ce2&part=A532">also has a fair amount to say</a> on details specific to the nematode; this article is better for augmenting preexisting knowledge of a vertebrate system.</div></html><br />
<br />
<h3>Locomotive Patterns</h3><br />
<br />
The worm’s movement through its environment is a result of systematic and rhythmic contractions and relaxations of the different sets of muscles in its body wall. The predictable, repeated behaviour of this movement is governed by a pattern generator, a neuron responsible for producing the rhythmic signal that causes muscle cells to contract and relax in a predictable fashion. There are probably two '''pattern generators''', one per side, which are out of phase with one another.<br />
<br />
The process of studying and mapping when neurons fire and what the consequences of those firings are is called '''electrophysiology''', and it is still largely in its infancy in ''C. elegans'', owing to the tiny size of its neurons. Much more work has been done in the related nematode ''Ascaris suum'', which is larger and has a very similar configuration of its neural net, but there are limitations to this similarity: for example, as of the writing of ''C. elegans II'', <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ce2&part=A780">it was believed</a></html> that the cells responsible for pattern generation in ''Ascaris'' for regular sinusoidal movement did not correspond to those in ''C. elegans''.<br />
<br />
However, what triggers these behaviors is better understood, and to synthetic biologists, the neurons responsible are targets of potentially great interest. The neurons AVB and PVC direct forward locomotion, while AVA and AVD direct backward locomotion. Activating these neurons can induce either direction of movement. This control could be combined with the mutant strain mentioned in <html><a href="http://www.ncbi.nlm.nih.gov/pubmed/4351805" target="_new">this</a></html> paper as E611 which causes the worm to always keep its head oriented towards a chemical gradient of interest, to allow fixed and predictable radial dispersion from a single source. More complex movement behavior is well-described at length in <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed/15689400">Gray <i>et al</i>. 2004</a></html>, especially turns and what triggers them.<html></div></html><br />
<br />
<html><a name="gpcr_table"></a><div class="section"><h2>G-Protein–Coupled Receptors (GPCRs)</h2></html><br />
<br />
<html><center><img src="http://upload.wikimedia.org/wikipedia/commons/thumb/c/c9/GPCR_cycle.jpg/800px-GPCR_cycle.jpg" style="width: 100%; max-width: 800px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://en.wikipedia.org/wiki/G_protein-coupled_receptor">From <i>Wikipedia</i></a></center></html><br />
<br />
GPCRs are common features of eukaryotes and, in multicellular organisms, are one of the major means by which tissues communicate with one another. In ''C. elegans'', they are also used for sensory purposes via the amphid. (We use some of them similarly in our noses.) Many projects have the potential to be highly dependent on GPCR importation and engineered GPCRs, although so far their use at iGEM has not been extensive owing to the high amount of work required to get them to function in prokaryotes.<br />
<br />
A GPCR is a transmembrane protein with seven distinctive alpha-helices that serve as transmembrane domains; because of this snake-like structure they are also known as seven-trans-membrane-domain receptors (7TM) or serpentine receptors. The amino terminus faces outward, and in conjunction with the loops of amino acids that extend beyond the membrane, functions as the recognition site. On the inside, the carboxy terminus and the interior loops participate in binding the '''G protein''', a three-subunit structure which is responsible for starting the messaging cascade inside of the cell.<br />
<br />
GPCRs are very highly portable between eukaryotes: <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550261/">Teng <i>et al</i>. 2006</a></html> showed that some human and mouse GPCRs could be put into ''C. elegans'' without modification. Because many organisms have so many GPCRs (humans have about 800 separate GPCR-coding genes; ''C. elegans'' has 1200; other related worms may have up to 1600), the evolutionary drift of the interface between these components between species is comparatively small. The primary factor as to whether or not a GPCR will work in a given ''C. elegans'' cell is a matter of the availability of the appropriate G protein complex.<br />
<br />
<html><div class="asideR" style="max-width: 40%">The “G” in “G protein” is for “guanine nucleotide-binding”. α subunits are attached to a GDP in the rest state; when the ligand binds to the receptor, this is swapped out for a GTP. Also, there’s a 22nd α subunit, gpa-18, but this is not well-documented and seems to be very divergent from all other G-protein α subunits.</div></html><br />
<br />
The three subunits of G proteins, α, β, and γ, each come in multiple varieties in the nematode, and only certain combinations of these varieties will bind to certain receptors. During signal transduction, the βγ complex is stable and functions as one unit, which simplifies matters slightly, but there are still four possible βγ complexes (two β and two γ subunits), and 21 α subunits. The composition of the βγ complex can be ignored because it does not interact directly with the receptor (only the α subunit does), but projects that involve changing the expression patterns of the α subunits may require bringing ''gpb-2'' or ''gpc-1'' genes with them. (''gpb-2'' is expressed in only neurons and muscle; ''gpc-1'' is only expressed in certain sensory neurons; ''gpb-1'' and ''gpc-2'' are expressed universally.)<br />
<br />
<h3>The Alpha Subunits</h3><br />
<br />
GSA-1 (analogous to vertebrate Gαs)<br />
* adenylate cyclase (''acy-1'') —› cAMP —› protein kinase A and cyclic nucleotide-gated ion channels<br />
* vertebrate version can activate calcium channels to contract muscle, inhibit sodium channels in heart<br />
* expressed in neurons and muscles in C. elegans<br />
<br />
EGL-30 (vertebrate Gαq/Gα11)<br />
* can couple to vertebrate α1-C adrenergic receptor<br />
* PIP2 —› IP3<br />
* triggers calcium release from internal stores (synapse firing)<br />
* normally localized near axon terminals in acetylcholine-releasing neurons<br />
<br />
GOA-1 (vertebrate Gαo)<br />
* counters EGL-30<br />
* expressed in nerves, pharynx, digestive, sex muscles, distal tip cells in hermaphrodites<br />
* partially redundant with GPA-16<br />
<br />
GPA-12 (vertebrate Gα12/13)<br />
* predominantly expressed in pharynx, hypodermal cells<br />
* protein kinase C (tpa-1)<br />
<br />
<html><div class="asideR" style="max-width: 40%;"><p><b>GPCRs, G-proteins, and worms</b></p><ul><br />
<li><a target="_new" href="http://www.google.com/url?q=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550261/&sa=D&sntz=1&usg=AFQjCNEHVNgnQkEfIEfWbZsEvxwiDwfqIg">Weng <i>et al</i>. 2006: Expression of mammalian GPCRs in C. elegans generates novel behavioural responses to human ligands</a><br />
<li><a target="_new" href="http://wormbook.org/chapters/www_heterotrimericGproteins/heterotrimericGproteins.html">WormBook: heterotrimeric G-proteins</a> (where most of this is from)<br />
<li><a target="_new" href="http://wormbook.org/chapters/www_putativechemoreceptorfam/putativechemoreceptorfam.html">WormBook: putitative chemoreceptor families</a><br />
<li><a target="_new" href="http://www.google.com/url?q=http://pharminfo.pharm.kyoto-u.ac.jp/services/glida/ligand_classification.php&sa=D&sntz=1&usg=AFQjCNG4gptqETlgBBdJmTvBMlssFqXYIQ">GLIDA: search for GPCRs by ligand</a><br />
<li><a target="_new" href="http://www.gpcr.org/7tm/search">GPCRDB: search for GPCRs by structure, function, or homology</a><br />
</ul><br />
</div></html><br />
<br />
The remaining 17 alpha subunits do not bear significant resemblance to those used in vertebrates, and most likely will not be useful for porting over GPCRs, but neurons with particularly large varieties of alpha subunits such as ASH, ASI, or AWA may still have improved chances. The majority are used heavily or exclusively in sensory neurons, where they may be involved in the decision to enter [[Team:Queens-Canada/reproductive#dauer|the dauer state]. Most of the information below is from WormBase.<br />
<br />
GPA-1<br />
* expressed in ADL, ASH, ASI (faint), ASJ, PHA, PHB<br />
* special male expression pattern: also in 1 or 2 pairs of p.c.s. neurons in the tail<br />
<br />
GPA-2<br />
* expressed in PVT, PHA, PHB, AIA cells, mu_sph, M1, M5, and I5<br />
* chemotaxis to water-soluable odorants<br />
<br />
GPA-3<br />
* expressed in PHA and PHB, and amphid cells, especially AWC and AWA<br />
<br />
GPA-4<br />
* expressed in ASI<br />
<br />
GPA-5<br />
* expressed in AWA, ASI (faint)<br />
<br />
GPA-6<br />
* expressed in AWA, PHB, ASI (faint)<br />
* does not localize to cilia<br />
<br />
GPA-7<br />
* expressed in most neurons and all muscle cells<br />
* also neurons in male tail<br />
<br />
GPA-8<br />
* expressed in URX, AQR, and PQR<br />
<br />
GPA-9<br />
* expressed in ASJ, PHB, PVQ, pharynx muscle, spermatheca<br />
<br />
GPA-10<br />
* expressed in ADF, ASI, ASJ, ALN, CAN, LUA, and the spermatheca<br />
<br />
GPA-11<br />
* expressed in ADL and ASH<br />
<br />
GPA-13<br />
* expressed in ADF, ASH, AWC, PHA, and PHB<br />
<br />
GPA-14<br />
* expressed in ASI, ASJ, ASH, ASK, ADE, PHA, PHB ALA, AVA, CAN, DVA, PVQ, RIA, and in vulval muscle<br />
<br />
GPA-15<br />
* expressed in ADL, ASH, ASK, PHA, PHB, the distal tip cell, the anchor cell, and many male-specific neurons<br />
<br />
GPA-16<br />
* expressed in AVM, PDE, PLM, BDU, PVC, and RIP, and weakly expressed in the pharynx, body-wall muscle, and vulval muscle<br />
<br />
GPA-17<br />
* expressed in intestine<br />
* no known use (or consequence for loss)<br />
* similar to Gαq (egl-30)<br />
<br />
ODR-3<br />
* expressed in AWA, AWB, AWC, ASH, and ADF<br />
* the AWC neurons consistently express GFP most strongly<br />
* localized to cilia<br />
* the AWB neurons express at lower levels<br />
* the AWA, ASH, and ADF neurons express only weakly<br />
<html></div></html><br />
<br />
'''[[Team:Queens-Canada/skin|Continue to the Worm's Exterior]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/nervousTeam:Queens-Canada/nervous2010-10-27T21:53:06Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>The Nervous System</h1><br />
<br />
<html><div class="section"><h2>The Amphid</h2></html><br />
<br />
<html><center><img src="http://wormatlas.org/hermaphrodite/nervous/Images/NeuroFIG14lr.jpg" style="width: 100%; max-width: 700px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/hermaphrodite/nervous/Images/neurofig14leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
The amphid of ''C. elegans'' is equivalent (and homologous) to the nose of a human. It is used for sensing volatile and water soluble chemicals, and is found in the anterior region of the worm, at the base of the lips. Consisting of 12 sensory neurons, some of which are ciliated, the amphid is the primary sensory organ of ''C. elegans''.<br />
<br />
There are only two responses that ''C. elegans'' has to olfactory stimuli: attraction and repulsion. These are governed by separate sensory neurons, which are hard-wired upstream to produce those responses, and use largely interchangeable sensors in the form of receptor proteins. The majority of these receptors are GPCRs and membrane-bound guanylyl cyclases. <html><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550261/" target="_new">It has been shown</a></html> that adding new GPCRs can create either reaction depending on where the GPCR is expressed, and that at least some GPCRs from humans and mice will work in ''C. elegans'' without modification.<br />
<br />
<html><div class="asideR">For more information on GPCRs, see <a href="#gpcr_table">below</a>.</div></html><br />
<br />
There are a large number of GPCRs and receptor GCs that occur natively in ''C. elegans''; over one thousand GPCR candidates have been identified through genomic analysis. However, their functions and stimuli are not well understood.<br />
<br />
The primary appeal of engineering the amphid is to add new receptors so that the worm is either attracted to, or repulsed by, some sensory input. The worm has very powerful chemotaxis, and could effectively replace bacteria in most biosensor applications. Below is a partial table from WormBook listing the neurons of the amphid, their functions, and some of the receptors that occur in each. The gpa family encodes not receptors, but G-protein α subunits, which are required for signal transduction from GPCRs and are the major influential factor in whether or not a specific GPCR can be used in a given neuron without modification.<br />
<br />
<html><div class="aside" style="width: auto; display: inline-block"></html><br />
{|<br />
|Neuron<br />
|Function<br />
|Receptors; G proteins<br />
|-<br />
|ASE<br />
|Water-soluble chemotaxis<br />
|Receptor guanylate cyclases?; gpa-3<br />
|-<br />
|AWC<br />
|Volatile chemotaxis, Lifespan, Navigation<br />
|GPCRs (str-2); odr-3 (major), gpa-3, gpa-2, gpa-5, gpa-13<br />
|-<br />
|AWA<br />
|Volatile chemotaxis, Lifespan (minor)<br />
|GPCRs (odr-10); odr-3 (major), gpa-3, gpa-5; gpa-13; gpa-6<br />
|-<br />
|AWB<br />
|Volatile avoidance<br />
|GPCRs; odr-3<br />
|-<br />
|ASH<br />
|Nociception: Osmotic avoidance, Nose touch avoidance, Chemical avoidance, Social feeding<br />
|GPCRs; odr-3 (major), gpa-3 (major), gpa-11, gpa-1, gpa-13, gpa-14, gpa-15<br />
|-<br />
|ASI<br />
|Dauer formation, Chemotaxis(minor), Navigation<br />
|GPCRs; gpa-1, gpa-3, gpa-4, gpa-5, gpa-6, gpa-10, gpa-14<br />
|-<br />
|ADF<br />
|Dauer formation, Chemotaxis (minor)<br />
|GPCRs; odr-3, gpa-3, gpa-10, gpa-13<br />
|-<br />
|ASG<br />
|Dauer formation (minor), Lifespan, Chemotaxis (minor)<br />
|GPCRs; gpa-3<br />
|-<br />
|ASJ<br />
|Dauer formation and recovery, Chemotaxis (minor), Lifespan<br />
|GPCRs; gpa-1, gpa-3, gpa-9, gpa-10, gpa-14<br />
|-<br />
|ASK<br />
|Avoidance (minor), Chemotaxis (minor), Lifespan, Navigation<br />
|GPCRs; gpa-2, gpa-3, gpa-14, gpa-15<br />
|-<br />
|ADL<br />
|Avoidance (minor), Social feeding<br />
|GPCRs; gpa-1, gpa-3, gpa-11, gpa-15<br />
|-<br />
|URX, AQR, PQR<br />
|Oxygen/aerotaxis, Social feeding<br />
|Soluble guanylate cyclases (gcy-35, gcy-36); gpa-8<br />
|-<br />
|PHA, PHB<br />
|Avoidance (antagonistic)<br />
|GPCRs; gpa-1, gpa-2, gpa-3, gpa-6, gpa-9, gpa-13, gpa-14, gpa-15<br />
|}<br />
<html></div></html><br />
<br />
The major object of desire when working with sensory neurons is the availability of a specific promoter that can target each individually. We have endeavored to provide a number of these; see [[Team:Queens-Canada/parts|our parts list]]. <html><a href="http://wormbook.org/chapters/www_chemosensation/chemosensation.html" target="_new">More detail on chemosensation in <i>C. elegans</i></a></html> is available from WormBook.<html></div></html><br />
<br />
<html><div class="section"><h2>Other Forms of Sensation</h2></html><br />
<br />
''C. elegans'' is also natively able to detect mechanical force exerted on its body as well as heat and cold. The potential applications for engineering these traits is minimal, although it is worth noting that ''C. elegans'' naturally moves towards temperatures at which it has known feeding conditions to be favorable, and away from those at which it was not able to feed. There are, however, mutants that habitually seek out cold (''cryophilic'') and heat (''thermophilic'') without regard for this process. These may be of use, and can be found in our article on getting strains.<html></div></html><br />
<br />
<html><div class="section"><h2>The Neuron</h2></html><br />
<br />
<html><center><img src="http://wormatlas.org/hermaphrodite/nervous/Images/NeuroFIG3lr.jpg" style="width: 100%; max-width: 800px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/hermaphrodite/nervous/Images/neurofig3leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
The neuron is a highly-specialized cell that provides the worm (and many other animals) with the ability to send signals more rapidly than chemical diffusion, by using localized ion gradients to generate an electrical current down the length of their exterior. There are 302 neurons in the adult hermaphroditic ''C. elegans'', which may seem like a tiny number compared to the hundred billion neurons that humans have in the brain alone, but in fact we have not yet fully deciphered their behavior; how the temperature-remembering behavior mentioned above works is not yet known.<br />
<br />
In order to be functional, a neuron must consist of one or more ‘inputs’, a cell body which contains the nucleus and also decides whether or not to take action based on a given pattern of these inputs, and an ‘output’, along which the final signal is sent. If a neuron is capable of receiving input from another neuron, the input portion is a '''dendrite''', so-named because introns in higher organisms often have many, many inputs and these portions of their structure resemble trees. The output portion is called an '''axon''', and is generally non-branching until its terminal region.<br />
<br />
Neurons meet at structures called '''synapses''', which are pockets of intercellular fluid into which special signalling molecules called '''neurotransmitters''' are released from the axon following a raise in calcium levels, received by surface receptors on the next dendrite, and then recycled. These receptors are often GPCRs or guanylate cyclases, making the evolution of sensory cells like the amphid a straight-forward affair. Reception by GPCRs is enough to start a '''post-synaptic potential''' (either ''excitatory'' or ''inhibitory'') which will be integrated at the cell body with input from any other dendrites to determine whether or not an '''action potential''' is appropriate.<br />
<br />
<html><div class="asideR" style="max-width: 40%"><br />
<b>Gap Junctions</b>: Not all neurons actually connect via traditional chemical synapses; a minority of connections use the faster gap junction method, where the electrical gradient from one neuron is able to flow directly into the next one. These are also known as electrical synapses. Different speeds of neuron function are important to ensure complex timing of processes.</div></html><br />
<br />
The details of how potentials and synapses work is a favorite subject of physiology and introductory biology courses, and most students are likely already familiar with their inner workings. The links below hopefully contain enough information to subsidize the mending of any discrepancies:<br />
<br />
<html><div class="asideL"><br />
<p><b>The Nervous System in C. elegans</b></p><ul><br />
<li><a target="_new" href="http://wormbook.org/chapters/www_synapticfunction/synapticfunction.html">WormBook: Synaptic function in C. elegans</a><br />
<li><a target="_new" href="http://ims.dse.ibaraki.ac.jp/ccep/">The C. elegans Neural Network</a><br />
<li><a target="_new" href="http://wormweb.org/neuralnet#c=BAG&m=1">WormWeb: C. elegans Interactive Neural Network</a></ul><br />
<br />
<p><b>Neurons (General Reading)</b></p><ul><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Action_potential">Wikipedia: Action Potential</a><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Excitatory_postsynaptic_potential">Wikipedia: Excitatory Post-Synaptic Potential</a><br />
<li><a target="_new" href="http://en.wikipedia.org/wiki/Inhibitory_postsynaptic_potential">Wikipedia: Inhibitory Postsynaptic Potential</a></ul><br />
</div></html><br />
<br />
The nervous system is one of the worm’s best-studied subsystems, and may potentially be engineerable to a much greater extent than other systems. <html><a target="_new" href="http://wormbook.org/chapters/www_specnervsys/specnervsys.html">“Specification of the nervous system”</a></html> in WormBook documents a considerable amount of material regarding the lineage of the worm’s neurons, and may help with targeting neurons other than those for which we have already prepared promoters.<br />
<br />
One simple way of engineering the worm’s nervous system is through '''optogenetics''', which involves placing light-gated ion channels in the membranes of nerve cells and then stimulating them to induce (or repress) action potentials in a controllable manner. We have endeavored to do this with our initial toolkit; the resultant channels and neuron-targeting promoters can be found on [[Team:Queens-Canada/parts|our parts list]].<html></div></html><br />
<br />
<html><div class="section"><h2>Locomotion: The Consequences of Sensation</h2></html><br />
<br />
Movement of the worm is controlled by interactions between the nervous and muscular systems. About 75 of the worm’s neurons innervate body wall muscles that control movement. The motor neurons of ''C. elegans'' are classified into groups by their anatomical position and by the neurotransmitters they release:<br />
<br />
<html><div class="aside" style="width: auto; display: inline-block"></html><br />
{|<br />
|<br />
|Ventral<br />
|Dorsal<br />
|-<br />
|Acetylcholine<br />
(excitatory neuron)<br />
|VA (backward movement)<br />
VB (forward movement)<br />
|DA (backward movement)<br />
DB (backward movement)<br />
AS<br />
|-<br />
|GABA (inhibitory neuron)<br />
|VD<br />
|DD<br />
|-<br />
|Other<br />
|VC (egg-laying behaviour)<br />
|<br />
|}<br />
<html></div></html><br />
<br />
The neurotransmitters from these motor neurons bind to very specific receptors on the muscle cell’s outer membrane (called the '''sarcolemma'''), which accordingly produces localized depolarization or repolarization at the area of binding. When sufficient depolarization occurs, an action potential is triggered, just like in interneurons, and this travels down the sarcolemma.<br />
<br />
<h3>Muscle Function</h3><br />
<br />
Action potentials produced in the sarcolemma eventually travel into the t-tubules, which are structures inside of the muscle cell that exist solely for the purpose of communicating these signals. The t-tubules connect directly to the muscle cell’s endoplasmic reticulum, known as the '''sarcoplasmic reticulum''', where the action potentials alter the conformation of dihydropyridine receptors (DHPR) and Ryanodine receptors (RyR) located thereon. These proteins then work together as voltage-sensitive calcium channels, their conformational change allowing calcium ions to be released from the SR into the muscle fibers.<br />
<br />
Muscle fibers consist of a chain of myosin proteins surrounded by six chains of actin proteins. '''Myosin''' is a motor protein that changes shape in response to the state of its binding sites and is capable of crawling up an '''actin filament''' (in this case, a molecular rope) if conditions are favorable. Six actin filaments are necessary because myosin heads align 60° to each other when stacked. There are two sets of actin filaments anchored to opposite walls of the muscle cell, accompanied by a myosin fiber that spans them, and switches directions in the middle (called the M-line). When the muscle contracts, the myosin chain pulls both sides of the muscle cell membrane towards the middle.<br />
<br />
<html><div class="asideL" style="max-width: 40%">The muscle fiber unit outlined here is actually repeated up to ten times in the adult down the length of a body-wall muscle cell.</div></html><br />
<br />
The myosin and actin filaments are not unaccompanied, however. The head domain of myosin is normally blocked from advancing up the actin filaments by '''tropomyosin''', a long molecule that wraps around the myosin filaments and covers their active sites. Tropomyosin is removed by '''troponin''' when calcium ions are available; thus, when the sarcoplasmic reticulum releases the necessary calcium ions, muscle contraction occurs. ATP is responsible for providing the energy to rhythmically change the conformation of the myosin heads. Returning to a blocked state causes the myosin fibres to lose their grip, and the actin fibres are pulled away as the cell returns to its natural elastic state.<br />
<br />
<html><div class="asideL" style="max-width: 40%"><a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed/18616971">This review article</a> is an exhaustive overview of invertebrate muscle fiber research. ''C. elegans II'' <a target="_new" href="http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ce2&part=A532">also has a fair amount to say</a> on details specific to the nematode; this article is better for augmenting preexisting knowledge of a vertebrate system.</div></html><br />
<br />
<h3>Locomotive Patterns</h3><br />
<br />
The worm’s movement through its environment is a result of systematic and rhythmic contractions and relaxations of the different sets of muscles in its body wall. The predictable, repeated behaviour of this movement is governed by a pattern generator, a neuron responsible for producing the rhythmic signal that causes muscle cells to contract and relax in a predictable fashion. There are probably two '''pattern generators''', one per side, which are out of phase with one another.<br />
<br />
The process of studying and mapping when neurons fire and what the consequences of those firings are is called '''electrophysiology''', and it is still largely in its infancy in ''C. elegans'', owing to the tiny size of its neurons. Much more work has been done in the related nematode ''Ascaris suum'', which is larger and has a very similar configuration of its neural net, but there are limitations to this similarity: for example, as of the writing of ''C. elegans II'', <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ce2&part=A780">it was believed</a></html> that the cells responsible for pattern generation in ''Ascaris'' for regular sinusoidal movement did not correspond to those in ''C. elegans''.<br />
<br />
However, what triggers these behaviors is better understood, and to synthetic biologists, the neurons responsible are targets of potentially great interest. The neurons AVB and PVC direct forward locomotion, while AVA and AVD direct backward locomotion. Activating these neurons can induce either direction of movement. This control could be combined with the mutant strain mentioned in <html><a href="http://www.ncbi.nlm.nih.gov/pubmed/4351805" target="_new">this</a></html> paper as E611 which causes the worm to always keep its head oriented towards a chemical gradient of interest, to allow fixed and predictable radial dispersion from a single source. More complex movement behavior is well-described at length in <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed/15689400">Gray <i>et al</i>. 2004</a></html>, especially turns and what triggers them.<html></div></html><br />
<br />
<html><a name="gpcr_table"></a><div class="section"><h2>G-Protein–Coupled Receptors (GPCRs)</h2></html><br />
<br />
GPCRs are common features of eukaryotes and, in multicellular organisms, are one of the major means by which tissues communicate with one another. In ''C. elegans'', they are also used for sensory purposes via the amphid. (We use some of them similarly in our noses.) Many projects have the potential to be highly dependent on GPCR importation and engineered GPCRs, although so far their use at iGEM has not been extensive owing to the high amount of work required to get them to function in prokaryotes.<br />
<br />
A GPCR is a transmembrane protein with seven distinctive alpha-helices that serve as transmembrane domains; because of this snake-like structure they are also known as seven-trans-membrane-domain receptors (7TM) or serpentine receptors. The amino terminus faces outward, and in conjunction with the loops of amino acids that extend beyond the membrane, functions as the recognition site. On the inside, the carboxy terminus and the interior loops participate in binding the '''G protein''', a three-subunit structure which is responsible for starting the messaging cascade inside of the cell.<br />
<br />
GPCRs are very highly portable between eukaryotes: <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550261/">Teng <i>et al</i>. 2006</a></html> showed that some human and mouse GPCRs could be put into ''C. elegans'' without modification. Because many organisms have so many GPCRs (humans have about 800 separate GPCR-coding genes; ''C. elegans'' has 1200; other related worms may have up to 1600), the evolutionary drift of the interface between these components between species is comparatively small. The primary factor as to whether or not a GPCR will work in a given ''C. elegans'' cell is a matter of the availability of the appropriate G protein complex.<br />
<br />
<html><div class="asideR" style="max-width: 40%">The “G” in “G protein” is for “guanine nucleotide-binding”. α subunits are attached to a GDP in the rest state; when the ligand binds to the receptor, this is swapped out for a GTP. Also, there’s a 22nd α subunit, gpa-18, but this is not well-documented and seems to be very divergent from all other G-protein α subunits.</div></html><br />
<br />
The three subunits of G proteins, α, β, and γ, each come in multiple varieties in the nematode, and only certain combinations of these varieties will bind to certain receptors. During signal transduction, the βγ complex is stable and functions as one unit, which simplifies matters slightly, but there are still four possible βγ complexes (two β and two γ subunits), and 21 α subunits. The composition of the βγ complex can be ignored because it does not interact directly with the receptor (only the α subunit does), but projects that involve changing the expression patterns of the α subunits may require bringing ''gpb-2'' or ''gpc-1'' genes with them. (''gpb-2'' is expressed in only neurons and muscle; ''gpc-1'' is only expressed in certain sensory neurons; ''gpb-1'' and ''gpc-2'' are expressed universally.)<br />
<br />
<h3>The Alpha Subunits</h3><br />
<br />
GSA-1 (analogous to vertebrate Gαs)<br />
* adenylate cyclase (''acy-1'') —› cAMP —› protein kinase A and cyclic nucleotide-gated ion channels<br />
* vertebrate version can activate calcium channels to contract muscle, inhibit sodium channels in heart<br />
* expressed in neurons and muscles in C. elegans<br />
<br />
EGL-30 (vertebrate Gαq/Gα11)<br />
* can couple to vertebrate α1-C adrenergic receptor<br />
* PIP2 —› IP3<br />
* triggers calcium release from internal stores (synapse firing)<br />
* normally localized near axon terminals in acetylcholine-releasing neurons<br />
<br />
GOA-1 (vertebrate Gαo)<br />
* counters EGL-30<br />
* expressed in nerves, pharynx, digestive, sex muscles, distal tip cells in hermaphrodites<br />
* partially redundant with GPA-16<br />
<br />
GPA-12 (vertebrate Gα12/13)<br />
* predominantly expressed in pharynx, hypodermal cells<br />
* protein kinase C (tpa-1)<br />
<br />
<html><div class="asideR" style="max-width: 40%;"><p><b>GPCRs, G-proteins, and worms</b></p><ul><br />
<li><a target="_new" href="http://www.google.com/url?q=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550261/&sa=D&sntz=1&usg=AFQjCNEHVNgnQkEfIEfWbZsEvxwiDwfqIg">Weng <i>et al</i>. 2006: Expression of mammalian GPCRs in C. elegans generates novel behavioural responses to human ligands</a><br />
<li><a target="_new" href="http://wormbook.org/chapters/www_heterotrimericGproteins/heterotrimericGproteins.html">WormBook: heterotrimeric G-proteins</a> (where most of this is from)<br />
<li><a target="_new" href="http://wormbook.org/chapters/www_putativechemoreceptorfam/putativechemoreceptorfam.html">WormBook: putitative chemoreceptor families</a><br />
<li><a target="_new" href="http://www.google.com/url?q=http://pharminfo.pharm.kyoto-u.ac.jp/services/glida/ligand_classification.php&sa=D&sntz=1&usg=AFQjCNG4gptqETlgBBdJmTvBMlssFqXYIQ">GLIDA: search for GPCRs by ligand</a><br />
<li><a target="_new" href="http://www.gpcr.org/7tm/search">GPCRDB: search for GPCRs by structure, function, or homology</a><br />
</ul><br />
</div></html><br />
<br />
The remaining 17 alpha subunits do not bear significant resemblance to those used in vertebrates, and most likely will not be useful for porting over GPCRs, but neurons with particularly large varieties of alpha subunits such as ASH, ASI, or AWA may still have improved chances. The majority are used heavily or exclusively in sensory neurons, where they may be involved in the decision to enter [[Team:Queens-Canada/reproductive#dauer|the dauer state]. Most of the information below is from WormBase.<br />
<br />
GPA-1<br />
* expressed in ADL, ASH, ASI (faint), ASJ, PHA, PHB<br />
* special male expression pattern: also in 1 or 2 pairs of p.c.s. neurons in the tail<br />
<br />
GPA-2<br />
* expressed in PVT, PHA, PHB, AIA cells, mu_sph, M1, M5, and I5<br />
* chemotaxis to water-soluable odorants<br />
<br />
GPA-3<br />
* expressed in PHA and PHB, and amphid cells, especially AWC and AWA<br />
<br />
GPA-4<br />
* expressed in ASI<br />
<br />
GPA-5<br />
* expressed in AWA, ASI (faint)<br />
<br />
GPA-6<br />
* expressed in AWA, PHB, ASI (faint)<br />
* does not localize to cilia<br />
<br />
GPA-7<br />
* expressed in most neurons and all muscle cells<br />
* also neurons in male tail<br />
<br />
GPA-8<br />
* expressed in URX, AQR, and PQR<br />
<br />
GPA-9<br />
* expressed in ASJ, PHB, PVQ, pharynx muscle, spermatheca<br />
<br />
GPA-10<br />
* expressed in ADF, ASI, ASJ, ALN, CAN, LUA, and the spermatheca<br />
<br />
GPA-11<br />
* expressed in ADL and ASH<br />
<br />
GPA-13<br />
* expressed in ADF, ASH, AWC, PHA, and PHB<br />
<br />
GPA-14<br />
* expressed in ASI, ASJ, ASH, ASK, ADE, PHA, PHB ALA, AVA, CAN, DVA, PVQ, RIA, and in vulval muscle<br />
<br />
GPA-15<br />
* expressed in ADL, ASH, ASK, PHA, PHB, the distal tip cell, the anchor cell, and many male-specific neurons<br />
<br />
GPA-16<br />
* expressed in AVM, PDE, PLM, BDU, PVC, and RIP, and weakly expressed in the pharynx, body-wall muscle, and vulval muscle<br />
<br />
GPA-17<br />
* expressed in intestine<br />
* no known use (or consequence for loss)<br />
* similar to Gαq (egl-30)<br />
<br />
ODR-3<br />
* expressed in AWA, AWB, AWC, ASH, and ADF<br />
* the AWC neurons consistently express GFP most strongly<br />
* localized to cilia<br />
* the AWB neurons express at lower levels<br />
* the AWA, ASH, and ADF neurons express only weakly<br />
<html></div></html><br />
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'''[[Team:Queens-Canada/skin|Continue to the Worm's Exterior]]'''<br />
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{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/digestiveTeam:Queens-Canada/digestive2010-10-27T21:47:29Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>The Digestive System</h1><br />
<br />
The ''C. elegans'' digestive system is divided into two components: the '''pharynx''', which breaks down food by grinding it up, and the '''intestine''', where food is digested, absorbed, and stored. The intestine is a major component of the worm, by mass a whole third of its somatic cells, and presents many interesting opportunities for synthetic biology through the prospects of enterosymbiosis with bacteria and the potential localization of catalytic pathways.<br />
<br />
<html><div class="section"><h2>The Pharynx</h2></html><br />
<br />
<html><center><img src="http://wormatlas.org/hermaphrodite/pharynx/Images/PhaFIG2lr.jpg" style="width: 100%; max-width: 700px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/hermaphrodite/pharynx/Images/phafig2leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
The pharynx is a complex structure that precedes the intestine. Quite unlike the human pharynx, it is a combination of pump and grinder, and it has been theorized to bear some homology to the heart of other organisms based on its selection of ion pumps, developmental independence, and the nature of its innervation.<br />
<br />
The pharynx normally takes in bacteria-filled water, pushes the water back out, grinds up the bacteria, and then passes them along to the intestine. The grinder, located in the terminal bulb, is very fine, and unless disrupted by the activity of a pathogenic bacterium, cuts up the food. The pump rate is dependent on the detection of food and recent periods of starvation, but it never stops.<br />
<br />
One of the primary interests synthetic biology may have with the ''C. elegans'' pharynx is in circumventing its behavior sufficiently for symbiotic, beneficial colonization of the worm’s intestine by an engineered bacterial strain (enterosymbiosis). While this goal may seem remote at first inspection, consider that [http://www.wormbook.org/chapters/www_intermicrobpath/intermicrobpath.html#d0e691 there are a number of bacteria] which already interfere with the nematode’s grinder and colonize the intestine as pathogens, and that this manner of symbiosis already exists stably in other nematode/bacteria systems, as documented in [http://dx.doi.org/10.1016/j.biocontrol.2005.11.016 Ciche ''et al''. 2006], for the ultimate purpose of infecting insects with the bacteria. Ciche ''et al''. also document a number of genes required for these systems to function properly; it might be possible to transfer some of them into C. elegans and ''E. coli'' to develop a reliable cooperativity.<br />
<br />
The pharynx of ''C. elegans'' is far removed from most other cell lineages developmentally, and so is easy to target in terms of promoters, although our initial chassis offering focuses more on the general nervous system. The pharynx’s nearly-isolated nervous system controls pump rate, innervates the muscle cells to pump, and is consulted (along with many other sources) in establishing the dauer decision, but most other communication with the rest of the organism is probably done through hormones (see [[Team:Queens-Canada/pseudocoelom|our section on the pseudocoelom]]). Other than as a component of enterosymbiosis projects, the potential usefulness of the pharynx in synthetic biology does not appear great to us, but there are surely many possibilities we have not considered.<html></div></html><br />
<br />
<html><div class="section"><h2>The Intestine</h2></html><br />
<br />
<html><center><img src="http://wormatlas.org/hermaphrodite/intestine/Images/IntFIG1lr.jpg" style="width: 100%; max-width: 800px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/hermaphrodite/intestine/Images/intfig1leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
The intestine is a long tube that spans most of the creature’s body. It is comprised of twenty cells which form rings down the length of the worm’s interior. With the exception of the grinding activity provided by the pharynx, and defecation provided by the anus, the intestine of ''C. elegans'' performs most of the digestive functions of higher organisms, including chemical breakdown of food, absorption of nutrient molecules, and synthesis and storage of fats. For a long time, the worm’s digestive system was very poorly understood, but this is steadily being corrected in combination with knowledge of the digestive systems of other nematodes.<br />
<br />
The pH of the intestine is not known precisely, but studies of various catabolic enymes known to be localized to the intestinal lumen suggests that it is probably somewhere between 4 and 5. (This is much more mild than the human stomach.) As one of the greatest promises of the intestine is to create a synthetic pathway of some sort within the organ, and to use the natural excretion process to separate the product from the worm, this could potentially complicate pathways that require proteins which function optimally at higher pHs, as most do. One way around this might be to compose the product inside of the intestinal cells and then export it. The promoter and 5' UTR of ''pho-1'' [http://www.ncbi.nlm.nih.gov/pubmed/15733671|are known] to be specific only to the later end of the intestine, which could reduce the exposure time to acid, or allow for a multi-stage synthesis process that progresses down the intestine. If pH and the presence of digestive enzymes could potentially pose a threat to the safe production of some product, however, it may make more sense to move such a project into [[Team:Queens-Canada/pseudocoelom|the excretory system]] instead.<br />
<br />
Several of the cells in the worm’s intestine have two nuclei after the L1 stage of larval development. The purpose and value of this is not known, and preventing it by knocking out the gene ''lin-14'' does not seem to affect the worm adversely. This property may be potentially exploitable as a means to insert a foreign molecule into the nucleus before telophase begins after this superfluous division. The intestine is something of a hot spot for peculiar nucleic acid activity—[[Team:Queens-Canada/rnai|dsRNA]] is normally taken up in the intestine, through the SID-2 channel lining the lumen, and then propagates throughout the worm (except neurons) via SID-1.<br />
<br />
Although nematodes in general typically grind up bacteria before they reach the intestine, there are some cases in which bacteria circumvent the normal pharynx function and successfully colonize the intestinal surface, with typically detrimental effects to the host worm. Several species are known to attack ''C. elegans'' in this way, and worms particularly vulnerable to intestinal colonization due to mutations are said to be of the ''BUS'' phenotype; a family of genes has been named ''bus'', appropriately. However, there are other nematode species where colonization isn’t doom for the worm: ''Steinernema carpocapsae'', a parasitic nematode that parasitizes wasp larvae, [http://www.ncbi.nlm.nih.gov/pubmed/17618298 forms an effective symbiosis with the bacterial species ''Xenorhabdus nematophila''], which is necessary for killing the insects and assisting the worm’s mode of reproduction.<br />
<br />
No immediate barrier is apparent to the thought of transferring the genes that make this colonization possible into ''C. elegans'' and ''E. coli'', although this is obviously a great deal of work, and the tendency to retain bacteria as engendered in ''bus'' mutants can most likely reproduce the effect at a sufficient rate and worm longevity for most projects. Like the exosymbiosis suggested in [[Team:Queens-Canada/skin|the cuticle article]], this system of enterosymbiosis presents a great opportunity for inter-kingdom cooperation and backward compatibility with the fundamental ''E. coli'' chassis.<html></div></html><br />
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'''[[Team:Queens-Canada/nervous|Continue to the Nervous System]]'''<br />
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{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/pseudocoelomTeam:Queens-Canada/pseudocoelom2010-10-27T21:45:07Z<p>Glh: </p>
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<div>{{:Team:Queens-Canada/head}}<br />
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<h1>The Pseudocoelom and the Excretory System</h1><br />
<br />
Like humans, the organs of ''C. elegans'' aren't simply pressed against one another with no gaps in between. Internal organs need access to nutrients, a means to eliminate metabolic waste, and inter-tissue communication. This system, referred to as the '''pericellular system''' by WormAtlas, is roughly equivalent to the circulatory and renal systems in humans.<br />
<br />
<html><center><img src="http://wormatlas.org/hermaphrodite/pericellular/Images/PeriFIG1lr.jpg" style="width: 100%; max-width: 700px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/hermaphrodite/pericellular/Images/perifig1leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
<html><div class="section"><h2>The Pseudocoelom</h2></html><br />
<br />
The '''pseudocoelom''' is a fluid-filled body which bathes all tissues. It is directly analogous to blood vessels in higher animals; however, it is not circulated, nor fully lined with specialized cells. Its major responsibility is providing cells with the oxygen, ions and nutrients that they require in order to function. It also acts as a conduit for intercellular signalling, and may potentially have great value to synthetic biology as a medium for sending messages to different parts of the worm without disrupting preexisting mechanisms.<br />
<br />
Signalling in the pseudocoelom is typically accomplished through '''hormones''', molecules that many multicellular organisms use to send messages via their circulatory systems. The majority of hormones are either peptide-based (in which case they are derived directly from a gene product, possibly with post-translational modification) or steroid hormones (in which case they are created by modifications to the molecule cholesterol.)<br />
<br />
<html><div class="asideR"><br />
<p><b>More on hormones and neuropeptides</b></p><ul><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_neuropeptides/neuropeptides.html">The neuropeptides of <i>C. elegans</i></a><br />
<li><a target="_new" href="http://wormbook.org/chapters/www_putativechemoreceptorfam/putativechemoreceptorfam.html">The chemoreceptor families of <i>C. elegans</i></a><br />
<li><a target="_new" href="http://wormbook.org/chapters/www_nuclearhormonerecep/nuclearhormonerecep.html">Hormone receptors in the nucleus</a><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_intracellulartrafficking/intracellulartrafficking.html">Intracellular trafficking of molecules</a><br />
</ul></div></html><br />
<br />
Steroid hormones offer a particular convenience in that, since they are largely non-polar, they pass through lipid bilayers easily and may not require a receptor outside of the nucleus itself, which binds directly to DNA and is affected by the presence of its effector hormone. However, engineering the pathway necessary to design and introduce a new steroid hormone that would be non-toxic and fully distinguishable to the organism would be greatly time-consuming. Instead, it may be more practical to design new peptide hormones, or to take them from other sources. They can be received via [[Team:Queens-Canada/nervous|GPCRs]] at the surface, and more conventional signalling pathways can be utilized intracellularly to activate or deactivate the genes in question. Some peptide hormones are released directly by neurons, and also used as neurotransmitters. These are known as neuropeptides. In some cases, such as serotonin, neurons may use the pseudocoelomic fluid as an intermediary for messaging.<br />
<br />
'''Exporting larger proteins into the pseudocoelom''': <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed/11560892">Fares and Greenwald 2001</a></html> used the first 79 amino acids of the protein-coding gene <html><a target="_new" href="http://www.wormbase.org/db/gene/gene?name=WBGene00004759;class=Gene"><i>sel-1</i></a></html> behind the promoter of <html><a target="_new" href="http://www.wormbase.org/db/gene/gene?name=WBGene00003515;class=Gene"><i>myo-3</i></a></html>, which is expressed in muscle cells that have direct contact to the pseudocoelom. By attaching the sequence for GFP to the 3' end of this construct, they were able to produce a form of GFP that was reliably secreted into the pseudocoelom. SEL-1 is normally a transmembrane protein.<html></div></html><br />
<br />
<html><div class="section"><h2>Coelomocytes</h2></html><br />
<br />
The '''coelomocytes''' are a group of six cells that serve as the worm’s innate immune system. They periodically endocytosize volumes of pseudocoelomic fluid and degrade foreign molecules in it. While this may seem like an obstacle to synthetic biology, they are potentially useful in that they act as a constraint on how long a signal can stay active. The coelomocytes are not necessary for normal, healthy C. elegans function under laboratory conditions.<html></div></html><br />
<br />
<html><div class="section"><h2>The Excretory System</h2></html><br />
<br />
<html><center><img src="http://wormatlas.org/hermaphrodite/excretory/Images/ExcFIG1lr.jpg" style="width: 100%; max-width: 500px; box-shadow: 1px 2px 3px #808080"><br><br />
<a target="_new" href="http://wormatlas.org/hermaphrodite/excretory/Images/excfig1leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
The excretory system functions as the worm’s kidneys, removing unwanted ions and byproducts of cellular metabolism from the pseudocoelom and releasing them into the environment. In addition, it permits the release molecules into the environment, possibly for signalling to other worms or other organisms.<br />
<br />
The excretory gland is a component of the excretory system. It consists of a pair of fused cells from which the worm releases pheromones to communicate with other worms in its environment. GFPWorm lists <html><a target="_new" href="http://worfdb.dfci.harvard.edu/promoteromedb/searchPromoterome.pl?by=name&sid=Y22D7AR.10+">the promoter</a></html> of the gene <html><a target="_new" href="http://www.google.com/url?q=http://www.wormbase.org/db/gene/gene%3Fname%3DWBGene00021263;class%3DGene&sa=D&sntz=1&usg=AFQjCNGD5zCR1Z6cqoGaWrIoZldWOq8R6g">Y22D7AR.10</a></html> as having expression only in the excretory gland cells. Unfortunately, it is not all that well-studied—as of this writing, only 131 papers indexed by Google Scholar mention ''C. elegans'' and the excretory gland; indeed, most attention relating to the secretion of proteins has been spent on the nervous system, specifically the release of neurotransmitters at the synaptic cleft. WormBook does have some general information on both <html><a target="_new" href="http://www.wormbook.org/chapters/www_intracellulartrafficking/intracellulartrafficking.html#d0e636">nonspecific</a></html> and <html><a target="_new" href="http://www.wormbook.org/chapters/www_intracellulartrafficking/intracellulartrafficking.html#d0e762">polar</a></html> (apical and basolateral) secretion, however, in the <html><a target="_new" href="http://www.wormbook.org/chapters/www_intracellulartrafficking/intracellulartrafficking.html">intracellular trafficking</a></html> chapter.<html></div></html><br />
<br />
'''[[Team:Queens-Canada/digestive|Continue to the Digestive System]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/pseudocoelomTeam:Queens-Canada/pseudocoelom2010-10-27T21:44:29Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>The Pseudocoelom and the Excretory System</h1><br />
<br />
Like humans, the organs of ''C. elegans'' aren't simply pressed against one another with no gaps in between. Internal organs need access to nutrients, a means to eliminate metabolic waste, and inter-tissue communication. This system, referred to as the '''pericellular system''' by WormAtlas, is roughly equivalent to the circulatory and renal systems in humans.<br />
<br />
<html><img src="http://wormatlas.org/hermaphrodite/pericellular/Images/PeriFIG1lr.jpg" style="width: 100%; max-width: 700px; box-shadow: 1px 2px 3px #808080"><br />
<center><a target="_new" href="http://wormatlas.org/hermaphrodite/pericellular/Images/perifig1leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
<html><div class="section"><h2>The Pseudocoelom</h2></html><br />
<br />
The '''pseudocoelom''' is a fluid-filled body which bathes all tissues. It is directly analogous to blood vessels in higher animals; however, it is not circulated, nor fully lined with specialized cells. Its major responsibility is providing cells with the oxygen, ions and nutrients that they require in order to function. It also acts as a conduit for intercellular signalling, and may potentially have great value to synthetic biology as a medium for sending messages to different parts of the worm without disrupting preexisting mechanisms.<br />
<br />
Signalling in the pseudocoelom is typically accomplished through '''hormones''', molecules that many multicellular organisms use to send messages via their circulatory systems. The majority of hormones are either peptide-based (in which case they are derived directly from a gene product, possibly with post-translational modification) or steroid hormones (in which case they are created by modifications to the molecule cholesterol.)<br />
<br />
<html><div class="asideR"><br />
<p><b>More on hormones and neuropeptides</b></p><ul><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_neuropeptides/neuropeptides.html">The neuropeptides of <i>C. elegans</i></a><br />
<li><a target="_new" href="http://wormbook.org/chapters/www_putativechemoreceptorfam/putativechemoreceptorfam.html">The chemoreceptor families of <i>C. elegans</i></a><br />
<li><a target="_new" href="http://wormbook.org/chapters/www_nuclearhormonerecep/nuclearhormonerecep.html">Hormone receptors in the nucleus</a><br />
<li><a target="_new" href="http://www.wormbook.org/chapters/www_intracellulartrafficking/intracellulartrafficking.html">Intracellular trafficking of molecules</a><br />
</ul></div></html><br />
<br />
Steroid hormones offer a particular convenience in that, since they are largely non-polar, they pass through lipid bilayers easily and may not require a receptor outside of the nucleus itself, which binds directly to DNA and is affected by the presence of its effector hormone. However, engineering the pathway necessary to design and introduce a new steroid hormone that would be non-toxic and fully distinguishable to the organism would be greatly time-consuming. Instead, it may be more practical to design new peptide hormones, or to take them from other sources. They can be received via [[Team:Queens-Canada/nervous|GPCRs]] at the surface, and more conventional signalling pathways can be utilized intracellularly to activate or deactivate the genes in question. Some peptide hormones are released directly by neurons, and also used as neurotransmitters. These are known as neuropeptides. In some cases, such as serotonin, neurons may use the pseudocoelomic fluid as an intermediary for messaging.<br />
<br />
'''Exporting larger proteins into the pseudocoelom''': <html><a target="_new" href="http://www.ncbi.nlm.nih.gov/pubmed/11560892">Fares and Greenwald 2001</a></html> used the first 79 amino acids of the protein-coding gene <html><a target="_new" href="http://www.wormbase.org/db/gene/gene?name=WBGene00004759;class=Gene"><i>sel-1</i></a></html> behind the promoter of <html><a target="_new" href="http://www.wormbase.org/db/gene/gene?name=WBGene00003515;class=Gene"><i>myo-3</i></a></html>, which is expressed in muscle cells that have direct contact to the pseudocoelom. By attaching the sequence for GFP to the 3' end of this construct, they were able to produce a form of GFP that was reliably secreted into the pseudocoelom. SEL-1 is normally a transmembrane protein.<html></div></html><br />
<br />
<html><div class="section"><h2>Coelomocytes</h2></html><br />
<br />
The '''coelomocytes''' are a group of six cells that serve as the worm’s innate immune system. They periodically endocytosize volumes of pseudocoelomic fluid and degrade foreign molecules in it. While this may seem like an obstacle to synthetic biology, they are potentially useful in that they act as a constraint on how long a signal can stay active. The coelomocytes are not necessary for normal, healthy C. elegans function under laboratory conditions.<html></div></html><br />
<br />
<html><div class="section"><h2>The Excretory System</h2></html><br />
<br />
<html><img src="http://wormatlas.org/hermaphrodite/excretory/Images/ExcFIG1lr.jpg" style="width: 100%; max-width: 500px; box-shadow: 1px 2px 3px #808080"><br />
<center><a target="_new" href="http://wormatlas.org/hermaphrodite/excretory/Images/excfig1leg.htm">From <i>WormAtlas</i></a></center></html><br />
<br />
The excretory system functions as the worm’s kidneys, removing unwanted ions and byproducts of cellular metabolism from the pseudocoelom and releasing them into the environment. In addition, it permits the release molecules into the environment, possibly for signalling to other worms or other organisms.<br />
<br />
The excretory gland is a component of the excretory system. It consists of a pair of fused cells from which the worm releases pheromones to communicate with other worms in its environment. GFPWorm lists <html><a target="_new" href="http://worfdb.dfci.harvard.edu/promoteromedb/searchPromoterome.pl?by=name&sid=Y22D7AR.10+">the promoter</a></html> of the gene <html><a target="_new" href="http://www.google.com/url?q=http://www.wormbase.org/db/gene/gene%3Fname%3DWBGene00021263;class%3DGene&sa=D&sntz=1&usg=AFQjCNGD5zCR1Z6cqoGaWrIoZldWOq8R6g">Y22D7AR.10</a></html> as having expression only in the excretory gland cells. Unfortunately, it is not all that well-studied—as of this writing, only 131 papers indexed by Google Scholar mention ''C. elegans'' and the excretory gland; indeed, most attention relating to the secretion of proteins has been spent on the nervous system, specifically the release of neurotransmitters at the synaptic cleft. WormBook does have some general information on both <html><a target="_new" href="http://www.wormbook.org/chapters/www_intracellulartrafficking/intracellulartrafficking.html#d0e636">nonspecific</a></html> and <html><a target="_new" href="http://www.wormbook.org/chapters/www_intracellulartrafficking/intracellulartrafficking.html#d0e762">polar</a></html> (apical and basolateral) secretion, however, in the <html><a target="_new" href="http://www.wormbook.org/chapters/www_intracellulartrafficking/intracellulartrafficking.html">intracellular trafficking</a></html> chapter.<html></div></html><br />
<br />
'''[[Team:Queens-Canada/digestive|Continue to the Digestive System]]'''<br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/safetyTeam:Queens-Canada/safety2010-10-27T21:41:05Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>Risk Assessment</h1><br />
Unlike some other nematodes, ''Caenorhabditis elegans'' is fully free-living and functions primarily as a digester of detritus, posing no threat except to the microbes which it eats. The worms are non-pathogenic organisms, and Biosafety level 1 practices are simple and safe; working with ''C. elegans'' carries a very low level of risk. Accordingly, concerns about researcher, public, and environmental safety are minimal. ''C. elegans'' can be safely cultured in a BSL-1 laboratory{{:Team:Queens-Canada/footnote-anchor|1}}, just like bacteria and yeast.<br />
<br />
The engineered ''C. elegans'' do not effect their environments, as our work focuses on proteins with intracellular effects that have no significant catalytic role. Thus, a malfunction in our BioBrick parts would pose minimal threat to humans, laboratory equipment, or other organisms. Further, our alterations do not grant the worms any advantages over the wildtype, making the spread of these alleles unlikely, should engineered worms be released into the environment: in fact, the high-copy extrachromosomal arrays used in microinjection induce stress on the worm and consequently lower its biological fitness.<br />
<br />
In principle, our work does pose some long-term risks in that it aims to make more advanced forms of synthetic biology more readily accessible, as well as providing a chassis that could potentially be used to create more dangerous projects such as catalytic mechanisms. These risks naturally arise from the advancement of the field of synthetic biology, and although they necessitate dilligence and thoughtfulness from the research community as the field develops, they do not represent undue or extraordinary threats.<br />
<br />
By itself, our project poses no threat to its environment greater than that presented by the standard ''E. coli'' chassis—in fact, even less, since nematodes are not capable of horizontal gene transfer and would disseminate engineered genes more slowly.<br />
<br />
Drs. Ian Chin-Sang, Kenton Ko, and Nancy Martin were three of the team’s Faculty Advisors this year, and provided the team with lab space in which we conducted all of our wet work. They are all members of the <html><a target="_new" href="http://www.safety.queensu.ca/biocom/">Queen’s Biohazards Committee</a></html> and ensured that we worked within the appropriate biosafety regulations.<br />
<br />
<html><div class="section"><h2>Footnotes and Citations</h2></html><br />
{{:Team:Queens-Canada/footnote|1|[http://www.ccac.ca/en/CCAC_Programs/ETCC/Module04/15.html Biosafety Guidelines and Levels of Containment]. Canadian Council on Animal Care. Accessed on 2010-06-22.}}<html></div></html><br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/safetyTeam:Queens-Canada/safety2010-10-27T21:40:08Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>Risk Assessment</h1><br />
Unlike some other nematodes, ''Caenorhabditis elegans'' is fully free-living and functions primarily as a digester of detritus, posing no threat except to the microbes which it eats. The worms are non-pathogenic organisms, and Biosafety level 1 practices are simple and safe; working with ''C. elegans'' carries a very low level of risk. Accordingly, concerns about researcher, public, and environmental safety are minimal. ''C. elegans'' can be safely cultured in a BSL-1 laboratory{{:Team:Queens-Canada/footnote-anchor|1}}, just like bacteria and yeast.<br />
<br />
The engineered ''C. elegans'' do not effect their environments, as our work focuses on proteins with intracellular effects that have no significant catalytic role. Thus, a malfunction in our BioBrick parts would pose minimal threat to humans, laboratory equipment, or other organisms. Further, our alterations do not grant the worms any advantages over the wildtype, making the spread of these alleles unlikely, should engineered worms be released into the environment: in fact, the high-copy extrachromosomal arrays used in microinjection induce stress on the worm and consequently lower its biological fitness.<br />
<br />
In principle, our work does pose some long-term risks in that it aims to make more advanced forms of synthetic biology more readily accessible, as well as providing a chassis that could potentially be used to create more dangerous projects such as catalytic mechanisms. These risks naturally arise from the advancement of the field of synthetic biology, and although they necessitate dilligence and thoughtfulness from the research community as the field develops, they do not represent undue or extraordinary threats.<br />
<br />
By itself, our project poses no threat to its environment greater than that presented by the standard ''E. coli'' chassis—in fact, even less, since nematodes are not capable of horizontal gene transfer and would disseminate engineered genes more slowly.<br />
<br />
Drs. Ian Chin-Sang, Ken Ko and Nancy Martin were three of the team's Faculty Advisors this year, and provided the team with lab space in which we conducted all of our wet work. They are all members of the <html><a target="_new" href="http://www.safety.queensu.ca//biocom/">Queen's Biohazards Committee</a></html> and ensured that we worked within the appropriate biosafety regulations<br />
<br />
<html><div class="section"><h2>Footnotes and Citations</h2></html><br />
{{:Team:Queens-Canada/footnote|1|[http://www.ccac.ca/en/CCAC_Programs/ETCC/Module04/15.html Biosafety Guidelines and Levels of Containment]. Canadian Council on Animal Care. Accessed on 2010-06-22.}}<html></div></html><br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/notebookTeam:Queens-Canada/notebook2010-10-27T21:35:42Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>Notebook</h1><br />
<html><style type="text/css"><br />
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<p>This describes what we did this Summer in painstaking detail. If you would like to see the images that correspond to the gels we ran, visit the [[Team:Queens-Canada/gels|Gels Page]].</p><br />
<div class="month">{{#calendar: |title=Team:Queens-Canada |year=2010 | month=06}}</div><br />
<div class="month">{{#calendar: |title=Team:Queens-Canada |year=2010 | month=07}}</div><br />
<div class="month">{{#calendar: |title=Team:Queens-Canada |year=2010 | month=08}}</div><br />
<div class="month">{{#calendar: |title=Team:Queens-Canada |year=2010 | month=09}}</div><br />
<div class="month">{{#calendar: |title=Team:Queens-Canada |year=2010 | month=10}}</div><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/gelsTeam:Queens-Canada/gels2010-10-27T21:34:35Z<p>Glh: New page: {{:Team:Queens-Canada/head}} <h1>Gels</h1> This is a complete list of all of the gel images we ran this summer. Dates are in format DD_MM_YY, with one or two messy exceptions. Each of th...</p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>Gels</h1><br />
<br />
This is a complete list of all of the gel images we ran this summer. Dates are in format DD_MM_YY, with one or two messy exceptions. Each of these images should correspond to a gel in the notebook.<br />
<br />
'''[[Team:Queens-Canada/notebook|Return to the Notebook]]'''<br />
<br />
<html><ul><br />
<li><a href="http://qgem.iri5.net/2010/gels/C Ladder.jpg">C Ladder.jpg</a></li><li><a href="http://qgem.iri5.net/2010/gels/C Plus Ladder.jpg">C Plus Ladder.jpg</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] 27_07_10.png">[1] 27_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] 29_07_10c.png">[1] 29_07_10c.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C-06_22_10.JPG">[1] C-06_22_10.JPG</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 01_09_10.tif">[1] C 01_09_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 02_09_10.tif">[1] C 02_09_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 03_06_10.bmp">[1] C 03_06_10.bmp</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 04_06_10.tif">[1] C 04_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 04_08_10.png">[1] C 04_08_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 05_07_10.tif">[1] C 05_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 05_08_10 labelled.png">[1] C 05_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 06_06_10.tif">[1] C 06_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 06_07_10.tif">[1] C 06_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 06_08_10 labelled.png">[1] C 06_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 07_07_10 a.tif">[1] C 07_07_10 a.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 07_07_10.tif">[1] C 07_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 08_07_10.png">[1] C 08_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 09_06_10.png">[1] C 09_06_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 09_07_10.jpg">[1] C 09_07_10.jpg</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 09_08_10 labelled.png">[1] C 09_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 10_08_10 labelled.png">[1] C 10_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 11_08_10 labelled.png">[1] C 11_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 12_07_10.jpg">[1] C 12_07_10.jpg</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 12_08_10 labelled.png">[1] C 12_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 13_07_10 (2).tif">[1] C 13_07_10 (2).tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] c 13_07_10.tif">[1] c 13_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 14_06_10.tif">[1] C 14_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 14_07_10.jpg">[1] C 14_07_10.jpg</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 14_09_10.tif">[1] C 14_09_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 14_10_10.tif">[1] C 14_10_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 15_06_10.tif">[1] C 15_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 15_07_10 (2).tif">[1] C 15_07_10 (2).tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 15_07_10.png">[1] C 15_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 16_07_10.png">[1] C 16_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 17_06_10 (2).tif">[1] C 17_06_10 (2).tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 17_06_10.tif">[1] C 17_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 17_08_10 labelled.png">[1] C 17_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 18_06_10.tif">[1] C 18_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 18_08_10 labelled.png">[1] C 18_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 19_07_10 legend.png">[1] C 19_07_10 legend.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 19_08_10 labelled.png">[1] C 19_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 20_07_10 labelled.png">[1] C 20_07_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 20_08_10 labelled.png">[1] C 20_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 21_07_10 labelled.png">[1] C 21_07_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 22_06_10.TIF">[1] C 22_06_10.TIF</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 22_07_10.tif">[1] C 22_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 23_06_10.jpg">[1] C 23_06_10.jpg</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 23_08_10 labelled.png">[1] C 23_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 24_06_10 (2).tif">[1] C 24_06_10 (2).tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 24_06_10.tif">[1] C 24_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 25_06_10.tif">[1] C 25_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 25_08_10 label.png">[1] C 25_08_10 label.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 26_07_10 labelled.png">[1] C 26_07_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 26_08_10 labelled.png">[1] C 26_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 27_05_10.tif">[1] C 27_05_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 27_08_10.tif">[1] C 27_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 28_06_10.jpg">[1] C 28_06_10.jpg</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 28_07_10.png">[1] C 28_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 29_06_10 (2).tif">[1] C 29_06_10 (2).tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 29_06_10.tif">[1] C 29_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 29_09_10 labelled (one lane missing).tif">[1] C 29_09_10 labelled (one lane missing).tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 30_06_10.png">[1] C 30_06_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 30_07_10.png">[1] C 30_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[1] C 31_08_10.tif">[1] C 31_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] 16_08_10 labelled.png">[2] 16_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] 16_08_10.tif">[2] 16_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] 29_07_10.png">[2] 29_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] 29_07_10.tif">[2] 29_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 01_09_10.tif">[2] C 01_09_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 02_09_10.tif">[2] C 02_09_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 04_08_10 legend.png">[2] C 04_08_10 legend.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 05_08_10 labelled.png">[2] C 05_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 05_08_10.tif">[2] C 05_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 06_07_10.jpg">[2] C 06_07_10.jpg</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] c 06_07_10.tif">[2] c 06_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 06_08_10 labelled.png">[2] C 06_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 06_08_10.tif">[2] C 06_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 08_07_10.png">[2] C 08_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 08_07_10.tif">[2] C 08_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 08_31_10.tif">[2] C 08_31_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 09_08_10.tif">[2] C 09_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 09_08_10legend.png">[2] C 09_08_10legend.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 10_06_10 (2).tif">[2] C 10_06_10 (2).tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 10_06_10.tif">[2] C 10_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 10_08_10.png">[2] C 10_08_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 10_08_10.tif">[2] C 10_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 11_08_10.tif">[2] C 11_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 14_07_10.png">[2] C 14_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 14_07_10.tif">[2] C 14_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 15_07_10.png">[2] C 15_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 15_07_10.tif">[2] C 15_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 17_06_10.tif">[2] C 17_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 17_08_10 labelled.png">[2] C 17_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 17_08_10.tif">[2] C 17_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 19_08_10 labelled.png">[2] C 19_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 19_08_10.tif">[2] C 19_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 20_07_10 no labels.png">[2] C 20_07_10 no labels.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 20_07_10.tif">[2] C 20_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 20_08_10 labelled.png">[2] C 20_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 20_08_10.tif">[2] C 20_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 21_07_10 labeled.png">[2] C 21_07_10 labeled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 21_07_10.tif">[2] C 21_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 21_07_10.tif">[2] C 21_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 23_08_10 labelled.png">[2] C 23_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 23_08_10.tif">[2] C 23_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 25_06_10.tif">[2] C 25_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2]C 25_08_10 labelled.png">[2]C 25_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 25_08_10 labelled.png">[2] C 25_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 25_08_10.tif">[2] C 25_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 26_08_10 labelled.png">[2] C 26_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 26_08_10.tif">[2] C 26_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 28_05_10.jpg">[2] C 28_05_10.jpg</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] c 28_07_10.png">[2] c 28_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 29_06_10-1.tif">[2] C 29_06_10-1.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 29_06_10.png">[2] C 29_06_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 30_06_10.png">[2] C 30_06_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 30_06_10.tif">[2] C 30_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 30_07_10.png">[2] C 30_07_10.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[2] C 30_07_10.tif">[2] C 30_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] 16_08_10 labelled.png">[3] 16_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] 16_08_10.tif">[3] 16_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 06_07_10.jpg">[3] C 06_07_10.jpg</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 09_08_10 labelled.png">[3] C 09_08_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 09_08_10.tif">[3] C 09_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 11_08_10 copy.png">[3] C 11_08_10 copy.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 11_08_10.tif">[3] C 11_08_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 17_06_10 (2).tif">[3] C 17_06_10 (2).tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 17_06_10.tif">[3] C 17_06_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 19_07_10 labelled.png">[3] C 19_07_10 labelled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 19_07_10.tif">[3] C 19_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 20_07_10 labeled.png">[3] C 20_07_10 labeled.png</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 20_07_10.tif">[3] C 20_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 30_07_10 label.jpg">[3] C 30_07_10 label.jpg</a></li><li><a href="http://qgem.iri5.net/2010/gels/[3] C 30_07_10.tif">[3] C 30_07_10.tif</a></li><li><a href="http://qgem.iri5.net/2010/gels/[4] C 28_05_10.jpg">[4] C 28_05_10.jpg</a></li></ul></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/partsTeam:Queens-Canada/parts2010-10-27T21:05:23Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks Parts List</h1><br />
<br />
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.<br />
<br />
<html><div class="section"><h2>Regulatory</h2></html><br />
<br />
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.<br />
<br />
<h3>Constitutive</h3><br />
* '''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.<br />
* '''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.<br />
* '''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>.<br />
<br />
<h3>Inducible</h3><br />
<br />
* '''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.<br />
<br />
<h3>Tissue-Specific</h3><br />
<br />
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]].<br />
<br />
* '''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>.<br />
* '''pOdr-1''': This targets the AWC sensory neuron.<br />
* '''pStr-1''': This targets the AWB sensory neuron.<br />
* '''pOsm-10''': This targets the ASH and ASI sensory neurons, as well as the PHA and PHB phasmid neurons.<br />
* '''pFlp-1''': This targets the AVA sensory neuron.<br />
* '''pSra-10''': This targets the AVB sensory neuron.<br />
* '''pStr-220''': This targets the AWC sensory neuron.<br />
<br />
<html></div><div class="section"><h2>Reporters</h2></html><br />
<br />
* '''eGFP''': excitation: 395 nm; emission: 509 nm.<br />
* '''eCFP''': excitation: 433 nm; emission: 475 nm.<br />
* '''eYFP''': excitation: ~485 nm; emission: ~700 nm.<br />
* '''mCherry''': excition: ~580 nm; emission: ~620 nm.<br />
<br />
<html></div><div class="section"><h2>Optogenetics Proteins</h2></html><br />
<br />
* '''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.<br />
* '''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.<br />
* ''Fusions'': ChR2::eYFP and NpHR::eCFP are both also provided. These are useful for ensuring that the channels localized successfully to their target.<br />
<br />
<html></div><div class="section"><h2>Constructs</h2></html><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/igem.org/thumb/2/29/Qgem_fluorescence_mec7_ecfp_utr.png/800px-Qgem_fluorescence_mec7_ecfp_utr.png" 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"><br />
</html><br />
<br />
Unlike the fusions listed under Optogenetics Proteins, these were assembled through BioBrick digestion/ligation.<br />
<br />
* '''pStr-220::eCFP::unc-54 3' UTR'''<br />
* '''pOdr-10::eCFP::unc-54 3' UTR'''<br />
* '''pGpd-2::eCFP::unc-54 3' UTR'''<br />
* '''pSip1::eCFP::unc-54 3' UTR'''<br />
* '''pFlp1::eCFP:unc-54 3' UTR'''<br />
* '''ChR2::eYFP::unc-54 3' UTR'''<br />
* '''pHsp3::eCFP::unc-54 3' UTR'''<br />
* '''pOsm-10::eCFP::unc-54 3' UTR'''<br />
* '''mCherry::unc-54 3' UTR'''<br />
* '''eYFP::unc-54 3' UTR'''<br />
* '''pOdr-1::eCFP::unc-54 3' UTR'''<br />
* '''pMec-7::eCFP::unc-54 3' UTR'''<br />
<br />
<html></div><div class="section"><h2>Getting the Parts</h2></html><br />
<br />
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.<br />
<br />
<html><script type="text/javascript"><br />
function toggle_visibility(id) {<br />
el = document.getElementById(id);<br />
if(el.style.display == "none") {<br />
el.style.display = "block";<br />
} else {<br />
el.style.display = "none";<br />
}<br />
}</script><br />
<div class="box"><br />
<div id="groupparts" style="display: block;"><br />
</html><groupparts>iGEM010 Queens-Canada</groupparts><html><br />
</div><br />
</div><br />
<br />
</div></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/partsTeam:Queens-Canada/parts2010-10-27T21:04:51Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks Parts List</h1><br />
<br />
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.<br />
<br />
<html><div class="section"><h2>Regulatory</h2></html><br />
<br />
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.<br />
<br />
<h3>Constitutive</h3><br />
* '''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.<br />
* '''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.<br />
* '''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>.<br />
<br />
<h3>Inducible</h3><br />
<br />
* '''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.<br />
<br />
<h3>Tissue-Specific</h3><br />
<br />
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]].<br />
<br />
* '''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>.<br />
* '''pOdr-1''': This targets the AWC sensory neuron.<br />
* '''pStr-1''': This targets the AWB sensory neuron.<br />
* '''pOsm-10''': This targets the ASH and ASI sensory neurons, as well as the PHA and PHB phasmid neurons.<br />
* '''pFlp-1''': This targets the AVA sensory neuron.<br />
* '''pSra-10''': This targets the AVB sensory neuron.<br />
* '''pStr-220''': This targets the AWC sensory neuron.<br />
<br />
<html></div><div class="section"><h2>Reporters</h2></html><br />
<br />
* '''eGFP''': excitation: 395 nm; emission: 509 nm.<br />
* '''eCFP''': excitation: 433 nm; emission: 475 nm.<br />
* '''eYFP''': excitation: ~485 nm; emission: ~700 nm.<br />
* '''mCherry''': excition: ~580 nm; emission: ~620 nm.<br />
<br />
<html></div><div class="section"><h2>Optogenetics Proteins</h2></html><br />
<br />
* '''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.<br />
* '''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.<br />
* ''Fusions'': ChR2::eYFP and NpHR::eCFP are both also provided. These are useful for ensuring that the channels localized successfully to their target.<br />
<br />
<html></div><div class="section"><h2>Constructs</h2></html><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/igem.org/thumb/2/29/Qgem_fluorescence_mec7_ecfp_utr.png/800px-Qgem_fluorescence_mec7_ecfp_utr.png" style="float: right; max-width: 800px; width: 40%; box-shadow: 1px 2px 3px #808080; margin-right: 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"><br />
</html><br />
<br />
Unlike the fusions listed under Optogenetics Proteins, these were assembled through BioBrick digestion/ligation.<br />
<br />
* '''pStr-220::eCFP::unc-54 3' UTR'''<br />
* '''pOdr-10::eCFP::unc-54 3' UTR'''<br />
* '''pGpd-2::eCFP::unc-54 3' UTR'''<br />
* '''pSip1::eCFP::unc-54 3' UTR'''<br />
* '''pFlp1::eCFP:unc-54 3' UTR'''<br />
* '''ChR2::eYFP::unc-54 3' UTR'''<br />
* '''pHsp3::eCFP::unc-54 3' UTR'''<br />
* '''pOsm-10::eCFP::unc-54 3' UTR'''<br />
* '''mCherry::unc-54 3' UTR'''<br />
* '''eYFP::unc-54 3' UTR'''<br />
* '''pOdr-1::eCFP::unc-54 3' UTR'''<br />
* '''pMec-7::eCFP::unc-54 3' UTR'''<br />
<br />
<html></div><div class="section"><h2>Getting the Parts</h2></html><br />
<br />
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.<br />
<br />
<html><script type="text/javascript"><br />
function toggle_visibility(id) {<br />
el = document.getElementById(id);<br />
if(el.style.display == "none") {<br />
el.style.display = "block";<br />
} else {<br />
el.style.display = "none";<br />
}<br />
}</script><br />
<div class="box"><br />
<div id="groupparts" style="display: block;"><br />
</html><groupparts>iGEM010 Queens-Canada</groupparts><html><br />
</div><br />
</div><br />
<br />
</div></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/partsTeam:Queens-Canada/parts2010-10-27T21:04:18Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks Parts List</h1><br />
<br />
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.<br />
<br />
<html><div class="section"><h2>Regulatory</h2></html><br />
<br />
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.<br />
<br />
<h3>Constitutive</h3><br />
* '''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.<br />
* '''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.<br />
* '''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>.<br />
<br />
<h3>Inducible</h3><br />
<br />
* '''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.<br />
<br />
<h3>Tissue-Specific</h3><br />
<br />
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]].<br />
<br />
* '''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>.<br />
* '''pOdr-1''': This targets the AWC sensory neuron.<br />
* '''pStr-1''': This targets the AWB sensory neuron.<br />
* '''pOsm-10''': This targets the ASH and ASI sensory neurons, as well as the PHA and PHB phasmid neurons.<br />
* '''pFlp-1''': This targets the AVA sensory neuron.<br />
* '''pSra-10''': This targets the AVB sensory neuron.<br />
* '''pStr-220''': This targets the AWC sensory neuron.<br />
<br />
<html></div><div class="section"><h2>Reporters</h2></html><br />
<br />
* '''eGFP''': excitation: 395 nm; emission: 509 nm.<br />
* '''eCFP''': excitation: 433 nm; emission: 475 nm.<br />
* '''eYFP''': excitation: ~485 nm; emission: ~700 nm.<br />
* '''mCherry''': excition: ~580 nm; emission: ~620 nm.<br />
<br />
<html></div><div class="section"><h2>Optogenetics Proteins</h2></html><br />
<br />
* '''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.<br />
* '''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.<br />
* ''Fusions'': ChR2::eYFP and NpHR::eCFP are both also provided. These are useful for ensuring that the channels localized successfully to their target.<br />
<br />
<html></div><div class="section"><h2>Constructs</h2></html><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/igem.org/thumb/2/29/Qgem_fluorescence_mec7_ecfp_utr.png/800px-Qgem_fluorescence_mec7_ecfp_utr.png" style="float: right; max-width: 800px; width: 40%; box-shadow: 1px 2px 3px #808080;" title="Our mec-7::eCFP::unc-54 3' UTR construct in action" alt="Our mec-7::eCFP::unc-54 3' UTR construct in action"><br />
</html><br />
<br />
Unlike the fusions listed under Optogenetics Proteins, these were assembled through BioBrick digestion/ligation.<br />
<br />
* '''pStr-220::eCFP::unc-54 3' UTR'''<br />
* '''pOdr-10::eCFP::unc-54 3' UTR'''<br />
* '''pGpd-2::eCFP::unc-54 3' UTR'''<br />
* '''pSip1::eCFP::unc-54 3' UTR'''<br />
* '''pFlp1::eCFP:unc-54 3' UTR'''<br />
* '''ChR2::eYFP::unc-54 3' UTR'''<br />
* '''pHsp3::eCFP::unc-54 3' UTR'''<br />
* '''pOsm-10::eCFP::unc-54 3' UTR'''<br />
* '''mCherry::unc-54 3' UTR'''<br />
* '''eYFP::unc-54 3' UTR'''<br />
* '''pOdr-1::eCFP::unc-54 3' UTR'''<br />
* '''pMec-7::eCFP::unc-54 3' UTR'''<br />
<br />
<html></div><div class="section"><h2>Getting the Parts</h2></html><br />
<br />
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.<br />
<br />
<html><script type="text/javascript"><br />
function toggle_visibility(id) {<br />
el = document.getElementById(id);<br />
if(el.style.display == "none") {<br />
el.style.display = "block";<br />
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}</script><br />
<div class="box"><br />
<div id="groupparts" style="display: block;"><br />
</html><groupparts>iGEM010 Queens-Canada</groupparts><html><br />
</div><br />
</div><br />
<br />
</div></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/partsTeam:Queens-Canada/parts2010-10-27T21:03:16Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks Parts List</h1><br />
<br />
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.<br />
<br />
<html><div class="section"><h2>Regulatory</h2></html><br />
<br />
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.<br />
<br />
<h3>Constitutive</h3><br />
* '''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.<br />
* '''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.<br />
* '''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>.<br />
<br />
<h3>Inducible</h3><br />
<br />
* '''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.<br />
<br />
<h3>Tissue-Specific</h3><br />
<br />
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]].<br />
<br />
* '''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>.<br />
* '''pOdr-1''': This targets the AWC sensory neuron.<br />
* '''pStr-1''': This targets the AWB sensory neuron.<br />
* '''pOsm-10''': This targets the ASH and ASI sensory neurons, as well as the PHA and PHB phasmid neurons.<br />
* '''pFlp-1''': This targets the AVA sensory neuron.<br />
* '''pSra-10''': This targets the AVB sensory neuron.<br />
* '''pStr-220''': This targets the AWC sensory neuron.<br />
<br />
<html></div><div class="section"><h2>Reporters</h2></html><br />
<br />
* '''eGFP''': excitation: 395 nm; emission: 509 nm.<br />
* '''eCFP''': excitation: 433 nm; emission: 475 nm.<br />
* '''eYFP''': excitation: ~485 nm; emission: ~700 nm.<br />
* '''mCherry''': excition: ~580 nm; emission: ~620 nm.<br />
<br />
<html></div><div class="section"><h2>Optogenetics Proteins</h2></html><br />
<br />
* '''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.<br />
* '''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.<br />
* ''Fusions'': ChR2::eYFP and NpHR::eCFP are both also provided. These are useful for ensuring that the channels localized successfully to their target.<br />
<br />
<html></div><div class="section"><h2>Constructs</h2></html><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/igem.org/thumb/2/29/Qgem_fluorescence_mec7_ecfp_utr.png/800px-Qgem_fluorescence_mec7_ecfp_utr.png" style="float: right; max-width: 800px; width: 40%; box-shadow: 1px 2px 3px #808080;" title="Our mec-7::eCFP::unc-54 3' UTR construct in action" alt="Our mec-7::eCFP::unc-54 3' UTR construct in action"><br />
</html><br />
<br />
* '''pStr-220::eCFP::unc-54 3' UTR'''<br />
* '''pOdr-10::eCFP::unc-54 3' UTR'''<br />
* '''pGpd-2::eCFP::unc-54 3' UTR'''<br />
* '''pSip1::eCFP::unc-54 3' UTR'''<br />
* '''pFlp1::eCFP:unc-54 3' UTR'''<br />
* '''ChR2::eYFP::unc-54 3' UTR'''<br />
* '''phsp3::eCFP::unc-54 3' UTR'''<br />
* '''pOsm-10::eCFP::unc-54 3' UTR'''<br />
* '''mCherry::unc-54 3' UTR'''<br />
* '''eYFP::unc-54 3' UTR'''<br />
* '''pOdr-1::eCFP::unc-54 3' UTR'''<br />
* '''pMec-7::eCFP::unc-54 3' UTR'''<br />
<br />
<html></div><div class="section"><h2>Getting the Parts</h2></html><br />
<br />
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.<br />
<br />
<html><script type="text/javascript"><br />
function toggle_visibility(id) {<br />
el = document.getElementById(id);<br />
if(el.style.display == "none") {<br />
el.style.display = "block";<br />
} else {<br />
el.style.display = "none";<br />
}<br />
}</script><br />
<div class="box"><br />
<div id="groupparts" style="display: block;"><br />
</html><groupparts>iGEM010 Queens-Canada</groupparts><html><br />
</div><br />
</div><br />
<br />
</div></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/partsTeam:Queens-Canada/parts2010-10-27T20:56:20Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks Parts List</h1><br />
<br />
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.<br />
<br />
<html><div class="section"><h2>Regulatory</h2></html><br />
<br />
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.<br />
<br />
<h3>Constitutive</h3><br />
* '''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.<br />
* '''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.<br />
* '''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>.<br />
<br />
<h3>Inducible</h3><br />
<br />
* '''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.<br />
<br />
<h3>Tissue-Specific</h3><br />
<br />
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]].<br />
<br />
* '''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>.<br />
* '''pOdr-1''': This targets the AWC sensory neuron.<br />
* '''pStr-1''': This targets the AWB sensory neuron.<br />
* '''pOsm-10''': This targets the ASH and ASI sensory neurons, as well as the PHA and PHB phasmid neurons.<br />
* '''pFlp-1''': This targets the AVA sensory neuron.<br />
* '''pSra-10''': This targets the AVB sensory neuron.<br />
* '''pStr-220''': This targets the AWC sensory neuron.<br />
<br />
<html></div><div class="section"><h2>Reporters</h2></html><br />
<br />
* '''eGFP''': excitation: 395 nm; emission: 509 nm.<br />
* '''eCFP''': excitation: 433 nm; emission: 475 nm.<br />
* '''eYFP''': excitation: ~485 nm; emission: ~700 nm.<br />
* '''mCherry''': excition: ~580 nm; emission: ~620 nm.<br />
<br />
<html></div><div class="section"><h2>Optogenetics Proteins</h2></html><br />
<br />
* '''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.<br />
* '''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.<br />
* ''Fusions'': ChR2::eYFP and NpHR::eCFP are both also provided. These are useful for ensuring that the channels localized successfully to their target.<br />
<br />
<html></div><div class="section"><h2>Constructs</h2></html><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/igem.org/thumb/2/29/Qgem_fluorescence_mec7_ecfp_utr.png/800px-Qgem_fluorescence_mec7_ecfp_utr.png" style="float: right; max-width: 800px; width: 40%; box-shadow: 1px 2px 3px #808080;" title="Our mec-7::eCFP::unc-54 3' UTR construct in action" alt="Our mec-7::eCFP::unc-54 3' UTR construct in action"><br />
</html><br />
<br />
* ''''''<br />
* ''''''<br />
* ''''''<br />
* ''''''<br />
* ''''''<br />
* ''''''<br />
* ''''''<br />
* ''''''<br />
* ''''''<br />
* ''''''<br />
* ''''''<br />
<br />
<html></div><div class="section"><h2>Getting the Parts</h2></html><br />
<br />
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.<br />
<br />
<html><script type="text/javascript"><br />
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el = document.getElementById(id);<br />
if(el.style.display == "none") {<br />
el.style.display = "block";<br />
} else {<br />
el.style.display = "none";<br />
}<br />
}</script><br />
<div class="box"><br />
<div id="groupparts" style="display: block;"><br />
</html><groupparts>iGEM010 Queens-Canada</groupparts><html><br />
</div><br />
</div><br />
<br />
</div></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/partsTeam:Queens-Canada/parts2010-10-27T20:24:55Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks Parts List</h1><br />
<br />
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.<br />
<br />
<html><div class="section"><h2>Regulatory</h2></html><br />
<br />
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.<br />
<br />
<h3>Constitutive</h3><br />
* '''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.<br />
* '''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.<br />
* '''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>.<br />
<br />
<h3>Inducible</h3><br />
<br />
* '''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.<br />
<br />
<h3>Tissue-Specific</h3><br />
<br />
* '''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>.<br />
* '''pOdr-1''': <br />
* '''pStr-1''':<br />
* '''pOsm-10''':<br />
* '''pFlp-1''':<br />
* '''pSra-10''':<br />
* '''pStr-220''':<br />
<br />
<html></div><div class="section"><h2>Reporters</h2></html><br />
<br />
* '''eGFP''':<br />
* '''eCFP''':<br />
* '''eYFP''':<br />
* '''mCherry''':<br />
<br />
<html></div><div class="section"><h2>Optogenetics Proteins</h2></html><br />
<br />
* '''ChR2''':<br />
* '''NpHR''':<br />
* ''Fusions'': ChR2::eYFP and NpHR::eCFP<br />
<br />
<html></div><div class="section"><h2>Constructs</h2></html><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/igem.org/thumb/2/29/Qgem_fluorescence_mec7_ecfp_utr.png/800px-Qgem_fluorescence_mec7_ecfp_utr.png" style="float: right; max-width: 800px; width: 40%; box-shadow: 1px 2px 3px #808080;" title="Our mec-7::eCFP::unc-54 3' UTR construct in action" alt="Our mec-7::eCFP::unc-54 3' UTR construct in action"><br />
</html><br />
<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
<br />
<html></div><div class="section"><h2>Getting the Parts</h2></html><br />
<br />
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.<br />
<br />
<html><script type="text/javascript"><br />
function toggle_visibility(id) {<br />
el = document.getElementById(id);<br />
if(el.style.display == "none") {<br />
el.style.display = "block";<br />
} else {<br />
el.style.display = "none";<br />
}<br />
}</script><br />
<div class="box"><br />
<div id="groupparts" style="display: block;"><br />
</html><groupparts>iGEM010 Queens-Canada</groupparts><html><br />
</div><br />
</div><br />
<br />
</div></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glhhttp://2010.igem.org/Team:Queens-Canada/partsTeam:Queens-Canada/parts2010-10-27T20:22:49Z<p>Glh: </p>
<hr />
<div>{{:Team:Queens-Canada/head}}<br />
<br />
<h1>WormWorks Parts List</h1><br />
<br />
<html><div class="section"><h2>Regulatory</h2></html><br />
<br />
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.<br />
<br />
<h3>Constitutive</h3><br />
* '''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.<br />
* '''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.<br />
* '''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>.<br />
<br />
<h3>Inducible</h3><br />
<br />
* '''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.<br />
<br />
<h3>Tissue-Specific</h3><br />
<br />
* '''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>.<br />
* '''pOdr-1''': <br />
* '''pStr-1''':<br />
* '''pOsm-10''':<br />
* '''pFlp-1''':<br />
* '''pSra-10''':<br />
* '''pStr-220''':<br />
<br />
<html></div><div class="section"><h2>Reporters</h2></html><br />
<br />
* '''eGFP''':<br />
* '''eCFP''':<br />
* '''eYFP''':<br />
* '''mCherry''':<br />
<br />
<html></div><div class="section"><h2>Optogenetics Proteins</h2></html><br />
<br />
* '''ChR2''':<br />
* '''NpHR''':<br />
* ''Fusions'': ChR2::eYFP and NpHR::eCFP<br />
<br />
<html></div><div class="section"><h2>Constructs</h2></html><br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/igem.org/thumb/2/29/Qgem_fluorescence_mec7_ecfp_utr.png/800px-Qgem_fluorescence_mec7_ecfp_utr.png" style="float: right; max-width: 800px; width: 40%; box-shadow: 1px 2px 3px #808080;" title="Our mec-7::eCFP::unc-54 3' UTR construct in action" alt="Our mec-7::eCFP::unc-54 3' UTR construct in action"><br />
</html><br />
<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
* '''''':<br />
<br />
<html></div><div class="section"><h2>Getting the Parts</h2></html><br />
<br />
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.<br />
<br />
<html><script type="text/javascript"><br />
function toggle_visibility(id) {<br />
el = document.getElementById(id);<br />
if(el.style.display == "none") {<br />
el.style.display = "block";<br />
} else {<br />
el.style.display = "none";<br />
}<br />
}</script><br />
<div class="box"><br />
<div id="groupparts" style="display: block;"><br />
</html><groupparts>iGEM010 Queens-Canada</groupparts><html><br />
</div><br />
</div><br />
<br />
</div></html><br />
<br />
{{:Team:Queens-Canada/foot}}</div>Glh