http://2010.igem.org/wiki/index.php?title=Special:Contributions/Willh&feed=atom&limit=50&target=Willh&year=&month=2010.igem.org - User contributions [en]2024-03-29T10:39:36ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Cambridge/Bioluminescence/Bacterial_LuciferasesTeam:Cambridge/Bioluminescence/Bacterial Luciferases2010-10-28T03:00:42Z<p>Willh: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#386abc|title=Project Vibrio: Introduction}}<br />
'''Project Vibrio''' was designed to create BioBricks from genes involved in bacterial bioluminescence. <br />
<br />
==Natural bioluminescent bacteria==<br />
{{:Team:Cambridge/Templates/RightImage|image=Tide.jpg|caption=Wave glowing with bioluminescent micro-organisms}}<br />
Bioluminescence is a trait found in a number of marine bacteria. Some strains use their ability to emit light to form symbiotic relationships. A number of deep sea fish and squids have specialised <em>light organs</em> which harbour populations of bacteria that help their hosts by emitting light. One such example is the partnership between the Hawaiian Bobtail squid <em>(Euprymna scolopes)</em> and the bacterium <em>Vibrio fischeri</em>. <br />
<br />
At night squid hunt high in the water column, attacking their prey from above. The squid uses the light produced by its symbionts to hide the shadow it casts when hunting in top waters in clear moonlit nights. An elaborate light sensing and shutter system adjusts the light output to the light that falls on the squid's back. <br />
<br />
Bioluminescent bacterial species can differ markedly in their lifestyles. Vibrio Harveyi is a free living marine bacterium, while Xenorhabdus luminescens is a symbiont of terrestrial nematodes.<br />
<br />
The light-generating chemical reactions in bioluminescent bacteria are catalysed by enzymes expressed from so-called lux genes. These genes encode not only the bacterial luciferase, but also the enzymes required for the synthesis of fatty aldehydes, which are the substrates for the reaction. In the bacterial genome, the lux genes occur clustered in the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Background lux operon].<br />
<br />
==Our work==<br />
{{:Team:Cambridge/Templates/RightImage|image=Phosphoreum_bright.jpg|caption=Our workspace illuminated by'' Vibrio phosphoreum'', a bacterium we investigated}}<br />
To complement 'Project Firefly', we intended to use the lux operon from ''Vibrio fischeri'' for the following three purposes:<br />
* Emission of blue light to complete our spectrum of emission wavelengths.<br />
* Substrate production within E. coli, avoiding the need for addition of external substrates, such as luciferin.<br />
* Design of a biosensor output device that can be combined with various input systems.<br />
<br />
Bacterial lux operons encode five enzymes involved in the light-generating pathway. In nature, the lux genes appear to be repressed by the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Background nucleoid protein, H-NS], and occur under [https://2010.igem.org/Team:Cambridge/Bioluminescence/Background quorum sensing control]. We wished to relieve repression by H-NS to achieve brighter light outputs. We furthermore removed quorum sensing control to facilitate use of the part in biosensors under different regulatory inputs. <br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/IntroductionTeam:Cambridge/Gibson/Introduction2010-10-27T20:47:39Z<p>Willh: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Introduction}}<br />
{{:Team:Cambridge/Templates/RightImage|image=GibsonAssembly.jpg|caption=Gibson Assembly joins any two lengths of DNA which have overlapping end regions}}<br />
'''Gibson Assembly''' is a cutting-edge DNA ligation technique developed by Dan Gibson at JCVI in 2009 [http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html]. <br />
<br />
It uses three enzymes to ligate two or more sequences of DNA when they have overlapping end sequences at their joining point (~40bp). These overlapping regions can be easily added to the ends of any length of DNA by using PCR with primers which have added "flaps". Thus PCR followed by Gibson allows you to join any two blunt ended pieces of DNA.<br />
<br />
== Advantages ==<br />
1) No scar created - useful for '''fusion proteins''' and '''adding an RBS''', where scars can be problematic.<br />
<br />
2) Can re-ligate linear DNA into a circle - useful for '''site-directed mutagenesis'''<br />
<br />
3) Any two blunt ended pieces of DNA can be joined, and DNA can be taken from any source which is accessible to PCR (for example, an organism's '''genome''')<br />
<br />
4) Although it is roughly the same speed as [http://partsregistry.org/Help:BioBrick_Assembly standard BioBrick assembly] when ligating two fragments, Gibson is perfect for '''assembling multi part systems''', since it takes the same amount of time to ligate n pieces of DNA together as it does for two pieces:<br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr> <br />
<td><b>Assembly Method</b> <br />
</td><td><b>Number of steps to ligate N pieces of DNA</b> <br />
</td></tr> <br />
<tr> <br />
<td><a href="http://partsregistry.org/Assembly:Standard_assembly" class="external text" title="http://partsregistry.org/Assembly:Standard_assembly" rel="nofollow">Standard Assembly</a> <br />
</td><td><a href="/Image:Steps-N.png" class="image" title="Image:Steps-N.png"><img alt="Image:Steps-N.png" src="/wiki/images/c/cc/Steps-N.png" border="0" /></a> <br />
</td></tr> <br />
<tr> <br />
<td><a href="http://partsregistry.org/Assembly:Rolling_assembly" class="external text" title="http://partsregistry.org/Assembly:Rolling_assembly" rel="nofollow">Parallel Assembly</a> <br />
</td><td><a href="/Image:Steps-Log2.png" class="image" title="Image:Steps-Log2.png"><img alt="Image:Steps-Log2.png" src="/wiki/images/6/6f/Steps-Log2.png" width="138" height="21" border="0" /></a> (rounded up)<br />
</td></tr> <br />
<tr> <br />
<td><a href="https://2010.igem.org/Team:Cambridge/Gibson/Mechanism" class="external text" title="https://2010.igem.org/Team:Cambridge/Gibson/Mechanism" rel="nofollow">Gibson Assembly</a> <br />
</td><td><a href="/Image:Steps1.png" class="image" title="Image:Steps1.png"><img alt="Image:Steps1.png" src="/wiki/images/1/10/Steps1.png" width="83" height="18" border="0" /></a> <br />
</td></tr></table> <br />
</div><br />
</html><br />
<br />
== Disadvantages ==<br />
1) Greater degree of planning required as primers must be ordered in advance<br />
<br />
2) More expensive than BioBrick assembly<br />
<br />
3) PCR is tricky<br />
<br />
==Important:==<br />
<br />
The flexibility that this assembly method offers is a great thing. However, this does not mean it replaces the need for standardised prefixes and suffixes. The BioBrick prefix and suffix and the use of standard well-characterised vectors with standard primer sites are crucial for standardised iGEM parts. If you use Gibson Assembly it is important that you still add the prefix and suffix to your DNA; in fact sometimes Gibson Assembly is a useful way to do this (using as template an existing standard BioBrick).<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/IntroductionTeam:Cambridge/Gibson/Introduction2010-10-27T20:46:48Z<p>Willh: /* Advantages */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Introduction}}<br />
{{:Team:Cambridge/Templates/RightImage|image=GibsonAssembly.jpg|caption=Gibson Assembly joins any two lengths of DNA which have overlapping end regions}}<br />
Gibson Assembly is a cutting-edge DNA ligation technique developed by Dan Gibson at JCVI in 2009 [http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html]. <br />
<br />
It uses three enzymes to ligate two or more sequences of DNA when they have overlapping end sequences at their joining point (~40bp). These overlapping regions can be easily added to the ends of any length of DNA by using PCR with primers which have added "flaps". Thus PCR followed by Gibson allows you to join any two blunt ended pieces of DNA.<br />
<br />
== Advantages ==<br />
1) No scar created - useful for '''fusion proteins''' and '''adding an RBS''', where scars can be problematic.<br />
<br />
2) Can re-ligate linear DNA into a circle - useful for '''site-directed mutagenesis'''<br />
<br />
3) Any two blunt ended pieces of DNA can be joined, and DNA can be taken from any source which is accessible to PCR (for example, an organism's '''genome''')<br />
<br />
4) Although it is roughly the same speed as [http://partsregistry.org/Help:BioBrick_Assembly standard BioBrick assembly] when ligating two fragments, Gibson is perfect for '''assembling multi part systems''', since it takes the same amount of time to ligate n pieces of DNA together as it does for two pieces:<br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr> <br />
<td><b>Assembly Method</b> <br />
</td><td><b>Number of steps to ligate N pieces of DNA</b> <br />
</td></tr> <br />
<tr> <br />
<td><a href="http://partsregistry.org/Assembly:Standard_assembly" class="external text" title="http://partsregistry.org/Assembly:Standard_assembly" rel="nofollow">Standard Assembly</a> <br />
</td><td><a href="/Image:Steps-N.png" class="image" title="Image:Steps-N.png"><img alt="Image:Steps-N.png" src="/wiki/images/c/cc/Steps-N.png" border="0" /></a> <br />
</td></tr> <br />
<tr> <br />
<td><a href="http://partsregistry.org/Assembly:Rolling_assembly" class="external text" title="http://partsregistry.org/Assembly:Rolling_assembly" rel="nofollow">Parallel Assembly</a> <br />
</td><td><a href="/Image:Steps-Log2.png" class="image" title="Image:Steps-Log2.png"><img alt="Image:Steps-Log2.png" src="/wiki/images/6/6f/Steps-Log2.png" width="138" height="21" border="0" /></a> (rounded up)<br />
</td></tr> <br />
<tr> <br />
<td><a href="https://2010.igem.org/Team:Cambridge/Gibson/Mechanism" class="external text" title="https://2010.igem.org/Team:Cambridge/Gibson/Mechanism" rel="nofollow">Gibson Assembly</a> <br />
</td><td><a href="/Image:Steps1.png" class="image" title="Image:Steps1.png"><img alt="Image:Steps1.png" src="/wiki/images/1/10/Steps1.png" width="83" height="18" border="0" /></a> <br />
</td></tr></table> <br />
</div><br />
</html><br />
<br />
== Disadvantages ==<br />
1) Greater degree of planning required as primers must be ordered in advance<br />
<br />
2) More expensive than BioBrick assembly<br />
<br />
3) PCR is tricky<br />
<br />
==Important:==<br />
<br />
The flexibility that this assembly method offers is a great thing. However, this does not mean it replaces the need for standardised prefixes and suffixes. The BioBrick prefix and suffix and the use of standard well-characterised vectors with standard primer sites are crucial for standardised iGEM parts. If you use Gibson Assembly it is important that you still add the prefix and suffix to your DNA; in fact sometimes Gibson Assembly is a useful way to do this (using as template an existing standard BioBrick).<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/IntroductionTeam:Cambridge/Gibson/Introduction2010-10-27T20:46:31Z<p>Willh: /* Advantages */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Introduction}}<br />
{{:Team:Cambridge/Templates/RightImage|image=GibsonAssembly.jpg|caption=Gibson Assembly joins any two lengths of DNA which have overlapping end regions}}<br />
Gibson Assembly is a cutting-edge DNA ligation technique developed by Dan Gibson at JCVI in 2009 [http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html]. <br />
<br />
It uses three enzymes to ligate two or more sequences of DNA when they have overlapping end sequences at their joining point (~40bp). These overlapping regions can be easily added to the ends of any length of DNA by using PCR with primers which have added "flaps". Thus PCR followed by Gibson allows you to join any two blunt ended pieces of DNA.<br />
<br />
== Advantages ==<br />
1) No scar created - useful for '''fusion proteins''' and '''adding an RBS''', where scars can be problematic.<br />
<br />
2) Can re-ligate linear DNA into a circle - useful for '''site-directed mutagenesis'''<br />
<br />
3) Any two blunt ended pieces of DNA can be joined, and DNA can be taken from any source which is accessible to PCR (for example, an organism's '''genome''')<br />
<br />
4) Although it is roughly the same speed as [http://partsregistry.org/Help:BioBrick_Assembly standard BioBrick assembly] when ligating two fragments, Gibson is perfect for '''assembling multi part systems''', since it takes the same amount of time to ligate n pieces of DNA together as it does for two pieces.<br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr> <br />
<td><b>Assembly Method</b> <br />
</td><td><b>Number of steps to ligate N pieces of DNA</b> <br />
</td></tr> <br />
<tr> <br />
<td><a href="http://partsregistry.org/Assembly:Standard_assembly" class="external text" title="http://partsregistry.org/Assembly:Standard_assembly" rel="nofollow">Standard Assembly</a> <br />
</td><td><a href="/Image:Steps-N.png" class="image" title="Image:Steps-N.png"><img alt="Image:Steps-N.png" src="/wiki/images/c/cc/Steps-N.png" border="0" /></a> <br />
</td></tr> <br />
<tr> <br />
<td><a href="http://partsregistry.org/Assembly:Rolling_assembly" class="external text" title="http://partsregistry.org/Assembly:Rolling_assembly" rel="nofollow">Parallel Assembly</a> <br />
</td><td><a href="/Image:Steps-Log2.png" class="image" title="Image:Steps-Log2.png"><img alt="Image:Steps-Log2.png" src="/wiki/images/6/6f/Steps-Log2.png" width="138" height="21" border="0" /></a> (rounded up)<br />
</td></tr> <br />
<tr> <br />
<td><a href="https://2010.igem.org/Team:Cambridge/Gibson/Mechanism" class="external text" title="https://2010.igem.org/Team:Cambridge/Gibson/Mechanism" rel="nofollow">Gibson Assembly</a> <br />
</td><td><a href="/Image:Steps1.png" class="image" title="Image:Steps1.png"><img alt="Image:Steps1.png" src="/wiki/images/1/10/Steps1.png" width="83" height="18" border="0" /></a> <br />
</td></tr></table> <br />
</div><br />
</html><br />
<br />
== Disadvantages ==<br />
1) Greater degree of planning required as primers must be ordered in advance<br />
<br />
2) More expensive than BioBrick assembly<br />
<br />
3) PCR is tricky<br />
<br />
==Important:==<br />
<br />
The flexibility that this assembly method offers is a great thing. However, this does not mean it replaces the need for standardised prefixes and suffixes. The BioBrick prefix and suffix and the use of standard well-characterised vectors with standard primer sites are crucial for standardised iGEM parts. If you use Gibson Assembly it is important that you still add the prefix and suffix to your DNA; in fact sometimes Gibson Assembly is a useful way to do this (using as template an existing standard BioBrick).<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/IntroductionTeam:Cambridge/Gibson/Introduction2010-10-27T20:45:28Z<p>Willh: /* Advantages */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Introduction}}<br />
{{:Team:Cambridge/Templates/RightImage|image=GibsonAssembly.jpg|caption=Gibson Assembly joins any two lengths of DNA which have overlapping end regions}}<br />
Gibson Assembly is a cutting-edge DNA ligation technique developed by Dan Gibson at JCVI in 2009 [http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html]. <br />
<br />
It uses three enzymes to ligate two or more sequences of DNA when they have overlapping end sequences at their joining point (~40bp). These overlapping regions can be easily added to the ends of any length of DNA by using PCR with primers which have added "flaps". Thus PCR followed by Gibson allows you to join any two blunt ended pieces of DNA.<br />
<br />
== Advantages ==<br />
1) No scar created - useful for '''fusion proteins''' and '''adding an RBS''', where scars can be problematic.<br />
<br />
2) Can re-ligate linear DNA into a circle - useful for '''site-directed mutagenesis'''<br />
<br />
3) Any two blunt ended pieces of DNA can be joined, and DNA can be taken from any source which is accessible to PCR (for example, an organism's '''genome''')<br />
<br />
4) Although it is roughly the same speed as [http://partsregistry.org/Help:BioBrick_Assembly standard BioBrick assembly] when ligating two fragments, Gibson is perfect for '''assembling multi part systems''', since it takes the same amount of time to ligate n pieces of DNA together as it does for two pieces.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr> <br />
<td><b>Assembly Method</b> <br />
</td><td><b>Number of steps to ligate N pieces of DNA</b> <br />
</td></tr> <br />
<tr> <br />
<td><a href="http://partsregistry.org/Assembly:Standard_assembly" class="external text" title="http://partsregistry.org/Assembly:Standard_assembly" rel="nofollow">Standard Assembly</a> <br />
</td><td><a href="/Image:Steps-N.png" class="image" title="Image:Steps-N.png"><img alt="Image:Steps-N.png" src="/wiki/images/c/cc/Steps-N.png" border="0" /></a> <br />
</td></tr> <br />
<tr> <br />
<td><a href="http://partsregistry.org/Assembly:Rolling_assembly" class="external text" title="http://partsregistry.org/Assembly:Rolling_assembly" rel="nofollow">Parallel Assembly</a> <br />
</td><td><a href="/Image:Steps-Log2.png" class="image" title="Image:Steps-Log2.png"><img alt="Image:Steps-Log2.png" src="/wiki/images/6/6f/Steps-Log2.png" width="138" height="21" border="0" /></a> (rounded up)<br />
</td></tr> <br />
<tr> <br />
<td><a href="https://2010.igem.org/Team:Cambridge/Gibson/Mechanism" class="external text" title="https://2010.igem.org/Team:Cambridge/Gibson/Mechanism" rel="nofollow">Gibson Assembly</a> <br />
</td><td><a href="/Image:Steps1.png" class="image" title="Image:Steps1.png"><img alt="Image:Steps1.png" src="/wiki/images/1/10/Steps1.png" width="83" height="18" border="0" /></a> <br />
</td></tr></table> <br />
</div><br />
</html><br />
<br />
== Disadvantages ==<br />
1) Greater degree of planning required as primers must be ordered in advance<br />
<br />
2) More expensive than BioBrick assembly<br />
<br />
3) PCR is tricky<br />
<br />
==Important:==<br />
<br />
The flexibility that this assembly method offers is a great thing. However, this does not mean it replaces the need for standardised prefixes and suffixes. The BioBrick prefix and suffix and the use of standard well-characterised vectors with standard primer sites are crucial for standardised iGEM parts. If you use Gibson Assembly it is important that you still add the prefix and suffix to your DNA; in fact sometimes Gibson Assembly is a useful way to do this (using as template an existing standard BioBrick).<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/MechanismTeam:Cambridge/Gibson/Mechanism2010-10-27T20:41:54Z<p>Willh: /* Creating overlapping DNA sequences */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Mechanism}}<br />
<br />
Gibson Assembly is a means to join overlapping DNA sequences, technically it does not describe the way in which these sequences are created. However since this will be of importance to iGEM teams, we will briefly discuss this.<br />
<br />
===Creating overlapping DNA sequences===<br />
Overlapping DNA sequences can be created by PCR. We can add twenty base-pairs to the end of a sequence by using a primer which runs as follows from 5' to 3'.<br />
<br />
[[Image:PCR.png|500px|center|template extension using primers]]<br />
<br />
If we wish to ligate two sequences called '''A''' and '''B''' (in that order) we need to ensure that there is an overlap of 40bp: i.e. the end of sequence A has the same 40 bp as the beginning of sequence B.<br />
<br />
We now perform two separate PCRs, one on A and one on B. The first adds the beginning 20 bp of B to sequence A by using a '''~40nt primer''' as detailed in the figure above (Note: the diagram shows both ends being extended -often necessary to perform multiple ligations at once). The second adds the final 20 bp of A to sequence B. The end of A and the beginning of B now are composed of the '''same 40bp sequence''' (end of A + beginning of B), and we are now ready to use Gibson Assembly. <br />
<br />
The Cambridge team have developed [http://www.gibthon.org/ Gibthon] to help you design primers for Gibson Assembly. The tool allows you to put in two sequences and choose 20bp of each to get a 40bp primer; it then analyses the melting temperature and secondary structure of this primer.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/a/a3/Cambridge-Gib1.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
== Gibson Assembly==<br />
Gibson Assembly master mix contains 3 enzymes:<br />
* T5 exonuclease<br />
* Phusion polymerase<br />
* Taq ligase<br />
<br />
The Gibson reaction relies on the action of the T5 exonuclease - this chews back at the 5' ends of both pieces of DNA<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/b5/Cambridge-Gib2.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/bf/Cambridge-Gib3.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Once it has chewed back far enough A-T G-C base pairing allows the two pieces to bind together.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/5/57/Cambridge-Gib5.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/1/1f/Cambridge-Gib6.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
We now have a single piece of DNA but it is not physically ligated together, it is merely held together by hydrogen bonding, also there are gaps in both single strands.<br />
<br />
Phusion is a DNA polymerase that repairs these gaps. It extends from the 3' end, so it does not interfere with T5 exonuclease which is acting at 5' ends.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/8f/Cambridge-Gib7.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Now we have DNA with no missing fragments but there is still a break in the phosphodiester bonds in the backbones of both single strands of DNA. This is corrected when Taq ligase action forms this bond.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/82/Cambridge-Gib8.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
And finally we have our finished piece of DNA. <br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/6/62/Cambridge-Gib9.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/MechanismTeam:Cambridge/Gibson/Mechanism2010-10-27T20:41:10Z<p>Willh: /* Creating overlapping DNA sequences */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Mechanism}}<br />
<br />
Gibson Assembly is a means to join overlapping DNA sequences, technically it does not describe the way in which these sequences are created. However since this will be of importance to iGEM teams, we will briefly discuss this.<br />
<br />
===Creating overlapping DNA sequences===<br />
Overlapping DNA sequences can be created by PCR. We can add twenty base-pairs to the end of a sequence by using a primer which runs as follows from 5' to 3'.<br />
<br />
[[Image:PCR.png|500px|center|template extension using primers]]<br />
<br />
If we wish to ligate two sequences called '''A''' and '''B''' (in that order) we need to ensure that there is an overlap of 40bp: i.e. the end of sequence A has the same 40 bp as the beginning of sequence B.<br />
<br />
We now perform two separate '''PCR'''s, one on A and one on B. The first adds the beginning 20 bp of B to sequence A by using a '''~40nt primer''' as detailed in the figure above (Note: the diagram shows both ends being extended -often necessary to perform multiple ligations at once). The second adds the final 20 bp of A to sequence B. The end of A and the beginning of B now are composed of the '''same 40bp sequence''' (end of A+beginning of B), and we are now ready to use Gibson Assembly. <br />
<br />
The Cambridge team have developed [http://www.gibthon.org/ Gibthon] to help you design primers for Gibson Assembly. The tool allows you to put in two sequences and choose 20bp of each to get a 40bp primer; it then analyses the melting temperature and secondary structure of this primer.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/a/a3/Cambridge-Gib1.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
== Gibson Assembly==<br />
Gibson Assembly master mix contains 3 enzymes:<br />
* T5 exonuclease<br />
* Phusion polymerase<br />
* Taq ligase<br />
<br />
The Gibson reaction relies on the action of the T5 exonuclease - this chews back at the 5' ends of both pieces of DNA<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/b5/Cambridge-Gib2.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/bf/Cambridge-Gib3.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Once it has chewed back far enough A-T G-C base pairing allows the two pieces to bind together.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/5/57/Cambridge-Gib5.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/1/1f/Cambridge-Gib6.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
We now have a single piece of DNA but it is not physically ligated together, it is merely held together by hydrogen bonding, also there are gaps in both single strands.<br />
<br />
Phusion is a DNA polymerase that repairs these gaps. It extends from the 3' end, so it does not interfere with T5 exonuclease which is acting at 5' ends.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/8f/Cambridge-Gib7.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Now we have DNA with no missing fragments but there is still a break in the phosphodiester bonds in the backbones of both single strands of DNA. This is corrected when Taq ligase action forms this bond.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/82/Cambridge-Gib8.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
And finally we have our finished piece of DNA. <br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/6/62/Cambridge-Gib9.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/MechanismTeam:Cambridge/Gibson/Mechanism2010-10-27T20:39:39Z<p>Willh: /* Creating overlapping DNA sequences */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Mechanism}}<br />
<br />
Gibson Assembly is a means to join overlapping DNA sequences, technically it does not describe the way in which these sequences are created. However since this will be of importance to iGEM teams, we will briefly discuss this.<br />
<br />
===Creating overlapping DNA sequences===<br />
Overlapping DNA sequences can be created by PCR. We can add twenty base-pairs to the end of a sequence by using a primer which runs as follows from 5' to 3'.<br />
<br />
[[Image:PCR.png|500px|center|template extension using primers]]<br />
<br />
If we wish to ligate two sequences called '''A''' and '''B''' (in that order) we need to ensure that there is an overlap of 40bp: i.e. the end of sequence A has the same 40 bp as the beginning of sequence B.<br />
<br />
We now perform two separate PCRs, one on A and one on B. The first adds the beginning 20 bp of B to sequence A by using a ~40nt primer as detailed in the figure above (note the diagram shows both ends being extended -often necessary to perform multiple ligations at once). The second adds the final 20 bp of A to sequence B. The end of A and the beginning of B now are composed of the same 40bp sequence (end of A+beginning of B), and we are now ready to use Gibson Assembly. <br />
<br />
The Cambridge team have developed [http://www.gibthon.org/ Gibthon] to help you design primers for Gibson Assembly. The tool allows you to put in two sequences and choose 20bp of each to get a 40bp primer; it then analyses the melting temperature and secondary structure of this primer.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/a/a3/Cambridge-Gib1.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
== Gibson Assembly==<br />
Gibson Assembly master mix contains 3 enzymes:<br />
* T5 exonuclease<br />
* Phusion polymerase<br />
* Taq ligase<br />
<br />
The Gibson reaction relies on the action of the T5 exonuclease - this chews back at the 5' ends of both pieces of DNA<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/b5/Cambridge-Gib2.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/bf/Cambridge-Gib3.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Once it has chewed back far enough A-T G-C base pairing allows the two pieces to bind together.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/5/57/Cambridge-Gib5.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/1/1f/Cambridge-Gib6.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
We now have a single piece of DNA but it is not physically ligated together, it is merely held together by hydrogen bonding, also there are gaps in both single strands.<br />
<br />
Phusion is a DNA polymerase that repairs these gaps. It extends from the 3' end, so it does not interfere with T5 exonuclease which is acting at 5' ends.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/8f/Cambridge-Gib7.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Now we have DNA with no missing fragments but there is still a break in the phosphodiester bonds in the backbones of both single strands of DNA. This is corrected when Taq ligase action forms this bond.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/82/Cambridge-Gib8.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
And finally we have our finished piece of DNA. <br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/6/62/Cambridge-Gib9.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/MechanismTeam:Cambridge/Gibson/Mechanism2010-10-27T20:33:39Z<p>Willh: /* Creating overlapping DNA sequences */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Mechanism}}<br />
<br />
Gibson Assembly is a means to join overlapping DNA sequences, technically it does not describe the way in which these sequences are created. However since this will be of importance to iGEM teams, we will briefly discuss this.<br />
<br />
===Creating overlapping DNA sequences===<br />
Overlapping DNA sequences can be created by PCR. We can add twenty base-pairs to the end of a sequence by using a primer which runs as follows from 5' to 3'.<br />
<br />
[[Image:PCR.png|500px|center|template extension using primers]]<br />
<br />
If we wish to ligate two sequences called A and B in that order, we need to ensure that there is an overlap of 40bp. I.e. the end of sequence A has the same 40 bp as the beginning of sequence B.<br />
<br />
By using two ~40nt oligonucleotides as primers, we can add the final 20 bp of sequence A to sequence B and the beginning 20 bp of sequence B to sequence A. The end of A and the beginning of B now are composed of the same 40bp sequence, and we are now ready to use Gibson Assembly. <br />
<br />
The Cambridge team have developed [http://www.gibthon.org/ Gibthon] to help you design primers for Gibson Assembly. The tool allows you to put in two sequences and choose 20bp of each to get a 40bp primer; it then analyses the melting temperature and secondary structure of this primer.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/a/a3/Cambridge-Gib1.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
== Gibson Assembly==<br />
Gibson Assembly master mix contains 3 enzymes:<br />
* T5 exonuclease<br />
* Phusion polymerase<br />
* Taq ligase<br />
<br />
The Gibson reaction relies on the action of the T5 exonuclease - this chews back at the 5' ends of both pieces of DNA<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/b5/Cambridge-Gib2.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/bf/Cambridge-Gib3.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Once it has chewed back far enough A-T G-C base pairing allows the two pieces to bind together.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/5/57/Cambridge-Gib5.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/1/1f/Cambridge-Gib6.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
We now have a single piece of DNA but it is not physically ligated together, it is merely held together by hydrogen bonding, also there are gaps in both single strands.<br />
<br />
Phusion is a DNA polymerase that repairs these gaps. It extends from the 3' end, so it does not interfere with T5 exonuclease which is acting at 5' ends.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/8f/Cambridge-Gib7.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Now we have DNA with no missing fragments but there is still a break in the phosphodiester bonds in the backbones of both single strands of DNA. This is corrected when Taq ligase action forms this bond.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/82/Cambridge-Gib8.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
And finally we have our finished piece of DNA. <br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/6/62/Cambridge-Gib9.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/MechanismTeam:Cambridge/Gibson/Mechanism2010-10-27T20:30:31Z<p>Willh: /* Creating overlapping DNA sequences */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Mechanism}}<br />
<br />
Gibson Assembly is a means to join overlapping DNA sequences, technically it does not describe the way in which these sequences are created. However since this will be of importance to iGEM teams, we will briefly discuss this.<br />
<br />
===Creating overlapping DNA sequences===<br />
Overlapping DNA sequences can be created by PCR. We can add twenty base-pairs to the end of a sequence by using a primer which runs as follows from 5' to 3'.<br />
<br />
[[Image:PCR.png|500px|center|template extension using primers]]<br />
<br />
By using two ~40nt oligonucleotides as primers, we can add the final 20 bp of sequence A to sequence B and the beginning 20 bp of sequence B to sequence A. We are then ready to use Gibson Assembly. <br />
<br />
The end of sequence A with the extension now looks like:<br />
<br />
.....endA+begB<br />
<br />
The beginning of sequence B looks like:<br />
<br />
endA+begB.......<br />
<br />
This results in there now being a total overlap of 40bp between the two fragments, enough to perform Gibson assembly.<br />
<br />
The Cambridge team have developed [http://www.gibthon.org/ Gibthon] to help you design primers for Gibson Assembly. The tool allows you to put in two sequences and choose 20bp of each to get a 40bp primer; it then analyses the melting temperature and secondary structure of this primer.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/a/a3/Cambridge-Gib1.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
== Gibson Assembly==<br />
Gibson Assembly master mix contains 3 enzymes:<br />
* T5 exonuclease<br />
* Phusion polymerase<br />
* Taq ligase<br />
<br />
The Gibson reaction relies on the action of the T5 exonuclease - this chews back at the 5' ends of both pieces of DNA<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/b5/Cambridge-Gib2.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/bf/Cambridge-Gib3.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Once it has chewed back far enough A-T G-C base pairing allows the two pieces to bind together.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/5/57/Cambridge-Gib5.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/1/1f/Cambridge-Gib6.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
We now have a single piece of DNA but it is not physically ligated together, it is merely held together by hydrogen bonding, also there are gaps in both single strands.<br />
<br />
Phusion is a DNA polymerase that repairs these gaps. It extends from the 3' end, so it does not interfere with T5 exonuclease which is acting at 5' ends.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/8f/Cambridge-Gib7.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Now we have DNA with no missing fragments but there is still a break in the phosphodiester bonds in the backbones of both single strands of DNA. This is corrected when Taq ligase action forms this bond.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/82/Cambridge-Gib8.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
And finally we have our finished piece of DNA. <br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/6/62/Cambridge-Gib9.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/MechanismTeam:Cambridge/Gibson/Mechanism2010-10-27T20:29:12Z<p>Willh: /* Creating overlapping DNA sequences */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Mechanism}}<br />
<br />
Gibson Assembly is a means to join overlapping DNA sequences, technically it does not describe the way in which these sequences are created. However since this will be of importance to iGEM teams, we will briefly discuss this.<br />
<br />
===Creating overlapping DNA sequences===<br />
Overlapping DNA sequences can be created by PCR. We can add twenty base-pairs to the end of a sequence by using a primer which runs as follows from 5' to 3'.<br />
<br />
[[Image:PCR.png|500px|center|template extension using primers]]<br />
<br />
By using two ~40nt oligonucleotides as primers, we can add 20 bp of sequence A to sequence B and 20 bp of sequence B to sequence A. We are then ready to use Gibson Assembly. The Cambridge team have developed [http://www.gibthon.org/ Gibthon] to help you design primers for Gibson Assembly. The tool allows you to put in two sequences and choose 20bp of each to get a 40bp primer; it then analyses the melting temperature and secondary structure of this primer.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/a/a3/Cambridge-Gib1.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Note:<br />
<br />
The end of sequence A with the extension now looks like:<br />
<br />
.....endA+begB<br />
<br />
The beginning of sequence B looks like:<br />
<br />
endA+begB.......<br />
<br />
This results in there now being a total overlap of 40bp between the two fragments, enough to perform Gibson assembly.<br />
<br />
== Gibson Assembly==<br />
Gibson Assembly master mix contains 3 enzymes:<br />
* T5 exonuclease<br />
* Phusion polymerase<br />
* Taq ligase<br />
<br />
The Gibson reaction relies on the action of the T5 exonuclease - this chews back at the 5' ends of both pieces of DNA<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/b5/Cambridge-Gib2.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/bf/Cambridge-Gib3.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Once it has chewed back far enough A-T G-C base pairing allows the two pieces to bind together.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/5/57/Cambridge-Gib5.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/1/1f/Cambridge-Gib6.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
We now have a single piece of DNA but it is not physically ligated together, it is merely held together by hydrogen bonding, also there are gaps in both single strands.<br />
<br />
Phusion is a DNA polymerase that repairs these gaps. It extends from the 3' end, so it does not interfere with T5 exonuclease which is acting at 5' ends.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/8f/Cambridge-Gib7.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Now we have DNA with no missing fragments but there is still a break in the phosphodiester bonds in the backbones of both single strands of DNA. This is corrected when Taq ligase action forms this bond.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/82/Cambridge-Gib8.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
And finally we have our finished piece of DNA. <br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/6/62/Cambridge-Gib9.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/MechanismTeam:Cambridge/Gibson/Mechanism2010-10-27T20:28:37Z<p>Willh: /* Creating overlapping DNA sequences */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Mechanism}}<br />
<br />
Gibson Assembly is a means to join overlapping DNA sequences, technically it does not describe the way in which these sequences are created. However since this will be of importance to iGEM teams, we will briefly discuss this.<br />
<br />
===Creating overlapping DNA sequences===<br />
Overlapping DNA sequences can be created by PCR. We can add twenty base-pairs to the end of a sequence by using a primer which runs as follows from 5' to 3'.<br />
<br />
[[Image:PCR.png|500px|center|template extension using primers]]<br />
<br />
By using two ~40nt oligonucleotides as primers, we can add 20 bp of sequence A to sequence B and 20 bp of sequence B to sequence A. We are then ready to use Gibson Assembly. The Cambridge team have developed [http://www.gibthon.org/ Gibthon] to help you design primers for Gibson Assembly. The tool allows you to put in two sequences and choose 20bp of each to get a 40bp primer; it then analyses the melting temperature and secondary structure of this primer.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/a/a3/Cambridge-Gib1.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Note:<br />
*The end of sequence A with the extension now looks like <br />
<br />
.....endA+begB<br />
<br />
*The beginning of sequence B looks like <br />
<br />
endA+begB.......<br />
<br />
*This results in there now being a total overlap of 40bp between the two fragments, enough to perform Gibson assembly.<br />
<br />
== Gibson Assembly==<br />
Gibson Assembly master mix contains 3 enzymes:<br />
* T5 exonuclease<br />
* Phusion polymerase<br />
* Taq ligase<br />
<br />
The Gibson reaction relies on the action of the T5 exonuclease - this chews back at the 5' ends of both pieces of DNA<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/b5/Cambridge-Gib2.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/b/bf/Cambridge-Gib3.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Once it has chewed back far enough A-T G-C base pairing allows the two pieces to bind together.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/5/57/Cambridge-Gib5.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/1/1f/Cambridge-Gib6.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
We now have a single piece of DNA but it is not physically ligated together, it is merely held together by hydrogen bonding, also there are gaps in both single strands.<br />
<br />
Phusion is a DNA polymerase that repairs these gaps. It extends from the 3' end, so it does not interfere with T5 exonuclease which is acting at 5' ends.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/8f/Cambridge-Gib7.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
Now we have DNA with no missing fragments but there is still a break in the phosphodiester bonds in the backbones of both single strands of DNA. This is corrected when Taq ligase action forms this bond.<br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/8/82/Cambridge-Gib8.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
And finally we have our finished piece of DNA. <br />
<br />
<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/6/62/Cambridge-Gib9.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html><br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:CambridgeTeam:Cambridge2010-10-27T18:12:47Z<p>Willh: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/boxesprototypenew}}<br />
<br />
<p>&nbsp;</p><br />
<br />
{{:Team:Cambridge/Templates/headerbar|colour=#386abc|title=The Project}}<br />
<html><img src="https://static.igem.org/mediawiki/2010/4/40/Cambridge-low.jpg" style="margin-left:4px; margin-bottom:5px; width:710px; margin-top:-2px;"><br />
<div style="height:30px; position:relative; font-size:12px; color:#6e6e6e; line-height:15px;"><br />
<div style="position:absolute; width:230px; left:5px; text-align:center;">A flask lit by E. coli transformed with one of our constructs </div><br />
<div style="position:absolute; width:230px; left:240px; text-align:center;">E. coli glowing with different coloured bioluminescent systems</div><br />
<div style="position:absolute; width:220px; left:485px; text-align:center;">Team members illuminated only by our bacteria</div><br />
</div><br />
</html><br />
<br />
{{:Team:Cambridge/Templates/Nolineheader2|header=Project Firefly}}<br />
Over the course of the summer we have built a number of BioBricks to allow <strong>bioluminescence</strong>. <br />
<br />
We adopted a number of strategies to extend the use of '''firefly luciferase''':<br />
* [https://2010.igem.org/Team:Cambridge/Codons '''Codon optimisation'''] for increased light output<br />
* Use of a [https://2010.igem.org/Team:Cambridge/Bioluminescence/Luciferin_Regeneration '''luciferin regenerating enzyme'''].<br />
* Mutagenesis to create a number of [https://2010.igem.org/Team:Cambridge/Bioluminescence/Colour '''different colours''']<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader2|header=Project Vibrio<br />
}}<br />
We complemented these firefly systems, which require the addition of the substrate luciferin, with light producing systems from '''Vibrio fischeri'''. We believe we have created the [https://2010.igem.org/Team:Cambridge/Bioluminescence/G28 '''first BioBrick'''] to emit light in normal E. coli strains without the addition of any external substrate.<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader2|header=Tools}}<br />
<br />
Over the summer we made extensive use of [https://2010.igem.org/Team:Cambridge/Gibson/Introduction '''Gibson Assembly'''] to manufacture our parts, and have submitted an [https://2010.igem.org/Team:Cambridge/Gibson/RFC '''RFC'''] to the [http://bbf.openwetware.org/ '''BioBricks Foundation'''] to help future teams make best use of this technique.<br />
<br />
Along with this, we also constructed a number of tools to assist the synthetic biologists of the future:<br />
* [https://2010.igem.org/Team:Cambridge/Tools/Gibson '''Gibthon Construct Designer'''] allows the user to enter a series of BioBrick or GenBank IDs in a specific order and computes the appropriate primers for [https://2010.igem.org/Team:Cambridge/Gibson/Introduction '''Gibson Assembly'''].<br />
* [https://2010.igem.org/Team:Cambridge/Tools/GenBank '''BioBrick → GenBank'''] allows parts from the registry to be downloaded in .gb format, making them compatible with a wide range of biological software.<br />
* The [https://2010.igem.org/Team:Cambridge/Tools/Ligate '''Ligation Calculator'''] is a small calculator to help you work out the proportions to use for ligation in BioBrick assembly without having to worry about units.<br />
* The [https://2010.igem.org/Team:Cambridge/Tools/Eglometer '''E.glometer'''] is a cheap, easily built, piece of electronics for measuring bioluminescence. It allows scientists without access to expensive plate readers to measure bacterial light output and has potential applications in [https://2010.igem.org/Team:Cambridge/Tools/microMeasure '''quantitative biosensors'''].<br />
<br />
<br />
<br />
[[Image:Cambridge_team_pictwo2010.jpg|center|frame|The team - in order - Anja Hohmann, Emily Knott, Hannah Copley, Will Handley, Theo Sanderson, Ben Reeve, Paul Masset, Peter Emmrich, Bill Collins]]<br />
<html></div></html>{{:Team:Cambridge/Templates/footerMinimal}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-27T16:35:09Z<p>Willh: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<br />
<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 2: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 2 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 3: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 4: The emission spectrum of V. Fischeri (shown in black)''}}<br />
<br />
We wrote a program ([[Team:Cambridge/luminanceSourceCode | source code]]) in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 4 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 2) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy which the tree absorbs in photosynthesis must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-27T15:54:39Z<p>Willh: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<br />
<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
<br />
We wrote a program ([[Team:Cambridge/luminanceSourceCode | source code]]) in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy which the tree absorbs in photosynthesis must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-27T15:51:18Z<p>Willh: /* Radiation out */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<br />
<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
<br />
We wrote a program ([[Team:Cambridge/luminanceSourceCode | source code]]) in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-27T15:50:05Z<p>Willh: /* Radiation out */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<br />
<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
<br />
We wrote a program ([[Team:Cambridge/luminanceSourceCode | source code]]) in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-27T15:49:41Z<p>Willh: /* Radiation out */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<br />
<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
We wrote a program ([[Team:Cambridge/luminanceSourceCode | source code]]) in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-27T15:48:51Z<p>Willh: /* Radiation out */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<br />
<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
We wrote a program ([[Team:Cambridge/luminanceSourceCode | source code]]) in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-27T15:46:55Z<p>Willh: /* Radiation out */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<br />
<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
We wrote a program ([[Team:Cambridge/luminanceSourceCode | source code]]) in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-27T15:46:09Z<p>Willh: /* Radiation out */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<br />
<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
We wrote a program ([[Team:Cambridge/luminanceSourceCode | source code]]) in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-27T15:45:00Z<p>Willh: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<br />
<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
We wrote a program ([[Team:Cambridge/luminanceSourceCode | source code]]) in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-26T20:40:09Z<p>Willh: /* Radiation out */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<html><br />
<div style="position:relative; height:500px;"><br />
<div style="position:absolute; left:0px; width:300px; top:0px; padding-bottom:50px;"> <br />
<a href="/Image:Solar_Spectrum.png" class="image" title="Solar Spectrum.png"><img alt="" src="/wiki/images/thumb/4/4c/Solar_Spectrum.png/300px-Solar_Spectrum.png" width="300" height="223" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; width:300px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.</i></div> <br />
</div> <br />
<br />
<div style="position:absolute; left:310px; width:300px; top:0px; padding-bottom:50px;"> <br />
<a href="/Image:Fischerispectrum.jpg" class="image" title="Fischerispectrum.jpg"><img alt="" src="/wiki/images/thumb/b/b3/Fischerispectrum.jpg/300px-Fischerispectrum.jpg" width="300" height="234" border="0" /></a> <br />
<br /> <br />
<br />
<div style="margin-top:5px; width:300px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 2: The emission spectrum of V. Fischeri (shown in black)</i></div> <br />
</div> <br />
<br />
<div style="position:absolute; top:280px; width:300px; padding-bottom:50px;"> <br />
<a href="/Image:Photopicscotopic.png" class="image" title="Photopicscotopic.png"><img alt="" src="/wiki/images/thumb/8/89/Photopicscotopic.png/300px-Photopicscotopic.png" width="300" height="205" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 3: Photopic (black) and scotopic (green) luminosity functions.</i></div> <br />
</div> <br />
<br />
<p><div style="float:right; padding-left:30px; width:300px; padding-bottom:50px;"> <br />
<a href="/Image:Luminosityfunction.png" class="image" title="Luminosityfunction.png"><img alt="" src="/wiki/images/thumb/3/3b/Luminosityfunction.png/300px-Luminosityfunction.png" width="300" height="44" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength</i></div> <br />
</div> <br />
</div><br />
</html><br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
We wrote a program ([[Team:Cambridge/luminanceSourceCode | source code]]) in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-26T20:39:01Z<p>Willh: /* Radiation out */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<html><br />
<div style="position:relative; height:500px;"><br />
<div style="position:absolute; left:0px; width:300px; top:0px; padding-bottom:50px;"> <br />
<a href="/Image:Solar_Spectrum.png" class="image" title="Solar Spectrum.png"><img alt="" src="/wiki/images/thumb/4/4c/Solar_Spectrum.png/300px-Solar_Spectrum.png" width="300" height="223" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; width:300px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.</i></div> <br />
</div> <br />
<br />
<div style="position:absolute; left:310px; width:300px; top:0px; padding-bottom:50px;"> <br />
<a href="/Image:Fischerispectrum.jpg" class="image" title="Fischerispectrum.jpg"><img alt="" src="/wiki/images/thumb/b/b3/Fischerispectrum.jpg/300px-Fischerispectrum.jpg" width="300" height="234" border="0" /></a> <br />
<br /> <br />
<br />
<div style="margin-top:5px; width:300px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 2: The emission spectrum of V. Fischeri (shown in black)</i></div> <br />
</div> <br />
<br />
<div style="position:absolute; top:280px; width:300px; padding-bottom:50px;"> <br />
<a href="/Image:Photopicscotopic.png" class="image" title="Photopicscotopic.png"><img alt="" src="/wiki/images/thumb/8/89/Photopicscotopic.png/300px-Photopicscotopic.png" width="300" height="205" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 3: Photopic (black) and scotopic (green) luminosity functions.</i></div> <br />
</div> <br />
<br />
<p><div style="float:right; padding-left:30px; width:300px; padding-bottom:50px;"> <br />
<a href="/Image:Luminosityfunction.png" class="image" title="Luminosityfunction.png"><img alt="" src="/wiki/images/thumb/3/3b/Luminosityfunction.png/300px-Luminosityfunction.png" width="300" height="44" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength</i></div> <br />
</div> <br />
</div><br />
</html><br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
We wrote a [[Team:Cambridge/luminanceSourceCode | program]] in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:31:41Z<p>Willh: /* Step 3: PCR */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer:<br />
<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="left"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="left"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:31:20Z<p>Willh: /* Step 4: Gibson Assembly */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="left"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="left"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:31:06Z<p>Willh: /* Step 4: Gibson Assembly */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="left"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="left"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:30:42Z<p>Willh: /* Step 3: PCR */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="left"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:30:27Z<p>Willh: /* Step 3: PCR */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="left"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<br />
<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:30:05Z<p>Willh: /* Step 3: PCR */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="left"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<br />
<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:29:32Z<p>Willh: /* Step 3: PCR */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="left"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
<br />
<br />
<br />
<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:28:38Z<p>Willh: /* Step 3: PCR */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
<br />
<br />
<br />
<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:28:14Z<p>Willh: /* Step 3: PCR */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:27:35Z<p>Willh: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
<div style="float:center; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:26:23Z<p>Willh: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:25:36Z<p>Willh: /* Step 1: Design Primers */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:25:22Z<p>Willh: /* Step 1: Design Primers */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]. We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T20:24:35Z<p>Willh: /* Step 1: Design Primers */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The[[Team:Cambridge/Gibson/Mechanism | standard]] way to do this is with PCR with specialised primers. We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-26T20:08:45Z<p>Willh: /* Bringing it all together */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<html><br />
<div style="position:relative; height:500px;"><br />
<div style="position:absolute; left:0px; width:300px; top:0px; padding-bottom:50px;"> <br />
<a href="/Image:Solar_Spectrum.png" class="image" title="Solar Spectrum.png"><img alt="" src="/wiki/images/thumb/4/4c/Solar_Spectrum.png/300px-Solar_Spectrum.png" width="300" height="223" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; width:300px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.</i></div> <br />
</div> <br />
<br />
<div style="position:absolute; left:310px; width:300px; top:0px; padding-bottom:50px;"> <br />
<a href="/Image:Fischerispectrum.jpg" class="image" title="Fischerispectrum.jpg"><img alt="" src="/wiki/images/thumb/b/b3/Fischerispectrum.jpg/300px-Fischerispectrum.jpg" width="300" height="234" border="0" /></a> <br />
<br /> <br />
<br />
<div style="margin-top:5px; width:300px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 2: The emission spectrum of V. Fischeri (shown in black)</i></div> <br />
</div> <br />
<br />
<div style="position:absolute; top:280px; width:300px; padding-bottom:50px;"> <br />
<a href="/Image:Photopicscotopic.png" class="image" title="Photopicscotopic.png"><img alt="" src="/wiki/images/thumb/8/89/Photopicscotopic.png/300px-Photopicscotopic.png" width="300" height="205" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 3: Photopic (black) and scotopic (green) luminosity functions.</i></div> <br />
</div> <br />
<br />
<p><div style="float:right; padding-left:30px; width:300px; padding-bottom:50px;"> <br />
<a href="/Image:Luminosityfunction.png" class="image" title="Luminosityfunction.png"><img alt="" src="/wiki/images/thumb/3/3b/Luminosityfunction.png/300px-Luminosityfunction.png" width="300" height="44" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength</i></div> <br />
</div> <br />
</div><br />
</html><br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Wattage</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>25-150<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>18-95<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>50-400<br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>50-400 <br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>18-180<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
We wrote a [[Team:Cambridge/luminanceSourceCode | program]] in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
'''Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)'''<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-26T20:08:28Z<p>Willh: /* Bringing it all together */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
<html><br />
<div style="position:relative; height:500px;"><br />
<div style="position:absolute; left:0px; width:300px; top:0px; padding-bottom:50px;"> <br />
<a href="/Image:Solar_Spectrum.png" class="image" title="Solar Spectrum.png"><img alt="" src="/wiki/images/thumb/4/4c/Solar_Spectrum.png/300px-Solar_Spectrum.png" width="300" height="223" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; width:300px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.</i></div> <br />
</div> <br />
<br />
<div style="position:absolute; left:310px; width:300px; top:0px; padding-bottom:50px;"> <br />
<a href="/Image:Fischerispectrum.jpg" class="image" title="Fischerispectrum.jpg"><img alt="" src="/wiki/images/thumb/b/b3/Fischerispectrum.jpg/300px-Fischerispectrum.jpg" width="300" height="234" border="0" /></a> <br />
<br /> <br />
<br />
<div style="margin-top:5px; width:300px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 2: The emission spectrum of V. Fischeri (shown in black)</i></div> <br />
</div> <br />
<br />
<div style="position:absolute; top:280px; width:300px; padding-bottom:50px;"> <br />
<a href="/Image:Photopicscotopic.png" class="image" title="Photopicscotopic.png"><img alt="" src="/wiki/images/thumb/8/89/Photopicscotopic.png/300px-Photopicscotopic.png" width="300" height="205" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 3: Photopic (black) and scotopic (green) luminosity functions.</i></div> <br />
</div> <br />
<br />
<p><div style="float:right; padding-left:30px; width:300px; padding-bottom:50px;"> <br />
<a href="/Image:Luminosityfunction.png" class="image" title="Luminosityfunction.png"><img alt="" src="/wiki/images/thumb/3/3b/Luminosityfunction.png/300px-Luminosityfunction.png" width="300" height="44" border="0" /></a> <br />
<br /> <br />
</p> <br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center"><i>Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength</i></div> <br />
</div> <br />
</div><br />
</html><br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Wattage</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>25-150<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>18-95<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>50-400<br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>50-400 <br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>18-180<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
We wrote a [[Team:Cambridge/luminanceSourceCode | program]] in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp ('''X=210''') and hypothesise a projected area of '''A=30m<sup>2</sup>''', and a '''day:night ratio 14:10''' then we find that the efficiency must be roughly '''0.02%'''. This means that 0.02% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/luminanceSourceCodeTeam:Cambridge/luminanceSourceCode2010-10-26T20:05:58Z<p>Willh: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Source Code for Luminance calculator}}<br />
<br />
This program calculates the conversion factor between your total radiant output intensity and your luminance output intensity.<br />
<br />
To use this program:<br />
<br />
*create a new folder<br />
*save the source code below into a file called LuminanceCalculator.cc<br />
*save the data under scotopic into a file called scotopic.dat<br />
*save the data under photopic into a file called photopic.dat<br />
*create your own spectra file by inputting the (x<sub>i</sub>,f(x<sub>i</sub>)) coordinates of the spectral distribution file, no spaces just carriage returns i.e. in the format:<br />
<br />
x<sub>1</sub><br />
<br />
f(x<sub>1</sub>)<br />
<br />
x<sub>2</sub><br />
<br />
f(x<sub>2</sub>)<br />
<br />
...<br />
<br />
x<sub>2</sub><br />
<br />
f(x<sub>2</sub>)<br />
<br />
*10 values is usually enough, but more would be better. x should be in nanometre (nm) f(x) should be in SI<br />
*save the above as spectra.dat<br />
*open a terminal<br />
*navigate to the folder which all of the above are in<br />
*compile using the command: g++ LuminanceCalculator.cc -o LuminanceCalculator.cc<br />
*run the program<br />
<br />
=Source Code=<br />
#include <iostream><br />
#include <vector><br />
#include <fstream><br />
#include <math.h><br />
<br />
#ifndef __mjdmatrix_h<br />
#define __mjdmatrix_h<br />
#include <iostream><br />
using namespace std;<br />
// generic object (class) definition of matrix:<br />
template <class D> class matrix{<br />
<br />
<br />
// NOTE: maxsize determines available memory storage, but<br />
// actualsize determines the actual size of the stored matrix in use<br />
// at a particular time.<br />
int maxsize; // max number of rows (same as max number of columns)<br />
int actualsize; // actual size (rows, or columns) of the stored matrix<br />
D* data; // where the data contents of the matrix are stored<br />
void allocate() {<br />
delete[] data;<br />
data = new D [maxsize*maxsize];<br />
};<br />
matrix() {}; // private ctor's<br />
matrix(int newmaxsize) {matrix(newmaxsize,newmaxsize);};<br />
<br />
<br />
public: <br />
<br />
void print()<br />
{<br />
for(int i=0; i<maxsize; i++){<br />
for(int j=0; j<maxsize; j++){<br />
<br />
cout << getvalue(i,j) << " ";<br />
}<br />
cout << endl;<br />
}<br />
<br />
};<br />
<br />
<br />
D getvalue(int row, int column){return data[ row * maxsize + column ];};<br />
matrix(int newmaxsize, int newactualsize) { // the only public ctor<br />
if (newmaxsize <= 0) newmaxsize = 5;<br />
maxsize = newmaxsize; <br />
if ((newactualsize <= newmaxsize)&&(newactualsize>0))<br />
actualsize = newactualsize;<br />
else <br />
actualsize = newmaxsize;<br />
// since allocate() will first call delete[] on data:<br />
data = 0;<br />
allocate();<br />
};<br />
~matrix() { delete[] data; };<br />
<br />
<br />
void comparetoidentity() {<br />
int worstdiagonal = 0;<br />
D maxunitydeviation = 0.0;<br />
D currentunitydeviation;<br />
for ( int i = 0; i < actualsize; i++ ) {<br />
currentunitydeviation = data[i*maxsize+i] - 1.;<br />
if ( currentunitydeviation < 0.0) currentunitydeviation *= -1.;<br />
if ( currentunitydeviation > maxunitydeviation ) {<br />
maxunitydeviation = currentunitydeviation;<br />
worstdiagonal = i;<br />
}<br />
}<br />
int worstoffdiagonalrow = 0;<br />
int worstoffdiagonalcolumn = 0;<br />
D maxzerodeviation = 0.0;<br />
D currentzerodeviation ;<br />
for ( int i = 0; i < actualsize; i++ ) {<br />
for ( int j = 0; j < actualsize; j++ ) {<br />
if ( i == j ) continue; // we look only at non-diagonal terms<br />
currentzerodeviation = data[i*maxsize+j];<br />
if ( currentzerodeviation < 0.0) currentzerodeviation *= -1.0;<br />
if ( currentzerodeviation > maxzerodeviation ) {<br />
maxzerodeviation = currentzerodeviation;<br />
worstoffdiagonalrow = i;<br />
worstoffdiagonalcolumn = j;<br />
}<br />
<br />
}<br />
}<br />
cout << "Worst diagonal value deviation from unity: " <br />
<< maxunitydeviation << " at row/column " << worstdiagonal << endl;<br />
cout << "Worst off-diagonal value deviation from zero: " <br />
<< maxzerodeviation << " at row = " << worstoffdiagonalrow <br />
<< ", column = " << worstoffdiagonalcolumn << endl;<br />
}<br />
<br />
<br />
void settoproduct(matrix& left, matrix& right) {<br />
actualsize = left.getactualsize();<br />
if ( maxsize < left.getactualsize() ) {<br />
maxsize = left.getactualsize();<br />
allocate();<br />
}<br />
for ( int i = 0; i < actualsize; i++ )<br />
for ( int j = 0; j < actualsize; j++ ) {<br />
D sum = 0.0;<br />
D leftvalue, rightvalue;<br />
bool success;<br />
for (int c = 0; c < actualsize; c++) {<br />
left.getvalue(i,c,leftvalue,success);<br />
right.getvalue(c,j,rightvalue,success);<br />
sum += leftvalue * rightvalue;<br />
}<br />
setvalue(i,j,sum);<br />
}<br />
}<br />
<br />
<br />
void copymatrix(matrix& source) {<br />
actualsize = source.getactualsize();<br />
if ( maxsize < source.getactualsize() ) {<br />
maxsize = source.getactualsize();<br />
allocate();<br />
}<br />
for ( int i = 0; i < actualsize; i++ )<br />
for ( int j = 0; j < actualsize; j++ ) {<br />
D value;<br />
bool success;<br />
source.getvalue(i,j,value,success);<br />
data[i*maxsize+j] = value;<br />
}<br />
};<br />
<br />
<br />
<br />
void setactualsize(int newactualsize) {<br />
if ( newactualsize > maxsize )<br />
{<br />
maxsize = newactualsize ; // * 2; // wastes memory but saves<br />
// time otherwise required for<br />
// operation new[]<br />
allocate();<br />
}<br />
if (newactualsize >= 0) actualsize = newactualsize;<br />
};<br />
<br />
<br />
<br />
int getactualsize() { return actualsize; };<br />
<br />
<br />
void getvalue(int row, int column, D& returnvalue, bool& success) {<br />
if ( (row>=maxsize) || (column>=maxsize) <br />
|| (row<0) || (column<0) )<br />
{ success = false;<br />
return; }<br />
returnvalue = data[ row * maxsize + column ];<br />
success = true;<br />
};<br />
<br />
<br />
bool setvalue(int row, int column, D newvalue) {<br />
if ( (row >= maxsize) || (column >= maxsize) <br />
|| (row<0) || (column<0) ) return false;<br />
data[ row * maxsize + column ] = newvalue;<br />
return true;<br />
};<br />
<br />
<br />
void invert() {<br />
if (actualsize <= 0) return; // sanity check<br />
if (actualsize == 1) return; // must be of dimension >= 2<br />
for (int i=1; i < actualsize; i++) data[i] /= data[0]; // normalize row 0<br />
for (int i=1; i < actualsize; i++) { <br />
for (int j=i; j < actualsize; j++) { // do a column of L<br />
D sum = 0.0;<br />
for (int k = 0; k < i; k++) <br />
sum += data[j*maxsize+k] * data[k*maxsize+i];<br />
data[j*maxsize+i] -= sum;<br />
}<br />
if (i == actualsize-1) continue;<br />
for (int j=i+1; j < actualsize; j++) { // do a row of U<br />
D sum = 0.0;<br />
for (int k = 0; k < i; k++)<br />
sum += data[i*maxsize+k]*data[k*maxsize+j];<br />
data[i*maxsize+j] = <br />
(data[i*maxsize+j]-sum) / data[i*maxsize+i];<br />
}<br />
}<br />
for ( int i = 0; i < actualsize; i++ ) // invert L<br />
for ( int j = i; j < actualsize; j++ ) {<br />
D x = 1.0;<br />
if ( i != j ) {<br />
x = 0.0;<br />
for ( int k = i; k < j; k++ ) <br />
x -= data[j*maxsize+k]*data[k*maxsize+i];<br />
}<br />
data[j*maxsize+i] = x / data[j*maxsize+j];<br />
}<br />
for ( int i = 0; i < actualsize; i++ ) // invert U<br />
for ( int j = i; j < actualsize; j++ ) {<br />
if ( i == j ) continue;<br />
D sum = 0.0;<br />
for ( int k = i; k < j; k++ )<br />
sum += data[k*maxsize+j]*( (i==k) ? 1.0 : data[i*maxsize+k] );<br />
data[i*maxsize+j] = -sum;<br />
}<br />
for ( int i = 0; i < actualsize; i++ ) // final inversion<br />
for ( int j = 0; j < actualsize; j++ ) {<br />
D sum = 0.0;<br />
for ( int k = ((i>j)?i:j); k < actualsize; k++ ) <br />
sum += ((j==k)?1.0:data[j*maxsize+k])*data[k*maxsize+i];<br />
data[j*maxsize+i] = sum;<br />
}<br />
};<br />
};<br />
#endif<br />
<br />
using namespace std;<br />
<br />
class Function<br />
{<br />
private:<br />
vector <double> wavelength;<br />
vector <double> function;<br />
vector <double> lambda;<br />
<br />
double RBF(double, double);<br />
double sigma;<br />
double width;<br />
int SIZE;<br />
<br />
public:<br />
void getData(const char*);<br />
void findLambda(double);<br />
void fix();<br />
<br />
double size(){return wavelength.size();}<br />
double lat(int i){return lambda.at(i);}<br />
double wat(int i){return wavelength.at(i);}<br />
double fat(int i){return function.at(i);}<br />
void wequals(int i, double value){wavelength.at(i)=value;}<br />
void fequals(int i, double value){function.at(i)=value;}<br />
<br />
void eraseBeginning(int);<br />
void erase(int);<br />
<br />
void print();<br />
void print(double,double,double);<br />
void printStore();<br />
<br />
double value(double);<br />
<br />
<br />
<br />
};<br />
<br />
double X(double j, double a, double h) {return a+j*h;}<br />
double H(double a,double b,double n) {return (b-a)/n;}<br />
<br />
double integral (double a, double b, Function);<br />
<br />
void runProgram(char*,char*);<br />
<br />
double luminousFlux(Function, Function);<br />
<br />
<br />
<br />
<br />
#define MINI 400<br />
#define MAXI 750<br />
<br />
#define CONSTANT 0.7<br />
#define N 10000<br />
<br />
#ifndef SPECTRA<br />
#define SPECTRA "spectra.dat"<br />
#endif<br />
<br />
<br />
int main()<br />
{<br />
char b;<br />
cout << "This program will tell you the conversion factor between Radiance and Luminance for your chosen object with known radiation spectrum" << endl;<br />
cout << endl << "Do you want scotopic (low light) [s] or photopic (normal light) [p] Luminance?" << endl;<br />
while(b!='p'&&b!='s'){cout << "please input s or p" << endl; cin >> b;}<br />
cout << endl;<br />
<br />
if(b=='s')<br />
runProgram("scotopic.dat",SPECTRA);<br />
if(b=='p')<br />
runProgram("photopic.dat",SPECTRA);<br />
<br />
return 0;<br />
}<br />
<br />
<br />
void runProgram(char* lumFile, char* specFile)<br />
{<br />
Function luminosity, spectra;<br />
<br />
cout << "Getting luminosity data..." << endl;<br />
luminosity.getData(lumFile);<br />
cout << "Calculating luminosity function..." << endl;<br />
luminosity.findLambda(CONSTANT);<br />
<br />
cout << "Getting spectral data..." << endl;<br />
spectra.getData(specFile);<br />
cout << "Calculating spectral function..." << endl;<br />
spectra.findLambda(CONSTANT);<br />
<br />
<br />
cout << "Calculating luminous flux..." << endl;<br />
double x=luminousFlux(luminosity,spectra) ;<br />
<br />
<br />
cout << "Conversion Factor is: " << x << endl << endl;<br />
}<br />
<br />
<br />
void testData(char* data,double CONST, char* output)<br />
{<br />
Function a;<br />
a.getData(data);<br />
a.findLambda(CONST);<br />
<br />
ofstream fout(output);<br />
<br />
if(!fout)<br />
{cout << "Could not open file " << output << " program terminated." << endl; fout.close(); return;}<br />
<br />
for(double i=MINI; i<=MAXI ; i+=0.1)<br />
fout << i << "\t" << a.value(i) << endl;<br />
<br />
// for(double i=0; i<a.size() ; i++)<br />
// cout << a.wat(i) << "\t" << a.fat(i)<<"\t" << a.lat(i)<< endl;<br />
<br />
<br />
fout.close();<br />
<br />
}<br />
<br />
<br />
<br />
<br />
double luminousFlux(Function x, Function y)<br />
{<br />
double sum1=0, sum2=0;<br />
int a=400,b=750;<br />
double h=H(a,b,N);<br />
<br />
<br />
for(double j=1; j<=(N/2)-1;j++)<br />
{sum1+=x.value(X(2*j,a,h))*y.value(X(2*j,a,h));}<br />
<br />
for(double j=1; j<=N/2; j++)<br />
{sum2+=x.value(X(2*j-1,a,h))*y.value(X(2*j-1,a,h));}<br />
<br />
return 683.002*((h/3)*( x.value(a)*y.value(a)+ 2*sum1 + 4*sum2 + x.value(b)*y.value(b) ))/integral(a,b,y);<br />
<br />
<br />
}<br />
<br />
<br />
<br />
double integral (double a, double b, Function func)<br />
{<br />
double sum1=0, sum2=0;<br />
double h=H(a,b,N);<br />
<br />
for(double j=1; j<=(N/2)-1;j++)<br />
{sum1+=func.value( X( 2*j , a , h ) );}<br />
<br />
for(double j=1; j<=N/2; j++)<br />
{sum2+=func.value( X( (2*j)-1 , a , h ) );}<br />
<br />
return (h/3)* ( func.value(a)+ 2*sum1 + 4*sum2 + func.value(b) );<br />
}<br />
<br />
//.............................................................................................................<br />
<br />
void Function::getData(const char* a)<br />
{<br />
double num; <br />
<br />
SIZE=200;<br />
<br />
ifstream fin(a);<br />
if(!fin)<br />
{cout << "Could not open file " << a << " program terminated." << endl; fin.close(); return;}<br />
<br />
fin >> num;<br />
for (int i=0; !fin.eof(); i++ ) <br />
{<br />
if(!(i%2)){ wavelength.push_back(num); fin >> num;}<br />
else { function.push_back(num); fin >> num;}<br />
}<br />
<br />
fin.close();<br />
<br />
int total=wavelength.size();<br />
vector <double> tempwavelength;<br />
vector <double> tempfunction; <br />
<br />
<br />
if(total>SIZE){<br />
for(int i=0; i<wavelength.size(); i++) {<br />
if(i%(total/SIZE)==0){<br />
tempwavelength.push_back(wavelength.at(i));<br />
tempfunction.push_back(function.at(i));<br />
}}<br />
wavelength.clear(); function.clear();<br />
for(int i=0; i<tempwavelength.size(); i++) { <br />
wavelength.push_back(tempwavelength.at(i));<br />
function.push_back(tempfunction.at(i));<br />
}<br />
}<br />
<br />
<br />
<br />
width=(wavelength.back()-wavelength.front())/wavelength.size();<br />
<br />
}<br />
<br />
void Function::print(double min, double max, double increment)<br />
{<br />
for(double i=min; i<=max ; i+=increment)<br />
cout << i << "\t" << value(i) << endl;<br />
}<br />
<br />
void Function::printStore()<br />
{<br />
for(double i=0; i<wavelength.size() ; i++)<br />
cout << wavelength.at(i) << "\t" << function.at(i) << endl;<br />
<br />
}<br />
<br />
void Function::print()<br />
{<br />
for(int i=0; i<wavelength.size(); i++)<br />
{<br />
cout << wavelength.at(i) << "\t" << function.at(i) << endl;<br />
}<br />
<br />
}<br />
<br />
double Function::RBF(double a, double b)<br />
{<br />
return exp(-(a-b)*(a-b)/(2*sigma*sigma));<br />
//return fabs(a-b);<br />
//return 1/sqrt(1+sigma*(a-b)*(a-b));<br />
}<br />
<br />
void Function::findLambda(double CONST)<br />
{ <br />
sigma=width*CONST;<br />
<br />
int M=wavelength.size();<br />
matrix <double> PHI(M,M);<br />
matrix <double> lamb(M,1);<br />
matrix <double> func(M,1); <br />
<br />
for (int i=0; i<M; i++) {<br />
func.setvalue(i,1,function.at(i));<br />
for (int j=0; j<M; j++) {<br />
PHI.setvalue(i,j,RBF(wavelength.at(i),wavelength.at(j)));<br />
<br />
}}<br />
<br />
PHI.invert(); lamb.settoproduct(PHI,func);<br />
<br />
<br />
double value;bool success;<br />
<br />
for(int i=0; i<M; i++){lambda.push_back(lamb.getvalue(i,1));}<br />
}<br />
<br />
double Function::value(double x)<br />
{<br />
double sum=0;<br />
for(int i=0; i<wavelength.size(); i++) sum+=lambda.at(i)*RBF(x,wavelength.at(i));<br />
<br />
return sum;<br />
}<br />
<br />
void Function::fix()<br />
{<br />
for(int i=0; i<wavelength.size(); i++){<br />
for(int j=0;j<i; j++){<br />
if(wavelength.at(i)==wavelength.at(j)) {wavelength.erase(wavelength.begin()+j); function.erase(function.begin()+j); j=j-1;}<br />
}}<br />
<br />
}<br />
<br />
<br />
void Function::eraseBeginning(int i)<br />
{<br />
i/=2;<br />
function.erase(function.begin(), function.begin()+i);<br />
wavelength.erase(wavelength.begin(),wavelength.begin()+i);<br />
}<br />
<br />
=Photopic=<br />
380<br />
<br />
3.9000e-005<br />
<br />
385<br />
<br />
6.4000e-005<br />
<br />
390<br />
<br />
1.2000e-004<br />
<br />
395<br />
<br />
2.1700e-004<br />
<br />
400<br />
<br />
3.9600e-004<br />
<br />
405<br />
<br />
6.4000e-004<br />
<br />
410<br />
<br />
1.2100e-003<br />
<br />
415<br />
<br />
2.1800e-003<br />
<br />
420<br />
<br />
4.0000e-003<br />
<br />
425<br />
<br />
7.3000e-003<br />
<br />
430<br />
<br />
1.1600e-002<br />
<br />
435<br />
<br />
1.6840e-002<br />
<br />
440<br />
<br />
2.3000e-002<br />
<br />
445<br />
<br />
2.9800e-002<br />
<br />
450<br />
<br />
3.8000e-002<br />
<br />
455<br />
<br />
4.8000e-002<br />
<br />
460<br />
<br />
6.0000e-002<br />
<br />
465<br />
<br />
7.3900e-002<br />
<br />
470<br />
<br />
9.0980e-002<br />
<br />
475<br />
<br />
1.1260e-001<br />
<br />
480<br />
<br />
1.3902e-001<br />
<br />
485<br />
<br />
1.6930e-001<br />
<br />
490<br />
<br />
2.0802e-001<br />
<br />
495<br />
<br />
2.5860e-001<br />
<br />
500<br />
<br />
3.2300e-001<br />
<br />
505<br />
<br />
4.0730e-001<br />
<br />
510<br />
<br />
5.0300e-001<br />
<br />
515<br />
<br />
6.0820e-001<br />
<br />
520<br />
<br />
7.1000e-001<br />
<br />
525<br />
<br />
7.9320e-001<br />
<br />
530<br />
<br />
8.6200e-001<br />
<br />
535<br />
<br />
9.1485e-001<br />
<br />
540<br />
<br />
9.5400e-001<br />
<br />
545<br />
<br />
9.8030e-001<br />
<br />
550<br />
<br />
9.9495e-001<br />
<br />
555<br />
<br />
1.0000e+000<br />
<br />
560<br />
<br />
9.9500e-001<br />
<br />
565<br />
<br />
9.7860e-001<br />
<br />
570<br />
<br />
9.5200e-001<br />
<br />
575<br />
<br />
9.1540e-001<br />
<br />
580<br />
<br />
8.7000e-001<br />
<br />
585<br />
<br />
8.1630e-001<br />
<br />
590<br />
<br />
7.5700e-001<br />
<br />
595<br />
<br />
6.9490e-001<br />
<br />
600<br />
<br />
6.3100e-001<br />
<br />
605<br />
<br />
5.6680e-001<br />
<br />
610<br />
<br />
5.0300e-001<br />
<br />
615<br />
<br />
4.4120e-001<br />
<br />
620<br />
<br />
3.8100e-001<br />
<br />
625<br />
<br />
3.2100e-001<br />
<br />
630<br />
<br />
2.6500e-001<br />
<br />
635<br />
<br />
2.1700e-001<br />
<br />
640<br />
<br />
1.7500e-001<br />
<br />
645<br />
<br />
1.3820e-001<br />
<br />
650<br />
<br />
1.0700e-001<br />
<br />
655<br />
<br />
8.1600e-002<br />
<br />
660<br />
<br />
6.1000e-002<br />
<br />
665<br />
<br />
4.4580e-002<br />
<br />
670<br />
<br />
3.2000e-002<br />
<br />
675<br />
<br />
2.3200e-002<br />
<br />
680<br />
<br />
1.7000e-002<br />
<br />
685<br />
<br />
1.1920e-002<br />
<br />
690<br />
<br />
8.2100e-003<br />
<br />
695<br />
<br />
5.7230e-003<br />
<br />
700<br />
<br />
4.1020e-003<br />
<br />
705<br />
<br />
2.9290e-003<br />
<br />
710<br />
<br />
2.0910e-003<br />
<br />
715<br />
<br />
1.4840e-003<br />
<br />
720<br />
<br />
1.0470e-003<br />
<br />
725<br />
<br />
7.4000e-004<br />
<br />
730<br />
<br />
5.2000e-004<br />
<br />
=Scotopic=<br />
380<br />
<br />
5.890e-004<br />
<br />
385<br />
<br />
1.108e-003<br />
<br />
390<br />
<br />
2.209e-003<br />
<br />
395<br />
<br />
4.530e-003<br />
<br />
400<br />
<br />
9.290e-003<br />
<br />
405<br />
<br />
1.852e-002<br />
<br />
410<br />
<br />
3.484e-002<br />
<br />
415<br />
<br />
6.040e-002<br />
<br />
420<br />
<br />
9.660e-002<br />
<br />
425<br />
<br />
1.436e-001<br />
<br />
430<br />
<br />
1.998e-001<br />
<br />
435<br />
<br />
2.625e-001<br />
<br />
440<br />
<br />
3.281e-001<br />
<br />
445<br />
<br />
3.931e-001<br />
<br />
450<br />
<br />
4.550e-001<br />
<br />
455<br />
<br />
5.130e-001<br />
<br />
460<br />
<br />
5.670e-001<br />
<br />
465<br />
<br />
6.200e-001<br />
<br />
470<br />
<br />
6.760e-001<br />
<br />
475<br />
<br />
7.340e-001<br />
<br />
480<br />
<br />
7.930e-001<br />
<br />
485<br />
<br />
8.510e-001<br />
<br />
490<br />
<br />
9.040e-001<br />
<br />
495<br />
<br />
9.490e-001<br />
<br />
500<br />
<br />
9.820e-001<br />
<br />
505<br />
<br />
9.980e-001<br />
<br />
510<br />
<br />
9.970e-001<br />
<br />
515<br />
<br />
9.750e-001<br />
<br />
520<br />
<br />
9.350e-001<br />
<br />
525<br />
<br />
8.800e-001<br />
<br />
530<br />
<br />
8.110e-001<br />
<br />
535<br />
<br />
7.330e-001<br />
<br />
540<br />
<br />
6.500e-001<br />
<br />
545<br />
<br />
5.640e-001<br />
<br />
550<br />
<br />
4.810e-001<br />
<br />
555<br />
<br />
4.020e-001<br />
<br />
560<br />
<br />
3.288e-001<br />
<br />
565<br />
<br />
2.639e-001<br />
<br />
570<br />
<br />
2.076e-001<br />
<br />
575<br />
<br />
1.602e-001<br />
<br />
580<br />
<br />
1.212e-001<br />
<br />
585<br />
<br />
8.990e-002<br />
<br />
590<br />
<br />
6.550e-002<br />
<br />
595<br />
<br />
4.690e-002<br />
<br />
600<br />
<br />
3.315e-002<br />
<br />
605<br />
<br />
2.312e-002<br />
<br />
610<br />
<br />
1.593e-002<br />
<br />
615<br />
<br />
1.088e-002<br />
<br />
620<br />
<br />
7.370e-003<br />
<br />
625<br />
<br />
4.970e-003<br />
<br />
630<br />
<br />
3.335e-003<br />
<br />
635<br />
<br />
2.235e-003<br />
<br />
640<br />
<br />
1.497e-003<br />
<br />
645<br />
<br />
1.005e-003<br />
<br />
650<br />
<br />
6.770e-004<br />
<br />
655<br />
<br />
4.590e-004<br />
<br />
660<br />
<br />
3.129e-004<br />
<br />
665<br />
<br />
2.146e-004<br />
<br />
670<br />
<br />
1.480e-004<br />
<br />
675<br />
<br />
1.026e-004<br />
<br />
680<br />
<br />
7.150e-005<br />
<br />
685<br />
<br />
5.010e-005<br />
<br />
690<br />
<br />
3.533e-005<br />
<br />
695<br />
<br />
2.501e-005<br />
<br />
700<br />
<br />
1.780e-005<br />
<br />
705<br />
<br />
1.273e-005<br />
<br />
710<br />
<br />
9.140e-006<br />
<br />
715<br />
<br />
6.600e-006<br />
<br />
720<br />
<br />
4.780e-006<br />
<br />
725<br />
<br />
3.482e-006<br />
<br />
730<br />
<br />
2.546e-006<br />
<br />
735<br />
<br />
1.870e-006<br />
<br />
740<br />
<br />
1.379e-006<br />
<br />
745<br />
<br />
1.022e-006<br />
<br />
750<br />
<br />
7.600e-007<br />
<br />
755<br />
<br />
5.670e-007<br />
<br />
760<br />
<br />
4.250e-007<br />
<br />
765<br />
<br />
3.196e-007<br />
<br />
770<br />
<br />
2.413e-007<br />
<br />
775<br />
<br />
1.829e-007<br />
<br />
780<br />
<br />
1.390e-007<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/luminanceSourceCodeTeam:Cambridge/luminanceSourceCode2010-10-26T20:03:52Z<p>Willh: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Source Code for Luminance calculator}}<br />
<br />
To use this program <br />
<br />
*create a new folder<br />
*save the source code below into a file called LuminanceCalculator.cc<br />
*save the data under scotopic into a file called scotopic.dat<br />
*save the data under photopic into a file called photopic.dat<br />
*create your own spectra file by inputting the (x<sub>i</sub>,f(x<sub>i</sub>)) coordinates of the spectral distribution file, no spaces just carriage returns i.e. in the format:<br />
x<sub>1</sub><br />
f(x<sub>1</sub>)<br />
x<sub>2</sub><br />
f(x<sub>2</sub>)<br />
...<br />
x<sub>2</sub><br />
f(x<sub>2</sub>)<br />
*10 values is usually enough, but more would be better. x should be in nanometre (nm) f(x) should be in SI<br />
*save the above as spectra.dat<br />
*open a terminal<br />
*navigate to the folder which all of the above are in<br />
*compile using the command: g++ LuminanceCalculator.cc -o LuminanceCalculator.cc<br />
*run the program<br />
<br />
=Source Code=<br />
#include <iostream><br />
#include <vector><br />
#include <fstream><br />
#include <math.h><br />
<br />
#ifndef __mjdmatrix_h<br />
#define __mjdmatrix_h<br />
#include <iostream><br />
using namespace std;<br />
// generic object (class) definition of matrix:<br />
template <class D> class matrix{<br />
<br />
<br />
// NOTE: maxsize determines available memory storage, but<br />
// actualsize determines the actual size of the stored matrix in use<br />
// at a particular time.<br />
int maxsize; // max number of rows (same as max number of columns)<br />
int actualsize; // actual size (rows, or columns) of the stored matrix<br />
D* data; // where the data contents of the matrix are stored<br />
void allocate() {<br />
delete[] data;<br />
data = new D [maxsize*maxsize];<br />
};<br />
matrix() {}; // private ctor's<br />
matrix(int newmaxsize) {matrix(newmaxsize,newmaxsize);};<br />
<br />
<br />
public: <br />
<br />
void print()<br />
{<br />
for(int i=0; i<maxsize; i++){<br />
for(int j=0; j<maxsize; j++){<br />
<br />
cout << getvalue(i,j) << " ";<br />
}<br />
cout << endl;<br />
}<br />
<br />
};<br />
<br />
<br />
D getvalue(int row, int column){return data[ row * maxsize + column ];};<br />
matrix(int newmaxsize, int newactualsize) { // the only public ctor<br />
if (newmaxsize <= 0) newmaxsize = 5;<br />
maxsize = newmaxsize; <br />
if ((newactualsize <= newmaxsize)&&(newactualsize>0))<br />
actualsize = newactualsize;<br />
else <br />
actualsize = newmaxsize;<br />
// since allocate() will first call delete[] on data:<br />
data = 0;<br />
allocate();<br />
};<br />
~matrix() { delete[] data; };<br />
<br />
<br />
void comparetoidentity() {<br />
int worstdiagonal = 0;<br />
D maxunitydeviation = 0.0;<br />
D currentunitydeviation;<br />
for ( int i = 0; i < actualsize; i++ ) {<br />
currentunitydeviation = data[i*maxsize+i] - 1.;<br />
if ( currentunitydeviation < 0.0) currentunitydeviation *= -1.;<br />
if ( currentunitydeviation > maxunitydeviation ) {<br />
maxunitydeviation = currentunitydeviation;<br />
worstdiagonal = i;<br />
}<br />
}<br />
int worstoffdiagonalrow = 0;<br />
int worstoffdiagonalcolumn = 0;<br />
D maxzerodeviation = 0.0;<br />
D currentzerodeviation ;<br />
for ( int i = 0; i < actualsize; i++ ) {<br />
for ( int j = 0; j < actualsize; j++ ) {<br />
if ( i == j ) continue; // we look only at non-diagonal terms<br />
currentzerodeviation = data[i*maxsize+j];<br />
if ( currentzerodeviation < 0.0) currentzerodeviation *= -1.0;<br />
if ( currentzerodeviation > maxzerodeviation ) {<br />
maxzerodeviation = currentzerodeviation;<br />
worstoffdiagonalrow = i;<br />
worstoffdiagonalcolumn = j;<br />
}<br />
<br />
}<br />
}<br />
cout << "Worst diagonal value deviation from unity: " <br />
<< maxunitydeviation << " at row/column " << worstdiagonal << endl;<br />
cout << "Worst off-diagonal value deviation from zero: " <br />
<< maxzerodeviation << " at row = " << worstoffdiagonalrow <br />
<< ", column = " << worstoffdiagonalcolumn << endl;<br />
}<br />
<br />
<br />
void settoproduct(matrix& left, matrix& right) {<br />
actualsize = left.getactualsize();<br />
if ( maxsize < left.getactualsize() ) {<br />
maxsize = left.getactualsize();<br />
allocate();<br />
}<br />
for ( int i = 0; i < actualsize; i++ )<br />
for ( int j = 0; j < actualsize; j++ ) {<br />
D sum = 0.0;<br />
D leftvalue, rightvalue;<br />
bool success;<br />
for (int c = 0; c < actualsize; c++) {<br />
left.getvalue(i,c,leftvalue,success);<br />
right.getvalue(c,j,rightvalue,success);<br />
sum += leftvalue * rightvalue;<br />
}<br />
setvalue(i,j,sum);<br />
}<br />
}<br />
<br />
<br />
void copymatrix(matrix& source) {<br />
actualsize = source.getactualsize();<br />
if ( maxsize < source.getactualsize() ) {<br />
maxsize = source.getactualsize();<br />
allocate();<br />
}<br />
for ( int i = 0; i < actualsize; i++ )<br />
for ( int j = 0; j < actualsize; j++ ) {<br />
D value;<br />
bool success;<br />
source.getvalue(i,j,value,success);<br />
data[i*maxsize+j] = value;<br />
}<br />
};<br />
<br />
<br />
<br />
void setactualsize(int newactualsize) {<br />
if ( newactualsize > maxsize )<br />
{<br />
maxsize = newactualsize ; // * 2; // wastes memory but saves<br />
// time otherwise required for<br />
// operation new[]<br />
allocate();<br />
}<br />
if (newactualsize >= 0) actualsize = newactualsize;<br />
};<br />
<br />
<br />
<br />
int getactualsize() { return actualsize; };<br />
<br />
<br />
void getvalue(int row, int column, D& returnvalue, bool& success) {<br />
if ( (row>=maxsize) || (column>=maxsize) <br />
|| (row<0) || (column<0) )<br />
{ success = false;<br />
return; }<br />
returnvalue = data[ row * maxsize + column ];<br />
success = true;<br />
};<br />
<br />
<br />
bool setvalue(int row, int column, D newvalue) {<br />
if ( (row >= maxsize) || (column >= maxsize) <br />
|| (row<0) || (column<0) ) return false;<br />
data[ row * maxsize + column ] = newvalue;<br />
return true;<br />
};<br />
<br />
<br />
void invert() {<br />
if (actualsize <= 0) return; // sanity check<br />
if (actualsize == 1) return; // must be of dimension >= 2<br />
for (int i=1; i < actualsize; i++) data[i] /= data[0]; // normalize row 0<br />
for (int i=1; i < actualsize; i++) { <br />
for (int j=i; j < actualsize; j++) { // do a column of L<br />
D sum = 0.0;<br />
for (int k = 0; k < i; k++) <br />
sum += data[j*maxsize+k] * data[k*maxsize+i];<br />
data[j*maxsize+i] -= sum;<br />
}<br />
if (i == actualsize-1) continue;<br />
for (int j=i+1; j < actualsize; j++) { // do a row of U<br />
D sum = 0.0;<br />
for (int k = 0; k < i; k++)<br />
sum += data[i*maxsize+k]*data[k*maxsize+j];<br />
data[i*maxsize+j] = <br />
(data[i*maxsize+j]-sum) / data[i*maxsize+i];<br />
}<br />
}<br />
for ( int i = 0; i < actualsize; i++ ) // invert L<br />
for ( int j = i; j < actualsize; j++ ) {<br />
D x = 1.0;<br />
if ( i != j ) {<br />
x = 0.0;<br />
for ( int k = i; k < j; k++ ) <br />
x -= data[j*maxsize+k]*data[k*maxsize+i];<br />
}<br />
data[j*maxsize+i] = x / data[j*maxsize+j];<br />
}<br />
for ( int i = 0; i < actualsize; i++ ) // invert U<br />
for ( int j = i; j < actualsize; j++ ) {<br />
if ( i == j ) continue;<br />
D sum = 0.0;<br />
for ( int k = i; k < j; k++ )<br />
sum += data[k*maxsize+j]*( (i==k) ? 1.0 : data[i*maxsize+k] );<br />
data[i*maxsize+j] = -sum;<br />
}<br />
for ( int i = 0; i < actualsize; i++ ) // final inversion<br />
for ( int j = 0; j < actualsize; j++ ) {<br />
D sum = 0.0;<br />
for ( int k = ((i>j)?i:j); k < actualsize; k++ ) <br />
sum += ((j==k)?1.0:data[j*maxsize+k])*data[k*maxsize+i];<br />
data[j*maxsize+i] = sum;<br />
}<br />
};<br />
};<br />
#endif<br />
<br />
using namespace std;<br />
<br />
class Function<br />
{<br />
private:<br />
vector <double> wavelength;<br />
vector <double> function;<br />
vector <double> lambda;<br />
<br />
double RBF(double, double);<br />
double sigma;<br />
double width;<br />
int SIZE;<br />
<br />
public:<br />
void getData(const char*);<br />
void findLambda(double);<br />
void fix();<br />
<br />
double size(){return wavelength.size();}<br />
double lat(int i){return lambda.at(i);}<br />
double wat(int i){return wavelength.at(i);}<br />
double fat(int i){return function.at(i);}<br />
void wequals(int i, double value){wavelength.at(i)=value;}<br />
void fequals(int i, double value){function.at(i)=value;}<br />
<br />
void eraseBeginning(int);<br />
void erase(int);<br />
<br />
void print();<br />
void print(double,double,double);<br />
void printStore();<br />
<br />
double value(double);<br />
<br />
<br />
<br />
};<br />
<br />
double X(double j, double a, double h) {return a+j*h;}<br />
double H(double a,double b,double n) {return (b-a)/n;}<br />
<br />
double integral (double a, double b, Function);<br />
<br />
void runProgram(char*,char*);<br />
<br />
double luminousFlux(Function, Function);<br />
<br />
<br />
<br />
<br />
#define MINI 400<br />
#define MAXI 750<br />
<br />
#define CONSTANT 0.7<br />
#define N 10000<br />
<br />
#ifndef SPECTRA<br />
#define SPECTRA "spectra.dat"<br />
#endif<br />
<br />
<br />
int main()<br />
{<br />
char b;<br />
cout << "This program will tell you the conversion factor between Radiance and Luminance for your chosen object with known radiation spectrum" << endl;<br />
cout << endl << "Do you want scotopic (low light) [s] or photopic (normal light) [p] Luminance?" << endl;<br />
while(b!='p'&&b!='s'){cout << "please input s or p" << endl; cin >> b;}<br />
cout << endl;<br />
<br />
if(b=='s')<br />
runProgram("scotopic.dat",SPECTRA);<br />
if(b=='p')<br />
runProgram("photopic.dat",SPECTRA);<br />
<br />
return 0;<br />
}<br />
<br />
<br />
void runProgram(char* lumFile, char* specFile)<br />
{<br />
Function luminosity, spectra;<br />
<br />
cout << "Getting luminosity data..." << endl;<br />
luminosity.getData(lumFile);<br />
cout << "Calculating luminosity function..." << endl;<br />
luminosity.findLambda(CONSTANT);<br />
<br />
cout << "Getting spectral data..." << endl;<br />
spectra.getData(specFile);<br />
cout << "Calculating spectral function..." << endl;<br />
spectra.findLambda(CONSTANT);<br />
<br />
<br />
cout << "Calculating luminous flux..." << endl;<br />
double x=luminousFlux(luminosity,spectra) ;<br />
<br />
<br />
cout << "Conversion Factor is: " << x << endl << endl;<br />
}<br />
<br />
<br />
void testData(char* data,double CONST, char* output)<br />
{<br />
Function a;<br />
a.getData(data);<br />
a.findLambda(CONST);<br />
<br />
ofstream fout(output);<br />
<br />
if(!fout)<br />
{cout << "Could not open file " << output << " program terminated." << endl; fout.close(); return;}<br />
<br />
for(double i=MINI; i<=MAXI ; i+=0.1)<br />
fout << i << "\t" << a.value(i) << endl;<br />
<br />
// for(double i=0; i<a.size() ; i++)<br />
// cout << a.wat(i) << "\t" << a.fat(i)<<"\t" << a.lat(i)<< endl;<br />
<br />
<br />
fout.close();<br />
<br />
}<br />
<br />
<br />
<br />
<br />
double luminousFlux(Function x, Function y)<br />
{<br />
double sum1=0, sum2=0;<br />
int a=400,b=750;<br />
double h=H(a,b,N);<br />
<br />
<br />
for(double j=1; j<=(N/2)-1;j++)<br />
{sum1+=x.value(X(2*j,a,h))*y.value(X(2*j,a,h));}<br />
<br />
for(double j=1; j<=N/2; j++)<br />
{sum2+=x.value(X(2*j-1,a,h))*y.value(X(2*j-1,a,h));}<br />
<br />
return 683.002*((h/3)*( x.value(a)*y.value(a)+ 2*sum1 + 4*sum2 + x.value(b)*y.value(b) ))/integral(a,b,y);<br />
<br />
<br />
}<br />
<br />
<br />
<br />
double integral (double a, double b, Function func)<br />
{<br />
double sum1=0, sum2=0;<br />
double h=H(a,b,N);<br />
<br />
for(double j=1; j<=(N/2)-1;j++)<br />
{sum1+=func.value( X( 2*j , a , h ) );}<br />
<br />
for(double j=1; j<=N/2; j++)<br />
{sum2+=func.value( X( (2*j)-1 , a , h ) );}<br />
<br />
return (h/3)* ( func.value(a)+ 2*sum1 + 4*sum2 + func.value(b) );<br />
}<br />
<br />
//.............................................................................................................<br />
<br />
void Function::getData(const char* a)<br />
{<br />
double num; <br />
<br />
SIZE=200;<br />
<br />
ifstream fin(a);<br />
if(!fin)<br />
{cout << "Could not open file " << a << " program terminated." << endl; fin.close(); return;}<br />
<br />
fin >> num;<br />
for (int i=0; !fin.eof(); i++ ) <br />
{<br />
if(!(i%2)){ wavelength.push_back(num); fin >> num;}<br />
else { function.push_back(num); fin >> num;}<br />
}<br />
<br />
fin.close();<br />
<br />
int total=wavelength.size();<br />
vector <double> tempwavelength;<br />
vector <double> tempfunction; <br />
<br />
<br />
if(total>SIZE){<br />
for(int i=0; i<wavelength.size(); i++) {<br />
if(i%(total/SIZE)==0){<br />
tempwavelength.push_back(wavelength.at(i));<br />
tempfunction.push_back(function.at(i));<br />
}}<br />
wavelength.clear(); function.clear();<br />
for(int i=0; i<tempwavelength.size(); i++) { <br />
wavelength.push_back(tempwavelength.at(i));<br />
function.push_back(tempfunction.at(i));<br />
}<br />
}<br />
<br />
<br />
<br />
width=(wavelength.back()-wavelength.front())/wavelength.size();<br />
<br />
}<br />
<br />
void Function::print(double min, double max, double increment)<br />
{<br />
for(double i=min; i<=max ; i+=increment)<br />
cout << i << "\t" << value(i) << endl;<br />
}<br />
<br />
void Function::printStore()<br />
{<br />
for(double i=0; i<wavelength.size() ; i++)<br />
cout << wavelength.at(i) << "\t" << function.at(i) << endl;<br />
<br />
}<br />
<br />
void Function::print()<br />
{<br />
for(int i=0; i<wavelength.size(); i++)<br />
{<br />
cout << wavelength.at(i) << "\t" << function.at(i) << endl;<br />
}<br />
<br />
}<br />
<br />
double Function::RBF(double a, double b)<br />
{<br />
return exp(-(a-b)*(a-b)/(2*sigma*sigma));<br />
//return fabs(a-b);<br />
//return 1/sqrt(1+sigma*(a-b)*(a-b));<br />
}<br />
<br />
void Function::findLambda(double CONST)<br />
{ <br />
sigma=width*CONST;<br />
<br />
int M=wavelength.size();<br />
matrix <double> PHI(M,M);<br />
matrix <double> lamb(M,1);<br />
matrix <double> func(M,1); <br />
<br />
for (int i=0; i<M; i++) {<br />
func.setvalue(i,1,function.at(i));<br />
for (int j=0; j<M; j++) {<br />
PHI.setvalue(i,j,RBF(wavelength.at(i),wavelength.at(j)));<br />
<br />
}}<br />
<br />
PHI.invert(); lamb.settoproduct(PHI,func);<br />
<br />
<br />
double value;bool success;<br />
<br />
for(int i=0; i<M; i++){lambda.push_back(lamb.getvalue(i,1));}<br />
}<br />
<br />
double Function::value(double x)<br />
{<br />
double sum=0;<br />
for(int i=0; i<wavelength.size(); i++) sum+=lambda.at(i)*RBF(x,wavelength.at(i));<br />
<br />
return sum;<br />
}<br />
<br />
void Function::fix()<br />
{<br />
for(int i=0; i<wavelength.size(); i++){<br />
for(int j=0;j<i; j++){<br />
if(wavelength.at(i)==wavelength.at(j)) {wavelength.erase(wavelength.begin()+j); function.erase(function.begin()+j); j=j-1;}<br />
}}<br />
<br />
}<br />
<br />
<br />
void Function::eraseBeginning(int i)<br />
{<br />
i/=2;<br />
function.erase(function.begin(), function.begin()+i);<br />
wavelength.erase(wavelength.begin(),wavelength.begin()+i);<br />
}<br />
<br />
=Photopic=<br />
380<br />
<br />
3.9000e-005<br />
<br />
385<br />
<br />
6.4000e-005<br />
<br />
390<br />
<br />
1.2000e-004<br />
<br />
395<br />
<br />
2.1700e-004<br />
<br />
400<br />
<br />
3.9600e-004<br />
<br />
405<br />
<br />
6.4000e-004<br />
<br />
410<br />
<br />
1.2100e-003<br />
<br />
415<br />
<br />
2.1800e-003<br />
<br />
420<br />
<br />
4.0000e-003<br />
<br />
425<br />
<br />
7.3000e-003<br />
<br />
430<br />
<br />
1.1600e-002<br />
<br />
435<br />
<br />
1.6840e-002<br />
<br />
440<br />
<br />
2.3000e-002<br />
<br />
445<br />
<br />
2.9800e-002<br />
<br />
450<br />
<br />
3.8000e-002<br />
<br />
455<br />
<br />
4.8000e-002<br />
<br />
460<br />
<br />
6.0000e-002<br />
<br />
465<br />
<br />
7.3900e-002<br />
<br />
470<br />
<br />
9.0980e-002<br />
<br />
475<br />
<br />
1.1260e-001<br />
<br />
480<br />
<br />
1.3902e-001<br />
<br />
485<br />
<br />
1.6930e-001<br />
<br />
490<br />
<br />
2.0802e-001<br />
<br />
495<br />
<br />
2.5860e-001<br />
<br />
500<br />
<br />
3.2300e-001<br />
<br />
505<br />
<br />
4.0730e-001<br />
<br />
510<br />
<br />
5.0300e-001<br />
<br />
515<br />
<br />
6.0820e-001<br />
<br />
520<br />
<br />
7.1000e-001<br />
<br />
525<br />
<br />
7.9320e-001<br />
<br />
530<br />
<br />
8.6200e-001<br />
<br />
535<br />
<br />
9.1485e-001<br />
<br />
540<br />
<br />
9.5400e-001<br />
<br />
545<br />
<br />
9.8030e-001<br />
<br />
550<br />
<br />
9.9495e-001<br />
<br />
555<br />
<br />
1.0000e+000<br />
<br />
560<br />
<br />
9.9500e-001<br />
<br />
565<br />
<br />
9.7860e-001<br />
<br />
570<br />
<br />
9.5200e-001<br />
<br />
575<br />
<br />
9.1540e-001<br />
<br />
580<br />
<br />
8.7000e-001<br />
<br />
585<br />
<br />
8.1630e-001<br />
<br />
590<br />
<br />
7.5700e-001<br />
<br />
595<br />
<br />
6.9490e-001<br />
<br />
600<br />
<br />
6.3100e-001<br />
<br />
605<br />
<br />
5.6680e-001<br />
<br />
610<br />
<br />
5.0300e-001<br />
<br />
615<br />
<br />
4.4120e-001<br />
<br />
620<br />
<br />
3.8100e-001<br />
<br />
625<br />
<br />
3.2100e-001<br />
<br />
630<br />
<br />
2.6500e-001<br />
<br />
635<br />
<br />
2.1700e-001<br />
<br />
640<br />
<br />
1.7500e-001<br />
<br />
645<br />
<br />
1.3820e-001<br />
<br />
650<br />
<br />
1.0700e-001<br />
<br />
655<br />
<br />
8.1600e-002<br />
<br />
660<br />
<br />
6.1000e-002<br />
<br />
665<br />
<br />
4.4580e-002<br />
<br />
670<br />
<br />
3.2000e-002<br />
<br />
675<br />
<br />
2.3200e-002<br />
<br />
680<br />
<br />
1.7000e-002<br />
<br />
685<br />
<br />
1.1920e-002<br />
<br />
690<br />
<br />
8.2100e-003<br />
<br />
695<br />
<br />
5.7230e-003<br />
<br />
700<br />
<br />
4.1020e-003<br />
<br />
705<br />
<br />
2.9290e-003<br />
<br />
710<br />
<br />
2.0910e-003<br />
<br />
715<br />
<br />
1.4840e-003<br />
<br />
720<br />
<br />
1.0470e-003<br />
<br />
725<br />
<br />
7.4000e-004<br />
<br />
730<br />
<br />
5.2000e-004<br />
<br />
=Scotopic=<br />
380<br />
<br />
5.890e-004<br />
<br />
385<br />
<br />
1.108e-003<br />
<br />
390<br />
<br />
2.209e-003<br />
<br />
395<br />
<br />
4.530e-003<br />
<br />
400<br />
<br />
9.290e-003<br />
<br />
405<br />
<br />
1.852e-002<br />
<br />
410<br />
<br />
3.484e-002<br />
<br />
415<br />
<br />
6.040e-002<br />
<br />
420<br />
<br />
9.660e-002<br />
<br />
425<br />
<br />
1.436e-001<br />
<br />
430<br />
<br />
1.998e-001<br />
<br />
435<br />
<br />
2.625e-001<br />
<br />
440<br />
<br />
3.281e-001<br />
<br />
445<br />
<br />
3.931e-001<br />
<br />
450<br />
<br />
4.550e-001<br />
<br />
455<br />
<br />
5.130e-001<br />
<br />
460<br />
<br />
5.670e-001<br />
<br />
465<br />
<br />
6.200e-001<br />
<br />
470<br />
<br />
6.760e-001<br />
<br />
475<br />
<br />
7.340e-001<br />
<br />
480<br />
<br />
7.930e-001<br />
<br />
485<br />
<br />
8.510e-001<br />
<br />
490<br />
<br />
9.040e-001<br />
<br />
495<br />
<br />
9.490e-001<br />
<br />
500<br />
<br />
9.820e-001<br />
<br />
505<br />
<br />
9.980e-001<br />
<br />
510<br />
<br />
9.970e-001<br />
<br />
515<br />
<br />
9.750e-001<br />
<br />
520<br />
<br />
9.350e-001<br />
<br />
525<br />
<br />
8.800e-001<br />
<br />
530<br />
<br />
8.110e-001<br />
<br />
535<br />
<br />
7.330e-001<br />
<br />
540<br />
<br />
6.500e-001<br />
<br />
545<br />
<br />
5.640e-001<br />
<br />
550<br />
<br />
4.810e-001<br />
<br />
555<br />
<br />
4.020e-001<br />
<br />
560<br />
<br />
3.288e-001<br />
<br />
565<br />
<br />
2.639e-001<br />
<br />
570<br />
<br />
2.076e-001<br />
<br />
575<br />
<br />
1.602e-001<br />
<br />
580<br />
<br />
1.212e-001<br />
<br />
585<br />
<br />
8.990e-002<br />
<br />
590<br />
<br />
6.550e-002<br />
<br />
595<br />
<br />
4.690e-002<br />
<br />
600<br />
<br />
3.315e-002<br />
<br />
605<br />
<br />
2.312e-002<br />
<br />
610<br />
<br />
1.593e-002<br />
<br />
615<br />
<br />
1.088e-002<br />
<br />
620<br />
<br />
7.370e-003<br />
<br />
625<br />
<br />
4.970e-003<br />
<br />
630<br />
<br />
3.335e-003<br />
<br />
635<br />
<br />
2.235e-003<br />
<br />
640<br />
<br />
1.497e-003<br />
<br />
645<br />
<br />
1.005e-003<br />
<br />
650<br />
<br />
6.770e-004<br />
<br />
655<br />
<br />
4.590e-004<br />
<br />
660<br />
<br />
3.129e-004<br />
<br />
665<br />
<br />
2.146e-004<br />
<br />
670<br />
<br />
1.480e-004<br />
<br />
675<br />
<br />
1.026e-004<br />
<br />
680<br />
<br />
7.150e-005<br />
<br />
685<br />
<br />
5.010e-005<br />
<br />
690<br />
<br />
3.533e-005<br />
<br />
695<br />
<br />
2.501e-005<br />
<br />
700<br />
<br />
1.780e-005<br />
<br />
705<br />
<br />
1.273e-005<br />
<br />
710<br />
<br />
9.140e-006<br />
<br />
715<br />
<br />
6.600e-006<br />
<br />
720<br />
<br />
4.780e-006<br />
<br />
725<br />
<br />
3.482e-006<br />
<br />
730<br />
<br />
2.546e-006<br />
<br />
735<br />
<br />
1.870e-006<br />
<br />
740<br />
<br />
1.379e-006<br />
<br />
745<br />
<br />
1.022e-006<br />
<br />
750<br />
<br />
7.600e-007<br />
<br />
755<br />
<br />
5.670e-007<br />
<br />
760<br />
<br />
4.250e-007<br />
<br />
765<br />
<br />
3.196e-007<br />
<br />
770<br />
<br />
2.413e-007<br />
<br />
775<br />
<br />
1.829e-007<br />
<br />
780<br />
<br />
1.390e-007<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-26T19:39:13Z<p>Willh: /* Modelling */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In==<br />
<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Wattage</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>25-150<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>18-95<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>50-400<br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>50-400 <br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>18-180<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
We wrote a [[Team:Cambridge/luminanceSourceCode | program]] in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp (X=210) and hypothesise a projected area of 20m<sup>2</sup>, and a day:night ratio of 14:10 then we find that the efficiency must be roughly '''0.03%'''. This means that 0.03% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/luminanceSourceCodeTeam:Cambridge/luminanceSourceCode2010-10-26T19:03:30Z<p>Willh: New page: {{:Team:Cambridge/Templates/headerMinimalprototype}} {{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Source Code for Luminance calculator}} To use this program *input this te...</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Source Code for Luminance calculator}}<br />
<br />
To use this program <br />
<br />
*input this text into a file named luminanceCalculator.cc<br />
*Open a terminal and navigate to the folder containing<br />
<br />
=Source Code=<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-26T18:55:57Z<p>Willh: /* Modelling */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
==Radiation In===<br />
<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
==Radiation out==<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Wattage</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>25-150<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>18-95<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>50-400<br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>50-400 <br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>18-180<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
We wrote a [[Team:Cambridge/luminanceSourceCode | program]] in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
==Bringing it all together==<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp (X=210) and hypothesise a projected area of 20m<sup>2</sup>, and a day:night ratio of 14:10 then we find that the efficiency must be roughly '''0.03%'''. This means that 0.03% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-26T18:53:31Z<p>Willh: /* Modelling */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
We had thought about bacteria being used for emergency lighting, and our advisor Fernan printed a fire exit sign, which was placed over a plate containing one of our strains of E. coli. In a real system we would imagine using anhydrobiosis to create bacteria 'in hibernation' which could be activated when needed.<br />
<html><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Fire-exit-horiz.jpg"></div><br />
<div style="margin-top:5px; font-size:12px; color:#6e6e6e; line-height:15px; text-align:center">Our <a href="https://2010.igem.org/Team:Cambridge/Bioluminescence/G28"> LuxBrick</a> with an overlay to give a meaningful message</div><br />
<div style="clear:both"></div><br />
</html><br />
<br />
=Modelling=<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,471 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
Since that's the energy which plants have available to, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td><b>Light Source</b><br />
</td><td><b>Wattage</b><br />
</td><td><b>Output(lumens)</b><br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>25-150<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>18-95<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>50-400<br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>50-400 <br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>18-180<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
The above table shows the luminous flux, measured in lumens for several different types of light. <br />
<br />
Lumens are a measure of luminous intensity. When it comes to "brightness" there are two distinct measurements;<br />
*'''Radiance''', which is measured in watts per square metre per steradian (W sr<sup>-1</sup> m<sup>-2</sup> and<br />
*'''Luminance''', which is measured in candelas per square metre (cd m<sup>-2</sup>).<br />
<br />
If you look at a black object and ask "how much light is coming out of that?" there are two answers. The first is obviously none, since it appears black, but the second relies on the fact that it is possible that the object is very hot and is emitting in the infra-red, or is a source of UV and is thus emitting a lot of light. The two measures above quantify this difference. Radiance measures the actual electromagnetic radiation coming from an object, whereas luminance adjusts for the perceptive abilities of the human eye. If you look at figure 3 you can see the '''luminosity functions''' which supply this weighting in the visible part of the spectrum.<br />
<br />
There are two curves, one for photopic vision, and the latter for scotopic. The former is normal vision in good light and covers the normal range of colours. The latter is for dark vision, when the (colour percieving) cone cells are unable to function. This part of the reason why you can only see in black and white in the dark.<br />
<br />
To convert from Radiance to Luminance you integrate the power spectrum weighted by the luminosity function so that wavelengths beyond that of human perception are cut out. Note that this transformation is therefore one-way. You can't convert from Luminance to Radiance.<br />
<br />
The measurements of light output taken above are in '''lumens''' (a measurement of Luminance) where 1lm=1cd*sr. The lumen thus quantifies the human-percieved total amount of light being emitted from an object. The above values use the scotopic luminosity function, since street lights operate in low-light conditions.<br />
<br />
We wrote a [[Team:Cambridge/luminanceSourceCode | program]] in c++ which allowed the user to input their own power spectrum and be told the resulting luminance measure. By inputting the curve shown in figure 2 which details the emission spectrum of the Vibrio Fischeri we found the formula:<br />
<br />
Total Bacterial Luminance(lm)=471.13 x Total Energy Output(W)<br />
<br />
It should be noted that this is actually a really good conversion factor, only about 33% of the radiant energy is lost to the outer regions of human perception (at best 1lm = 683.002 W). This is due to the fact that (as you can see from the scotopic luminosity function in figure 3) the human eye is better at seeing blue light in the dark than red light, and our bacterial light is clearly blue.<br />
<br />
Now we combined the above calculations. If we assume that the tree absorbs all light passing through its projected area '''A''' (not a bad approximation, since only a few percent is able to make it through the canopy) then the total radiant light energy falling on it during the day time per second is '''60*A''' W. If the total radiant energy outputted at night per second is '''E''', then the luminous flux is '''471.13E''' lm, which we desire to be as bright as a street lamp '''X''' lm, then combining these we find that the total efficiency of our plant has to be:<br />
<br />
Efficiency = (X*T<sub>night</sub>) / (471.13*60A*T<sub>day</sub>)<br />
<br />
where '''T<sub>day</sub>''' and '''T<sub>night</sub>''' are the hours of daylight and night time respectively. If we choose the least bright street lamp (X=210) and hypothesise a projected area of 20m<sup>2</sup>, and a day:night ratio of 14:10 then we find that the efficiency must be roughly '''0.03%'''. This means that 0.03% of the total energy hitting the tree must be converted eventually into light output, a potentially achievable target.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-26T17:02:52Z<p>Willh: /* Modelling */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
=Modelling=<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,413 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
That's what plants have available to them, we then considered what they need to be putting out in order to function as street lights.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>'''Light Source'''<br />
</td><td>'''Wattage'''<br />
</td><td>'''Output(lumens)'''<br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>25-150<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>18-95<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<td>Metal Halide <br />
</td><td>50-400<br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>50-400 <br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>18-180<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
1,413 – 1,321W/m<sup>2</sup><br />
"Chapter 8 – Measurement of sunshine duration" (PDF). CIMO Guide. World Meteorological Organization. http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. Retrieved 2008-12-01.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-26T17:00:53Z<p>Willh: /* Modelling */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
=Modelling=<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,413 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). <br />
<br />
This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Light Source<br />
</td><td>Wattage<br />
</td><td>Output(lumens)<br />
</td></tr><br />
<tr><br />
<td>Incandescent<br />
</td><td>25-150<br />
</td><td>210-2700 <br />
</td></tr><br />
<tr><br />
<td>Fluorescent<br />
</td><td>18-95<br />
</td><td>1000-7500 <br />
</td></tr><br />
<tr><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<td>Metal Halide <br />
</td><td>50-400<br />
</td><td>1900-30000<br />
</td></tr><br />
<tr><br />
<td>High-Pressure Sodium<br />
</td><td>50-400 <br />
</td><td>3600-46000 <br />
</td></tr><br />
<tr><br />
<td>Low-Pressure Sodium<br />
</td><td>18-180<br />
</td><td>1800-33000<br />
</td></tr><br />
<tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
1,413 – 1,321W/m<sup>2</sup><br />
"Chapter 8 – Measurement of sunshine duration" (PDF). CIMO Guide. World Meteorological Organization. http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. Retrieved 2008-12-01.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T16:55:49Z<p>Willh: /* Step 1: Design Primers */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers. We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Gibson/ProtocolTeam:Cambridge/Gibson/Protocol2010-10-26T16:55:32Z<p>Willh: /* Step 1: Design Primers */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}<br />
<br />
The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].<br />
<br />
==Step 1: Design Primers==<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Gibthon.png|caption=New and improved Gibson oligo design}}<br />
<br />
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.<br />
<br />
The standard way to do this is with PCR with specialised primers. We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]<br />
<br />
==Step 2: Order Primers==<br />
<br />
This step can take a while, so Gibson Assembly requires some planning ahead<br />
<br />
<br />
==Step 3: PCR ==<br />
<div style="float:right; clear:both">&nbsp;</div><br />
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}<br />
PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<br />
<tr><br />
<td>Step<br />
</td><td>Temp<br />
</td><td>Time<br />
</td></tr><br />
<tr><br />
<td>1:Initial Melting<br />
</td><td>98°C<br />
</td><td>30s<br />
</td></tr><br />
<tr><br />
<td>2:Melting<br />
</td><td>98°C<br />
</td><td>10s<br />
</td></tr><br />
<tr><br />
<br />
<td>3:Annealing<br />
</td><td>T<sub>m</sub>°C<br />
</td><td>15s<br />
</td></tr><br />
<tr><br />
<td>4:Elongation<br />
</td><td>72<br />
</td><td>45s per kb DNA<br />
</td></tr><br />
<tr><br />
<td>5:GoTo step 2<br />
</td><td><br />
</td><td>30 times<br />
</td></tr><br />
<tr><br />
<br />
<td>6:Final Elongation<br />
</td><td>72°C<br />
</td><td>7m30<br />
</td></tr><br />
<tr><br />
<td>7:Final Hold<br />
</td><td>4°C<br />
</td><td>∞<br />
</td></tr><br />
<br />
<br />
<br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
<br />
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.<br />
<br />
The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].<br />
<br />
==Step 4: Gibson Assembly==<br />
<br />
1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]<br />
<br />
2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3<br />
<br />
3) Incubate for 1 hour at 50°C<br />
<br />
<br />
<i>e.g. If you were ligating two fragments (A and B) you could put:</i><br />
<br />
<br />
<html><br />
<style><br />
table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}<br />
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}<br />
</style><br />
<div align="center"><br />
<table class="vistable" padding="0" cellspacing="0"> <br />
<tr><br />
<td><i>2.5µl</i><br />
</td><td><i>fragment A</i><br />
</td></tr><br />
<tr><br />
<td><i>2.5µl</i><br />
<br />
</td><td><i>fragment B</i><br />
</td></tr><br />
<tr><br />
<td><i>15µl</i><br />
</td><td><i>Gibson Master Mix</i><br />
</td></tr><br />
<br />
</table> <br />
</div><br />
</html><br />
<br />
==Step 5: Transformation==<br />
The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willhhttp://2010.igem.org/Team:Cambridge/Tools/LightingTeam:Cambridge/Tools/Lighting2010-10-26T16:47:07Z<p>Willh: /* Modelling */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Future applications: Lighting}}<br />
We had a number of workshops considering the potential broader societal implications of our work. Given the energy crisis facing our planet, lighting, which accounts for 8% of our use of electricity, seemed an interesting application of our work.<br />
=Bioluminescent street lamps=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-City.jpg|caption=''We created a 3D model to try to visualise a city lit by bioluminescent trees''}}<br />
<br />
In order to provide any solution to the problem, a biological solution must tap into a currently unused energy resource. For this reason we decided to consider the use of '''bioluminescent trees''' to replace conventional street lamps. <br />
<br />
A tree in this position would be able to photosynthesise during the day, building up reserves of energy. We then imagined it emitting light by night, using the bacterial luciferase system, under the control of the inherent circadian clock based gene regulation systems.<br />
<div style="clear:both"><br />
<br />
=Putting it into practice=<br />
{{:Team:Cambridge/Templates/RightImage|image=Jungle_book.jpg|caption=''Ben proving it is possible to read by bioluminescent light''}}<br />
We wanted to provide some proof of concept of these ideas. We built the [http://www.youtube.com/watch?v=tUFscEVK5Ks bacterial bubble lamp] to investigate this, and also read the Jungle Book using a flask containing E. coli expressing our bacterial luciferase.<br />
<br />
=Modelling=<br />
{{:Team:Cambridge/Templates/RightImage|image=Solar_Spectrum.png|caption=''Figure 1: The solar radiation spectrum for direct light at both the top of the Earth's atmosphere and at sea level.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Fischerispectrum.jpg|caption=''Figure 2: The emission spectrum of V. Fischeri (shown in black)''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=photopicscotopic.png|caption=''Figure 3: Photopic (black) and scotopic (green) luminosity functions.''}}<br />
{{:Team:Cambridge/Templates/RightImage|image=Luminosityfunction.png|caption=''Figure 4: The formula for calculating luminance. (F:Luminous Flux, y:Luminosity Function,J:Spectral Power Distribution,λ:Wavelength''}}<br />
<br />
<br />
<br />
To show that it may one day indeed be possible to have bioluminescent trees replacing street lamps we considered how efficicient the plants would have to be in order to match a low-intensity street lamp.<br />
<br />
Figure 1 shows the radiation spectrum recieved from the sun at sea level. In total this gives about '''1,321 – 1,413 W/m<sup>2</sup>''' of radiant energy for regions in North America. (Data supplied by American Society for Testing and Materials ([http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. ASTM]) Terrestrial Reference Spectra, a common standard used in photovoltaics). This is a lot of energy, but of course most of it is not accessible to plants. They can only absorb in the visible region (corresponding to roughy 45% of total solar energy), radiation known as PAR (photosynthetically active radiation). In addition, there are other constraints, such as reflectivity of leaves and the absorption spectrum of chlorophyll. The net result is that in general plants are only able to take between '''3''' and '''6%''' of total solar radiation, corresponding to roughly '''60 W/m<sup>2</sup>''' (Figures from Hall, D.O. and House, J.I., Biomass and Bioenergy, 6,11-30 (1994).)<br />
<br />
<br />
<br />
<br />
American Society for Testing and Materials (ASTM) Terrestrial Reference Spectra, a common standard used in photovoltaics<br />
<br />
<br />
1,413 – 1,321W/m<sup>2</sup><br />
"Chapter 8 – Measurement of sunshine duration" (PDF). CIMO Guide. World Meteorological Organization. http://www.wmo.int/pages/prog/www/IMOP/publications/CIMO-Guide/CIMO%20Guide%207th%20Edition,%202008/Part%20I/Chapter%208.pdf. Retrieved 2008-12-01.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>Willh