http://2011.igem.org/wiki/index.php?title=Special:Contributions/Cat&feed=atom&limit=50&target=Cat&year=&month=2011.igem.org - User contributions [en]2024-03-28T22:20:42ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:Cambridge/TeamTeam:Cambridge/Team2011-09-22T03:30:18Z<p>Cat: /* Attribution */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
Our core team was made of 9 Cambridge University [[Team:Cambridge/Team/Students | undergraduates]] from a wide range of courses. There were 4 biologists, three engineers, one physicist and one mathematician. While every member of the group broadened their knowledge, our different areas of expertise allowed us to simultaneously approach problems from many angles. <br />
<br />
We were also aided by a student from the Royal College of Art (RCA) who was able to provide us with a completely different perspective on our work. Her advice and creative input was invaluable to our project.<br />
<br />
We were advised by several [[Team:Cambridge/Team/Academics | academics]] (both from within the university and externally), both professors and PhD students, who provided technical assistance and insight. They all did a fantastic job of providing positive feedback, without guiding the direction of our project.<br />
<br />
With the team's budget especially tight this year due to the increased cost of the competition, our [[Team:Cambridge/Team/Sponsors | sponsors]] were invaluable - they contributed the reagents, kits and cash which enabled us to take part.<br />
==Attribution==<br />
<br />
'''Work carried out primarily by our advisors is clearly labelled as such. In particular we are grateful to Paul Grant for assistance operating the confocal and for his advice during [[Team:Cambridge/Project/Microscopy| our microscopy]], and to Matthwe Hawkeye (or collaborator in the Nanophotonics Dept) for assisting Felix in making [[Team:Cambridge/Project/In_Vitro | thin films]] and in measuring spectral properties of reflectin. Unless otherwise stated, all work recorded on the wiki was carried out by the students. Credit for the wiki code to team member Haydn King'''<br />
<br />
[[File:Team_photo.png|center|frame|From left to right: Haydn, Jonathan, Marta, Heather, Katy, Cat, Matt, Joe and Felix]]<br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/TeamTeam:Cambridge/Team2011-09-22T03:29:52Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
Our core team was made of 9 Cambridge University [[Team:Cambridge/Team/Students | undergraduates]] from a wide range of courses. There were 4 biologists, three engineers, one physicist and one mathematician. While every member of the group broadened their knowledge, our different areas of expertise allowed us to simultaneously approach problems from many angles. <br />
<br />
We were also aided by a student from the Royal College of Art (RCA) who was able to provide us with a completely different perspective on our work. Her advice and creative input was invaluable to our project.<br />
<br />
We were advised by several [[Team:Cambridge/Team/Academics | academics]] (both from within the university and externally), both professors and PhD students, who provided technical assistance and insight. They all did a fantastic job of providing positive feedback, without guiding the direction of our project.<br />
<br />
With the team's budget especially tight this year due to the increased cost of the competition, our [[Team:Cambridge/Team/Sponsors | sponsors]] were invaluable - they contributed the reagents, kits and cash which enabled us to take part.<br />
==Attribution==<br />
<br />
'''Work carried out primarily by our advisors is clearly labelled as such. In particular we are grateful to Paul Grant for assistance operating the confocal and for his advice during [[Team:Cambridge/Project/Microscopy| our microscopy]], and to Matthwe Hawkeye (or collaborator in the Nanophotonics Dept) for assisting Felix in making [[Team:Cambridge/Project/In_Vitro | thin films]] and in measuring spectral properties of reflectin.''' Unless otherwise stated, all work recorded on the wiki was carried out by the students. Credit for the wiki code to team member Haydn King'''<br />
<br />
[[File:Team_photo.png|center|frame|From left to right: Haydn, Jonathan, Marta, Heather, Katy, Cat, Matt, Joe and Felix]]<br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Protocols/Gibson_AssemblyTeam:Cambridge/Protocols/Gibson Assembly2011-09-22T03:22:14Z<p>Cat: /* Theory */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_PROTOCOL_HEAD}}<br />
==Gibson Assembly==<br />
<br />
===Theory===<br />
Gibson Assembly is a scar-free method of DNA recombination that is highly efficient and surpasses standard assembly in utility by easily assembling multiple fragments simultaneously. This protocol is adapted from [http://www.cambridgeigem.org/RFC57.pdf RFC57] by the 2010 Cambridge iGEM team.<br />
<br />
===Practice===<br />
<br />
Master Mix for Gibson Assembly<br />
{| border="1px"<br />
! scope="col" width="220" style="text-align:center;"|Reagent<br />
! scope="col" width="150" style="text-align:center;" |Volume (µl)<br />
|-<br />
|Taq ligase (40u/µl) <br />
|50<br />
|-<br />
|5x isothermal buffer <br />
|100<br />
|-<br />
|T5 exonuclease (1u/µl) <br />
|2<br />
|-<br />
|Phusion polymerase (2u/µl) <br />
|6.25<br />
|-<br />
|Nuclease-free water <br />
|216.75<br />
|-<br />
|'''Total''' <br />
|'''375'''<br />
|-<br />
|}<br />
<br />
Master Mix is 1.33x concentrated<br />
<br />
DNA and Gibson Master Mix should be combined with a volumetric ration of 1:3 in a PCR tube. The total volume can be 20-50µl.<br />
<br />
Set thermocycler containing the PCR tubes to 50 degrees C for 1 hour.<br />
<br />
===Safety===<br />
No bacteria are used during the reaction there is therefore little or no biological hazard. Nevertheless, it is important to observe correct laboratory procedure and wear appropriate clothing and gloves; nucleases are present on human skin. <br />
<br />
{{Template:Team:Cambridge/CAM_2011_PROTOCOL_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Project/In_VitroTeam:Cambridge/Project/In Vitro2011-09-22T03:03:50Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
=Isolating Reflectin and Making Thin Films=<br />
<br />
[[File:Cam crazy multilayer3.jpg | thumb | 250px | right | A PDMS on reflectin multilayer thin film]]<br />
<br />
Previous in vitro investigations of reflectin showed that it was possible to use a method called [[Team:Cambridge/Protocols/Flow_coating | flow coating]] to deposit a thin layer of reflectin onto a silicon substrate which would then demonstrate structural colour.<br />
<br />
We wanted to investigate using a different coating method – [[Team:Cambridge/Protocols/Spin_Coating | spin coating]] – and to try out different methods for [[Team:Cambridge/Protocols/Protein_Purification | protein purification]]. A polyhis-tagged variant of the protein was overexpressed and purified using a polyhis affinity column. Thin films were made by precipitating with either [[Team:Cambridge/Protocols/Acetone_Precipitation_of_Proteins | acetone]] or [[Team:Cambridge/Protocols/Ethanol_Precipitation_of_Proteins | ethanol]] and resuspending in HFIP before either spin coating or flow coating onto a silicon wafer.<br />
<br />
We also investigated an [[Team:Cambridge/Protocols/Inclusion_Body_Prep | inclusion body prep]], as we found that the protein formed inclusion bodies upon over-expression. By varying our purification and coating methods, we were able to create vibrant thin films.<br />
<br />
==[[Team:Cambridge/Experiments/Thin_Films | Thin Films]]==<br />
In order to demonstrate structural colour, thin films were created by re-suspending the purified protein in HFIP and [[Team:Cambridge/Protocols/Spin_Coating | spin coating]] or [[Team:Cambridge/Protocols/Flow_coating | flow coating]] the re-suspended protein onto a silicon substrate. This gave us brightly coloured thin films which responded to water vapour in the air by changing colour. We performed controls by making HFIP films and films with Bovine Serum Albumin (BSA, a generic protein) neither of which showed any structural colour.<br />
<br />
<center><br />
<gallery widths=150px caption='An initial thin film (left), alongside its controls'><br />
File:Cam Reflectin Thin Film2.jpg | ''Reflectin HFIP solution showed iridescence after coating''<br />
File:Cam HFIP only control thinfilm.jpg | ''HFIP (solvent) only control does not exhibit iridescence''<br />
File:BSAcontrolfilm1.jpg | ''Bovine Serum Albumin makes a dull, striated thin film''<br />
</gallery><br />
</center><br />
<br />
Our initial films were not uniform in colour and would form crystals when allowed to dry out. We improved this by increasing the purity of our protein as well as refining our coating technique.<br />
<br />
We first tried to reduce the urea content of our sample, in the belief that this would reduce the tendency to crystallise, however, this did not work as our sample became too dilute. Almost by accident, we tried using a [[Team:Cambridge/Experiments/Reflectin_Thin_Films_IV | lower volume of protein]] and found - to our great surprise - that the quality of the thin films produced increased greatly, and the crystallisation was greatly reduced.<br />
<br />
The reflectin thin films were dynamic - they responded to water vapour in the air. The video below shows the effect of breathing several times on a thin film - the colour change is quite dramatic.<br />
<br />
<center><br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/X3pexBYWOrY" frameborder="0" allowfullscreen></iframe></html><br />
</center><br />
<br />
Flushed with success, we then pushed on to make [[Team:Cambridge/Experiments/Reflectin_Thin_Films_VI | multi layers]] of Reflectin, hoping that this would again increase the brightness and uniformity of the thin films. The beauty of the results was astonishing.<br />
<br />
<center><br />
<gallery caption='A sample of the microscope images taken of multi-layers.' widths=140px><br />
File:Cam Multilayer drop 1.jpg <br />
File:Cam Crazy multilayer single AP 2k spin2nd.jpg<br />
File:Cam crazy multilayer2.jpg<br />
File:Cam_crazy_multilayer3.jpg<br />
</gallery><br />
</center><br />
<br />
==Polyhis-Tagging Reflectin==<br />
<br />
The reflectin genes were polyhis-tagged by incorporating the sequence of part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K128005 BBa_K128005] into the primers used to clone the gene. Polyhis tagging was chosen because it allows proteins to be purified with relatively simple apparatus, an affinity column, to which a metal containing resin is added traps the protein when cell lysate is added.<br />
<br />
==Over-Expression==<br />
<br />
Reflectin was placed on a high copy plasmid ([http://partsregistry.org/Part:pSB1A2 PSB1A2]) under an arabinose inducible promoter ([http://partsregistry.org/Part:BBa_I0500 pBad]) in order to express reflectin at a high level. As well as a negative control, we used an sfGFP-reflectin fusion as a control for reflecitn production.<br />
<br />
In the non-GFP cells we found that there were brightly lit points at the ends of nearly every cell, which were not present in the negative control. In the GFP fusion cells, these spots glowed strongly, while the rest of the cell was relatively dark. This indicated that reflectin produces inclusion bodies when expressed at high level.<br />
<br />
[[File:Cam Reflectin-GFP-inclusionbodies.jpg | thumb| 400px| center| ''E. coli'' transformed with our pBAD-ReflectinA1-GFP construct, induced by adding 1mM arabinose, demonstrating localisation of the protein.]]<br />
<br />
==Extraction & Purification==<br />
We investigated two principal methods for extracting the protein from the transformed <i>E. coli</i> - we lysed the cells and ran the lysate through a polyhis affinity column and we tried a proprietary inclusion body prep.<br />
<br />
===[[Team:Cambridge/Protocols/Protein_Purification | Polyhis-Tag Affinity Column]]===<br />
The polyhis affinity column allowed us to extract our tagged protein from the lysate without too much difficulty. In order to increase the purity of our sample we tried using an [[Team:Cambridge/Protocols/Acetone_Precipitation_of_Proteins | acetone]] or [[Team:Cambridge/Protocols/Ethanol_Precipitation_of_Protein | ethanol]] precipitation step to remove chemicals retained in the elution buffer and to concentrate the protein for downstream processing.<br />
<br />
Having done this, we ran our sample on an SDS Page protein gel, to verify that we had in fact purified reflectin. We were expecting to see a thick band of reflectin at around 43 kDa, which – as the image shows – was the case.<br />
<br />
[[File:CAM_SDS_POLYHIS.png | center | thumb | 500px | The result of the Polyhis affinity column SDS Page gel]]<br />
<br />
===[[Team:Cambridge/Protocols/Inclusion_Body_Prep | Inclusion Body Prep]]===<br />
<br />
The Norgen Proteospin inclusion body prep kit was experimented with as a commercial alternative to the previous purification method. The kit was simple to use, but required the use of an ultracentrifuge capable of spinning up to 25ml of fluid at 27,000g. It was found that the spinning forces specified in the protocol for these steps weren't capable of moving fluid through the necessary columns and higher forces were needed. <br />
<br />
On analysing the end-product of the purification with SDS-PAGE approximately 20 discrete bands were observed from 10-200 kda with 2 particularly bold bands, both of which are too short for reflectin. However, a reasonably thick band of protein was present at 43kDA (the correct length for reflctin).<br />
<br />
[[File:CAM_SDS_INCLUSION_BODY.png | center | thumb | 450px | The result of the inclusion body SDS Page gel]]<br />
<br />
As the gel shows, the resulting protein is nowhere near as pure as the result of the previous method as the inclusion bodies do not contain only reflectin. However, greater purity might have been achieved by using the result of the inclusion body prep as the first stage of the polyhis affinity protocol, as it would reduce the amount of non-polyhis protein going in to the column. This was not tested in practice, however, as our protein appeared to be pure enough for processing, and we did not have the time to experiment further.<br />
<br />
<br />
<br />
<br />
===Future Work===<br />
As well as further improving our thin film's colour, we also wanted to improve their long-term stability, as well as investigate controlling their colour electrically.<br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/desc/overview/homeTeam:Cambridge/desc/overview/home2011-09-22T03:02:05Z<p>Cat: </p>
<hr />
<div><p>Welcome to the Cambridge 2011 iGEM team's homepage!</p><br />
<p>From here, you can find out more about our <a href='/Team:Cambridge/Project'>project</a>, see the <a href='/Team:Cambridge/Parts'>parts</a> we submitted, browse the <a href='/Team:Cambridge/Experiments'>experiments</a> we performed to characterise them and read up on the <a href='/Team:Cambridge/Team'>team</a>. The <a href='/Team:Cambridge/Media'>media page</a> contains our most impressive images and videos.</p><br />
<p>You can also take a look at what we've done to help the wider <a href='/Team:Cambridge/Project/Gibthon'> SynBio community</a>, as well as read our <a href='/Team:Cambridge/Society'>report</a> on what effect iGEM has on its participants.</p></div>Cathttp://2011.igem.org/Team:Cambridge/desc/society/interviewsTeam:Cambridge/desc/society/interviews2011-09-22T02:04:45Z<p>Cat: </p>
<hr />
<div><p>Some previous iGEMers were generous enough to grant us interviews, allowing us to record their experiences of life after iGEM.</p><br />
<p>Their insights provided another perspective for <a href='/Team:Cambridge/Society/report'>our reflections upon the competition</a>. We are very grateful for their assistance.</p></div>Cathttp://2011.igem.org/Team:Cambridge/desc/society/interviewsTeam:Cambridge/desc/society/interviews2011-09-22T02:03:15Z<p>Cat: </p>
<hr />
<div><p>Some previous iGEMers were generous enough to grant us interviews, allowing us to record their experiences of life after iGEM.</p><br />
<p>Their insights provided another perspective for <a href='Team:Cambridge/Society/report'>our reflections upon the competition</a>. We are very grateful for their assistance.</p></div>Cathttp://2011.igem.org/Team:Cambridge/desc/society/interviewsTeam:Cambridge/desc/society/interviews2011-09-22T02:02:04Z<p>Cat: Created page with "<p>Some previous iGEMers were generous enough to grant us interviews, allowing us to record their experiences of life after iGEM.</p> <p>Their insights provided another perspecti..."</p>
<hr />
<div><p>Some previous iGEMers were generous enough to grant us interviews, allowing us to record their experiences of life after iGEM.</p><br />
<p>Their insights provided another perspective for <a herf='Team:Cambridge/Society/report'>our reflections upon the competition</a>. We are very grateful for their assistance.</p></div>Cathttp://2011.igem.org/Team:Cambridge/Experiments/Squid_Dissection_and_Tissue_SampleTeam:Cambridge/Experiments/Squid Dissection and Tissue Sample2011-09-22T01:43:11Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_EXPERIMENT_HEAD}}<br />
==Amplification of Reflectin Genes from Squid Genomic DNA==<br />
Before we could begin with the rest of our project, we needed the reflectin coding region. [http://en.wikipedia.org/wiki/Loligo ''Loligo''] tissue was sourced from fishing bait suppliers and culinary wholesalers in order to attempt genomic DNA extraction. Our literature search indicated that the reflectin gene from ''E. scolopes'' contains no introns, so genomic DNA should be suitable for expression in ''E. coli''.<br />
<br />
==Summary==<br />
We aimed to [[Team:Cambridge/Protocols/Extraction_of_genomic_DNA_from_squid | extract DNA]] from dissected squid tissue, amplify the relevant gene by [[Team:Cambridge/Protocols/PCR | PCR]] and perform [[Team:Cambridge/Protocols/Gel_Electrophoresis | gel electrophoresis]] to verify that we had successfully extracted reflectin.<br />
<br />
Our literature search suggested that (for reasons unknown) cephalopod DNA is difficult to isolate and clone from. Since we did not have any other way of getting our reflectin gene, we tried two different [[Team:Cambridge/Protocols/Extraction_of_genomic_DNA_from_squid | DNA extraction protocols]], in the hope that one would work successfully. One extraction protocol appeared to work, while the other left us with DNA which was pink – a sure sign that it was heavily contaminated.<br />
<br />
We proceeded with PCR on the successful extraction. By designing multiple sets of primers for each different DNA sample, it was hoped that we would see some successful amplification. However, by running [[Team:Cambridge/Protocols/Gel_Electrophoresis | gel electrophoresis]] we found that none of our samples had been successfully amplified.<br />
<br />
Whether this was due to our DNA extraction or due to a PCR problem soon became a moot point, as we were kindly sent synthetic reflectin genes by Wendy Crookes-Goodson, author of many of the papers on reflectin.<br />
<br />
==Detail==<br />
<br />
===Squid Dissection===<br />
<br />
Specimens of what we identified as [http://www.marlin.ac.uk/speciesinformation.php?speciesID=3718 ''Loligo vulgaris''] and [http://en.wikipedia.org/wiki/Opalescent_Inshore_Squid ''Loligo opalescens''] were dissected for examination. Samples of skin tissue and eye cups were taken from ''L. vulgaris'' for further imaging. No obvious iridescence was seen in the skin sample under a dissection microscope or by confocal microscopy, (though this is known to require the neurotransmitter acetyl choline in order to be activated). In accordance with [http://rsif.royalsocietypublishing.org/content/early/2011/02/14/rsif.2010.0702.full Holt ''et al''], we found clearly visible static iridescence in the tissue surrounding the eye lens in the form of the silvery tissue exhibiting a sheen of colours from across the visible spectrum.<br />
<center><br />
<gallery><br />
File:CAM_Cat_Squid.jpg | Preparing squid samples<br />
File:CAM_L_opalescens.jpg | ''Loligo opalescens''<br />
<!-- File:CAM_L_vulgaris.jpg |''Loligo vulgaris'' specimen dissected --><br />
File:CAM Iridescent.JPG | ''L. vulgaris'' eye cup, showing Bragg reflectance<br />
</gallery><br />
</center><br />
<br />
===DNA Extraction===<br />
<br />
To isolate squid genomic DNA as a template for PCR, samples of squid tissue were cut from representative specimens. Internal organs and tissue were dissected with the aim of minimizing external contamination. ''L. vulgaris'' specimens were obtained from two different sources, so samples were taken from both. <br />
<br />
A [[Team:Cambridge/Protocols/Extraction_of_genomic_DNA_from_squid | DNA extraction protocol]] was followed to isolate genomic DNA from tissue.<br />
<br />
===Design of Primers===<br />
<br />
We decided to perform PCR in order to isolate and amplify reflectin coding sequences from genomic DNA of ''Loligo vulgaris'' and ''Loligo opalescens''.<br />
As no reflectin gene sequences from the two species that we worked with are known, we designed primers relying on the published sequences of A1, A2 and B1 reflectin mRNAs from ''Loligo pealei''. <br />
<br />
The assumptions that we made and the way of our reasoning are the following: <br />
<br />
'''1. Because of close evolutionary relationship and an important role of reflectin proteins in the animal physiology and survival, we expect other squids from the Loligo genus to express the same, or very similar, set of reflectin proteins.''' <br />
<br />
:Although the phylogeny of the loliginid squids has not been fully revealed yet, a fairly consistent picture of evolutionary relationships within the taxon has emerged from several studies, which included:<br />
:*comparison of sequences of two mitochondrial genes (the 16S rRNA and the cytochrome c oxidase subunit I genes) between members of 19 loliginid species and several outgroups (Anderson, 2000 [1]);<br />
:*comparison of multiple data sets, such as morphology, allozymes and DNA seqence data for the two mitochondrial genes (Anderson, 2000 [2]);<br />
:The fairly reliable phylogenetic tree of the loliginid squids is presented below:<br />
[[File:cam_phylogenetictree_ofloligo.jpg | center | 700px | ]]<br />
<br />
:According to the phylogenetic tree, ''L. pealei'' is fairly closely related to ''L. opalescens'', and ''L. vulgaris'' shows close evolutionary relationship with ''L. forbesi'', whose sequence of reflectin-like protein mRNA is also published in the GenBank database. However, the exact evolutionary history of the two taxons is not resolved yet. <br />
<br />
'''2. As the amino acid sequence of proteins is generally more conserved than nucleotide sequence, we considered designing degenerate primers, using [http://www.kazusa.or.jp/codon/cgi-bin/spsearch.cgi?species=loligo&c=i codon usage tables] to maximize the likelihood of amplifying ''L. opalescens'' and ''L. vulgaris'' reflectin genes.''' <br />
:However, comparison of reflectin mRNA sequences of ''Loligo pealei'' and reflectin-like methionine-rich repeat protein 1 mRNA sequence from ''Loligo forbesi'' showed quite high conservation of the nucleotide sequence.<br />
:*We performed BLAST analysis using the methionine-rich repeat protein as a query and comparing it to A1 and A2 reflectin sequences from ''Loligo pealei''. <br />
:*After that, we analysed conservation of nucleotide sequences of the highly conserved protein regions.<br />
[[File:cam_blastalignment_A2.jpg | left | 320px | thumb | ''Loligo pealei'' reflectin-like A1 protein]]<br />
[[File:cam_blastalignment_A1.jpg | right | 320px | thumb | ''Loligo pealei'' reflectin-like A2 protein]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Consequently, we decided not to take codon bias into consideration and design primers complementary to terminal sequences of reflectin mRNAs from ''L. pealei'', so that they would allow us to amplify the entire coding sequence, from the START to the STOP codon. <br />
<br />
{| border="1px" align="center" style="text-align:center;"<br />
!scope="col" width="80" | Primer<br />
!scope="col" width="220" | Reflectin A1<br />
!scope="col" width="220" | Reflectin A2<br />
!scope="col" width="220" | Reflectin B1<br />
|-<br />
| '''Forward'''<br />
| 5’ ATGAATCGAT ATCTGAATCG 3’<br />
| 5’ ATGAATCGCT ACATGATGAG 3’ <br />
| 5’ ATGTCTTCTT TTATGGATCC 3’ <br />
|-<br />
| '''Reverse'''<br />
| 5’ TTAATACATG TGATAGTCGT 3’<br />
| 5’ CTAATACCAA GAATTGTAAT 3’<br />
| 5’ TTAGGCTGAA TCTGTGAGCT 3’ <br />
|}<br />
<br />
It is worth emphasizing that the published sequences are mRNA sequences, giving us no information about endogenous promoters or 5'UTR and 3'UTR regions. Additionally, they do not provide any information about the presence and position of introns either. <br />
*However, according to the literature (Crookes, 2004), the bobtail squid reflectin genes do not contain introns, and thus, assuming the common evolutionary origin, reflectin genes from other squid species are likely not to include introns as well. <br />
*Thus, high deviation of PCR products from the expected length will give us a clue about the presence of intro sequences, although this might be also caused by other factors, such as:<br />
**interspecific variation<br />
**misannealing and amplification of other sequences than reflectin genes<br />
<br />
===PCR Reaction===<br />
The next step involved PCR reaction with Phusion Hot Start II DNA Polymerase, according to the following [[Team:Cambridge/Protocols/PCR | protocol]]. We chose a modified version of PCR - gradient PCR, which allowed us to test different annealing temperatures. As we did not expect the primers to show complete fidelity to the coding sequences of the analyzed species, different conditions of annealing would ensure that we would amplify the most matching sequences from the genomic DNA template.<br />
<br />
We prepared 72 PCR reactions:<br />
{| align="center" style="text-align:center;"<br />
|scope="col" width="300" | '''genomic DNA template''' from three different squids: two ''L. vulgaris'' individuals and one ''L. opalescens'' individual<br />
|scope="col" width="20" |×<br />
|scope="col" width="300" | '''three sets of primers: ''' A1, A2 and B1<br />
|scope="col" width="20" |×<br />
|scope="col" width="300" | '''eight different annealing temperatures''' ranging from 72°C to 50°C<br />
|}<br />
<br />
===Gel Electrophoresis===<br />
<br />
We performed [[Team:Cambridge/Protocols/Gel_Electrophoresis | gel electrophoresis]] of the products of PCR reaction in order to check:<br />
*how effective the reaction of amplification was,<br />
*if the size of PCR products roughly matched the length of reflectin mRNAs from Loligo pealei, and<br />
*how successful the DNA extraction protocol was.<br />
<br />
Samples run on the first gel included:<br />
<br />
{| border="1" style="text-align:center;"<br />
! scope="col" width="120" | Well<br />
! scope="col" width="120" | 1<br />
! scope="col" width="120" | 2<br />
! scope="col" width="120" | 3<br />
! scope="col" width="120" | 4<br />
! scope="col" width="120" | 5<br />
! scope="col" width="120" | 6<br />
|- align="center"<br />
|'''Sample'''<br />
|HyperLadder I<br />
|''L. opalescens''<br/> A1 primers <br/> 72.0°C<br />
|''L. opalescens''<br/> A1 primers <br/> 68.9°C<br />
|''L. opalescens''<br/> A1 primers <br/> 65.7°C<br />
|''L. opalescens''<br/> A1 primers <br/> 62.6°C<br />
|''L. opalescens''<br/> A1 primers <br/> 59.4°C<br />
|- align="center"<br />
!'''7'''<br />
!'''8'''<br />
!'''9'''<br />
!'''10'''<br />
!'''11'''<br />
!'''12'''<br />
!'''13'''<br />
|- align="center"<br />
|''L. opalescens''<br/> A1 primers <br/> 56.3°C<br />
|''L. opalescens''<br/> A1 primers <br/> 53.1°C<br />
|''L. opalescens''<br/> A1 primers <br/> 50.0°C<br />
|HyperLadder I<br />
|''L. opalescens''<br/> A2 primers <br/> 72.0°C<br />
|''L. opalescens''<br/> A2 primers <br/> 68.9°C<br />
|''L. opalescens''<br/> A2 primers <br/> 65.7°C<br />
|- align="center"<br />
!'''14'''<br />
!'''15'''<br />
!'''16'''<br />
!'''17'''<br />
!'''18'''<br />
!'''19'''<br />
!'''20'''<br />
|- align="center"<br />
|''L. opalescens''<br/> A2 primers <br/> 62.6°C<br />
|''L. opalsecens''<br/> A2 primers <br/> 59.4°C<br />
|''L. opalescens''<br/> A2 primers <br/> 56.3°C<br />
|''L. opalescens''<br/> A2 primers <br/> 53.1°C<br />
|''L. opalescens''<br/> A2 primers <br/> 50.0°C<br />
|HyperLadder I<br />
|''L. opalescens''<br/> genomic DNA<br />
|}<br />
Note: HyperLadder I is a set of molecular weight markers that allow for quantification and size determination within the 200bp - 10,000bp range.<br />
<br />
As we can see from the picture of the gel:<br />
*No bands are visible in the lanes loaded with the PCR products, and surprisingly neither primers nor template DNA can be detected either.<br />
*There is no trace of DNA in the last lane (numer 20) which was loaded with the supernatant obtained during the squid DNA extraction process <br />
<br />
[[File:cam_gelelectrophoresis_2107.jpg | center | thumb | 400px | A picture of the first gel with wells numbered from the left to the right. Only three lanes with HyperLadder are visible. ]]<br />
<br />
<br />
These observations suggest low efficiency of the extraction method. To check if this problem was unique to DNA from ''Loligo opalescens'', we ran a second gel which included genomic DNA from the two ''Loligo vulgaris'' individuals as well as an array of PCR reactions conducted at different temperatures (65.7°C and 56.3°C) and with different sets of primers.<br />
<br />
{| border="1" style="text-align:center;"<br />
! scope="col" width="120" | Well<br />
! scope="col" width="120" | 1<br />
! scope="col" width="120" | 2<br />
! scope="col" width="120" | 3<br />
! scope="col" width="120" | 4<br />
! scope="col" width="120" | 5<br />
! scope="col" width="120" | 6<br />
|- align="center"<br />
|'''Sample'''<br />
|HyperLadder I<br />
|''L. vulgaris lf'' <br/> genomic DNA <br/> <br />
|''L. vulgaris ba'' <br/> genomic DNA <br/> <br />
|''L. opalescens'' <br/> B1 primers <br/> 65.7°C<br />
|''L. vulgaris lf'' <br/> A1 primers <br/> 65.7°C<br />
|''L. vulgaris lf'' <br/> A2 primers <br/> 65.7°C<br />
|- align="center"<br />
!'''7'''<br />
!'''8'''<br />
!'''9'''<br />
!'''10'''<br />
!'''11'''<br />
!'''12'''<br />
!'''13'''<br />
|- align="center"<br />
|''L. vulgaris lf''<br/> B1 primers <br/> 65.7°C<br />
|''L. vulgaris ba'' <br/> A1 primers <br/> 65.7°C<br />
|''L. vulgaris ba'' <br/> A2 primers <br/> 65.7°C<br />
|''L. vulgaris ba'' <br/> B1 primers <br/> 65.7°C<br />
|HyperLadder I<br />
|''L. opalescens''<br/> B1 primers <br/> 56.3°C<br />
|''L. vulgaris lf'' <br/> A1 primers <br/> 56.3°C<br />
|- align="center"<br />
!'''14'''<br />
!'''15'''<br />
!'''16'''<br />
!'''17'''<br />
!'''18'''<br />
!'''19'''<br />
!'''20'''<br />
|- align="center"<br />
|''L. vulgaris lf'' <br/> A2 primers <br/> 56.3°C<br />
|''L. vulgaris lf'' <br/> B1 primers <br/> 56.3°C<br />
|''L. vulgaris ba'' <br/> A1 primers <br/> 56.3°C<br />
|''L. vulgaris ba'' <br/> A2 primers <br/> 56.3°C<br />
|''L. vulgaris ba'' <br/> B1 primers <br/> 56.3°C<br />
|HyperLadder I<br />
|''L.opalescens''<br/> genomic DNA<br />
|}<br />
Note: ''L. vulgaris ba'' stands for the ''Loligo'' squid obtained from fishing bait suppliers, while ''L. vulgaris lf'' stands for the ''Loligo'' squid bought from the local culinary wholesaler. <br />
<br />
We could see a similar pattern in the second gel:<br />
*no products of PCR reactions, as well as primers and template DNA were visible,<br />
*no smear of genomic DNA from ''L. opalescens'' and ''L. vulgaris ba'' was detected,<br />
*however, we noticed a thin faint band of genomic DNA from ''L. vulgaris lf'', but the detectable amount of the template DNA did not improve the efficiency of corresponding PCR reactions.<br />
<br />
[[File:cam_gelelectrophoresisI_2107.jpg | center | thumb | 400px | A close-up of the second gel with the HyperLadder I on the leftmost lane and a faint band of ''L. vulgaris'' genomic DNA in the adjacent lane. ]]<br />
<br />
We concluded that the DNA extraction protocol applied in this experiment was not very efficient and that this was the main reason why we failed to obtain any amplified reflectin or reflectin-related genes.<br />
<br />
===References===<br />
#'''Anderson F.E. (2000)''' Phylogeny and historical biogeography of the loliginid squids (Mollusca: Cephalopoda) based on mitochondrial DNA sequence data. ''Molecular Phylogenetics and Evolution'', 15: 191-214.<br />
#'''Anderson F.E. (2000)''' Phylogenetic relationships among loliginid squids (Cephalopoda: Myopsida) based on analyses of multiple data sets. ''Zoological Journal of the Linnean Society'', 130: 603–633.<br />
#'''Crookes W.J., Ding L., Huang Q.L., Kimbell J.R., Horwitz J., McFall-Ngai M.J. (2004)''' Reflectins: the unusual proteins of squid reflective tissues. ''Science'', 303: 235–238.<br />
<br />
<br />
{{Template:Team:Cambridge/CAM_2011_EXPERIMENT_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Society/interviewsTeam:Cambridge/Society/interviews2011-09-22T01:42:34Z<p>Cat: /* The impact of iGEM – an interview with Justin Pahara */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==Background==<br />
On our online questionnaire, participants were asked if they were willing to be contacted with some further questions about their iGEM experience and their subsequent careers. It was good to see that a large proportion of our survey participants answered in the affirmative to this.<br />
<br />
<br />
<html><div style='float:right;margin-left:20px;'><iframe width="425" height="349" src="http://www.youtube.com/embed/bmk576dEXts?hl=en&fs=1" frameborder="0" allowfullscreen></iframe></div></html>Four respondents were contacted. Two (Shuna Gould and Justin Pahara) were working in Cambridge and were willing to drop by the lab for a filmed interview. Kim de Mora was in Edinburgh and recorded via skype. Joel Grim was working in the U.S. and contacted via Google Chat.<br />
<br style='clear:both' /><br />
==The impact of iGEM – an interview with Justin Pahara==<br />
[[File:Justin1.jpg |frameless|200px|left| ]]<br />
Justin Pahara was first involved in iGEM shortly after finishing his first degree in Immunology and Infection. He met Andrew Hessle, who he describes as an evangelist for Synthetic Biology. Through this meeting Justin was introduced to synthetic biology which not only changed his views, but lead him to cross the globe, pursue a PHD in biotechnology and found several start up companies including one orientated towards catalysing interdisciplinary work in synthetic biology.<br />
[[File:CAM_Justin1.png |frameless|200px|right| ]]<br />
In 2007, Justin was part of Andrew Hessle’s Alberta University team as iGEM was brought to Canada for the first time. The team focussed on engineering biobutanol production, as they believed it could be a biofuel superior to bioethanol, hence their slogan “save the ethanol for drinking”. In 2008 Justin was also an iGEM advisor on the 2008 team.<br />
<br />
We asked Justin if iGEM changed the kind of people you expected to work with. With some conviction he told us the iGEM most definitely had; he is now a strong advocate of interdisciplinary work having seen the benefits that can come from bringing expertise from artists, businesspeople, and engineers together to push science forward. He stressed that the solution to a problem will often be better when found in conjunction with a team from diverse backgrounds than when limited to colleagues in your own academic circle - a step outside the scientific comfort zone.<br />
[[File:CAM Justin2.png |frameless|200px|left|]]<br />
Perhaps one of the biggest influences on an alumnus iGEM could ever have is convincing them that they could make a living in synthetic biology. This is what iGEM has done for Justin Pahara. His company Synbiota, founded with colleagues who work in bioinformatics and software development, will provide tools and services to enable and equip scientists, particularly those in synthetic biology, with a grand aim of making a web of information and idea sharing which they hope will improve the productivity of the field from grass-roots school projects to field leaders.<br />
<br />
We probed him to find out how much of iGEM had rubbed off on him and his business ventures. He replied that Synbiota is adhering to synthetic biology principles. They agree with abstraction as a valuable process by which to remove unnecessary complexity and strongly support crowd-sourcing of information as an engine with which to power progress. Justin firmly believes that it is only a matter of time until the rest of the world adopts these principles in broader contexts.<br />
<br />
Justin claims that he would always have headed into biotechnology to make his name, but that iGEM resulted in a profound change in his ideas about science and engineering. Although he believes he would have realised the benefits of concepts such as abstraction and crowd-sourcing eventually, he feels he has had a head-start on colleagues working in biotech as a direct result of his involvement in iGEM and being introduced to synthetic biology. For Justin, the most important thing he learnt was the parallels that can be drawn between synthetic biology and the software industry as markets.<br />
[[File:CAM_Justin3.png |frameless|200px|right|]]<br />
In particular Justin envisages a future where the cost of DNA synthesis comes down to the point where, like when the personal computer entered peoples homes, making DNA will be not only relatively quick and cheap, but a technology with a rapidly growing user base. In fact he predicts that the design and ‘light tinkering’ of DNA is a field that will literally explode. He paints an inspiring picture one of DNA modification by those in academia, corporations, DIY bio, even junior high school kids will be just chilling out, writing DNA code.<br />
<br />
Questions by Cambridge iGEM 2011 Team, Interview by Joe Harvey, Write-up by Matt Jones.<br />
<br />
==The impact of iGEM – an interview with Shuna Gould==<br />
[[File:Cam Shuna1b.JPG|frameless|200px|right]]<br />
Shuna gould heard about iGEM in her second year of studying Natural sciences at Cambridge and was attracted partly because of the exciting work that previous Cambridge teams had done. Having done a couple of summer research projects before she was particularly interested in iGEM because of the independent nature of the research.<br />
<br />
Shuna is therefore in an excellent position to compare iGEM to other summer research projects. She points out that it is much more challenging, intense and a powerful learning curve. Several of our team, and postgraduates we’ve spoken to agree and we’re sure that throughout iGEM there will be countless other people who recognise that this is no ordinary summer research project.<br />
<br />
We heard how iGEM has influenced Shuna’s career not only in terms of providing opportunities within science, but also how she feels ready to make a leap into completely different lines of work after her PHD, thanks to skills at least partly developed during iGEM.<br />
<br />
Soon after iGEM Shuna was fortunate to be able to continue her teams work with two of the principal investigators involved in supervising her team, Jim Haseloff and Jim Ajioka from Cambridge University. iGEM has also impacted on her path since then, and she is now embarking on a PHD in which she plans to use techniques she was first taught during iGEM.<br />
[[File:CAM_Shuna2.png|frameless|200px|left]]<br />
One key influence for Shuna was that iGEM opened her mind to PHD topics that she had not previously considered and that her chosen PHD supervisor was excited when he heard that she had had training in synthetic biology techniques. Furthermore, together they have discussed in depth how Gibson assembly can be used to aid her in reliably cutting and pasting genomes during her project which will be dealing with much larger lengths of DNA than she used during her bacterial plasmid work in iGEM. This is at least one example of a situation where techniques taught in iGEM are infiltrating into more in-depth research like a PHD thesis as iGEM students progress in academia.<br />
[[File:CAM_Shuna3.png|frameless|200px|right]]<br />
It is fascinating to hear how iGEM has affected peoples professional lives, but we wanted to know how synthetic biology has affected the mindsets of iGEM alumni. Shuna said she was initially sceptical about the use of abstraction in biology, as her biochemistry training taught her that all biological systems are wonderfully complex. However, through iGEM she learnt that engineering principles allow a fresh, common sense approach to biology, and she also commented on the power of being able to take components out of systems in order to explore their properties and that being encouraged to ‘play around’ with genes is not only exciting but often a very useful thought process. Moreover, iGEM pushed Shuna to get to grips with modelling and to explore the use of computational tools in her research, which will undoubtedly be useful in the future.<br />
[[File:CAM_Shuna4.png|frameless|200px|left]]<br />
When we asked Shuna about the open source nature of iGEM we unearthed a conflict. Shuna said that she loves the open source ethos of iGEM, the fact that her team made genes for bacterial pigments that are now widely available for anyone else to use is fantastic, she told us. However, she did say that if something profitable were to come from her PHD research, she would look into making money from it, and recognises that research in an open source world lacks that reinvestment of profits that commercialised research has.<br />
<br />
Finally we asked about the impact of synthetic biology in the wider world. Shuna told us that she is most excited about what synthetic biology can do for medicine, mentioning the ease with which microorganisms can synthesise drugs like antibiotics which are otherwise require a complex and costly chemical synthesis.<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Experiments/Squid_Dissection_and_Tissue_SampleTeam:Cambridge/Experiments/Squid Dissection and Tissue Sample2011-09-22T01:39:03Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_EXPERIMENT_HEAD}}<br />
==Amplification of Reflectin Genes from Squid Genomic DNA==<br />
Before we could begin with the rest of our project, we needed the reflectin coding region. [http://en.wikipedia.org/wiki/Loligo ''Loligo''] tissue was sourced from fishing bait suppliers and culinary wholesalers in order to attempt genomic DNA extraction. Our literature search indicated that the reflectin gene from ''E. scolopes'' contains no introns, so genomic DNA should be suitable for expression in ''E. coli''.<br />
<br />
==Summary==<br />
We aimed to [[Team:Cambridge/Protocols/Extraction_of_genomic_DNA_from_squid | extract DNA]] from dissected squid tissue, amplify the relevant gene by [[Team:Cambridge/Protocols/PCR | PCR]] and perform [[Team:Cambridge/Protocols/Gel_Electrophoresis | gel electrophoresis]] to verify that we had successfully extracted reflectin.<br />
<br />
Our literature search suggested that (for reasons unknown) cephalopod DNA is difficult to isolate and clone from. Since we did not have any other way of getting our reflectin gene, we tried two different [[Team:Cambridge/Protocols/Extraction_of_genomic_DNA_from_squid | DNA extraction protocols]], in the hope that one would work successfully. One extraction protocol appeared to work, while the other left us with DNA which was pink – a sure sign that it was heavily contaminated.<br />
<br />
We proceeded with PCR on the successful extraction. By designing multiple sets of primers for each different DNA sample, it was hoped that we would see some successful amplification. However, by running [[Team:Cambridge/Protocols/Gel_Electrophoresis | gel electrophoresis]] we found that none of our samples had been successfully amplified.<br />
<br />
Whether this was due to our DNA extraction or due to a PCR problem soon became a moot point, as we were kindly sent synthetic reflectin genes by Wendy Crookes-Goodson, author of many of the papers on reflectin.<br />
<br />
==Detail==<br />
<br />
===Squid Dissection===<br />
<br />
Specimens of what we identified as [http://www.marlin.ac.uk/speciesinformation.php?speciesID=3718 ''Loligo vulgaris''] and [http://en.wikipedia.org/wiki/Opalescent_Inshore_Squid ''Loligo opalescens''] were dissected for examination. Samples of skin tissue and eye cups were taken from ''L. vulgaris'' for further imaging. No obvious iridescence was seen in the skin sample under a dissection microscope or by confocal microscopy, (though this is known to require the neurotransmitter acetyl choline in order to be activated). In accordance with [http://rsif.royalsocietypublishing.org/content/early/2011/02/14/rsif.2010.0702.full Holt ''et al''], we found clearly visible static iridescence in the tissue surrounding the eye lens in the form of the silvery tissue exhibiting a sheen of colours from across the visible spectrum.<br />
<center><br />
<gallery><br />
File:CAM_Cat_Squid.jpg | Preparing squid samples<br />
File:CAM_L_opalescens.jpg | ''Loligo opalescens''<br />
<!-- File:CAM_L_vulgaris.jpg |''Loligo vulgaris'' specimen dissected --><br />
File:CAM Iridescent.JPG | ''L. vulgaris'' eye cup, showing Bragg reflectance<br />
</gallery><br />
</center><br />
<br />
===DNA Extraction===<br />
<br />
To isolate squid genomic DNA as a template for PCR, samples of squid tissue were cut from representative specimens. Internal organs and tissue were dissected with the aim of minimizing external contamination. ''L. vulgaris'' specimens were obtained from two different sources, so samples were taken from both. <br />
<br />
A [[Team:Cambridge/Protocols/Extraction_of_genomic_DNA_from_squid | DNA extraction protocol]] was followed to isolate genomic DNA from tissue.<br />
<br />
===Design of Primers===<br />
<br />
We decided to perform PCR in order to isolate and amplify reflectin coding sequences from genomic DNA of ''Loligo vulgaris'' and ''Loligo opalescens''.<br />
As no reflectin gene sequences from the two species that we worked with are known, we designed primers relying on the published sequences of A1, A2 and B1 reflectin mRNAs from ''Loligo pealei''. <br />
<br />
The assumptions that we made and the way of our reasoning are the following: <br />
<br />
'''1. Because of close evolutionary relationship and an important role of reflectin proteins in the animal physiology and survival, we expect other squids from the Loligo genus to express the same, or very similar, set of reflectin proteins.''' <br />
<br />
:Although the phylogeny of the loliginid squids has not been fully revealed yet, a fairly consistent picture of evolutionary relationships within the taxon has emerged from several studies, which included:<br />
:*comparison of sequences of two mitochondrial genes (the 16S rRNA and the cytochrome c oxidase subunit I genes) between members of 19 loliginid species and several outgroups (Anderson, 2000 [1]);<br />
:*comparison of multiple data sets, such as morphology, allozymes and DNA seqence data for the two mitochondrial genes (Anderson, 2000 [2]);<br />
:The fairly reliable phylogenetic tree of the loliginid squids is presented below:<br />
[[File:cam_phylogenetictree_ofloligo.jpg | center | 700px | ]]<br />
<br />
:According to the phylogenetic tree, ''L. pealei'' is fairly closely related to ''L. opalescens'', and ''L. vulgaris'' shows close evolutionary relationship with ''L. forbesi'', whose sequence of reflectin-like protein mRNA is also published in the GenBank database. However, the exact evolutionary history of the two taxons is not resolved yet. <br />
<br />
'''2. As the amino acid sequence of proteins is generally more conserved than nucleotide sequence, we considered designing degenerate primers, using [http://www.kazusa.or.jp/codon/cgi-bin/spsearch.cgi?species=loligo&c=i codon usage tables] to maximize the likelihood of amplifying ''L. opalescens'' and ''L. vulgaris'' reflectin genes.''' <br />
:However, comparison of reflectin mRNA sequences of ''Loligo pealei'' and reflectin-like methionine-rich repeat protein 1 mRNA sequence from ''Loligo forbesi'' showed quite high conservation of the nucleotide sequence.<br />
:*We performed BLAST analysis using the methionine-rich repeat protein as a query and comparing it to A1 and A2 reflectin sequences from ''Loligo pealei''. <br />
:*After that, we analysed conservation of nucleotide sequences of the highly conserved protein regions.<br />
[[File:cam_blastalignment_A2.jpg | left | 320px | thumb | ''Loligo pealei'' reflectin-like A1 protein]]<br />
[[File:cam_blastalignment_A1.jpg | right | 320px | thumb | ''Loligo pealei'' reflectin-like A2 protein]]<br />
<br />
<br style="clear:both;"/><br />
<br />
Consequently, we decided not to take codon bias into consideration and design primers complementary to terminal sequences of reflectin mRNAs from ''L. pealei'', so that they would allow us to amplify the entire coding sequence, from the START to the STOP codon. <br />
<br />
{| border="1px" align="center" style="text-align:center;"<br />
!scope="col" width="80" | Primer<br />
!scope="col" width="220" | Reflectin A1<br />
!scope="col" width="220" | Reflectin A2<br />
!scope="col" width="220" | Reflectin B1<br />
|-<br />
| '''Forward'''<br />
| 5’ ATGAATCGAT ATCTGAATCG 3’<br />
| 5’ ATGAATCGCT ACATGATGAG 3’ <br />
| 5’ ATGTCTTCTT TTATGGATCC 3’ <br />
|-<br />
| '''Reverse'''<br />
| 5’ TTAATACATG TGATAGTCGT 3’<br />
| 5’ CTAATACCAA GAATTGTAAT 3’<br />
| 5’ TTAGGCTGAA TCTGTGAGCT 3’ <br />
|}<br />
<br />
It is worth emphasizing that the published sequences are mRNA sequences, giving us no information about endogenous promoters or 5'UTR and 3'UTR regions. Additionally, they do not provide any information about the presence and position of introns either. <br />
*However, according to the literature (Crookes, 2004), the bobtail squid reflectin genes do not contain introns, and thus, assuming the common evolutionary origin, reflectin genes from other squid species are likely not to include introns as well. <br />
*Thus, high deviation of PCR products from the expected length will give us a clue about the presence of intro sequences, although this might be also caused by other factors, such as:<br />
**interspecific variation<br />
**misannealing and amplification of other sequences than reflectin genes<br />
<br />
===PCR Reaction===<br />
The next step involved PCR reaction with Phusion Hot Start II DNA Polymerase, according to the following [[Team:Cambridge/Protocols/PCR | protocol]]. We chose a modified version of PCR - gradient PCR, which allowed us to test different annealing temperatures. As we did not expect the primers to show complete fidelity to the coding sequences of the analyzed species, different conditions of annealing would ensure that we would amplify the most matching sequences from the genomic DNA template.<br />
<br />
We prepared 72 PCR reactions:<br />
{| align="center" style="text-align:center;"<br />
|scope="col" width="300" | '''genomic DNA template''' from three different squids: two ''L. vulgaris'' individuals and one ''L. opalescens'' individual<br />
|scope="col" width="20" |×<br />
|scope="col" width="300" | '''three sets of primers: ''' A1, A2 and B1<br />
|scope="col" width="20" |×<br />
|scope="col" width="300" | '''eight different annealing temperatures''' ranging from 72°C to 50°C<br />
|}<br />
<br />
===Gel Electrophoresis===<br />
<br />
We performed [[Team:Cambridge/Protocols/Gel_Electrophoresis | gel electrophoresis]] of the products of PCR reaction in order to check:<br />
*how effective the reaction of amplification was,<br />
*if the size of PCR products roughly matched the length of reflectin mRNAs from Loligo pealei, and<br />
*how successful the DNA extraction protocol was.<br />
<br />
Samples run on the first gel included:<br />
<br />
{| border="1" style="text-align:center;"<br />
! scope="col" width="120" | Well<br />
! scope="col" width="120" | 1<br />
! scope="col" width="120" | 2<br />
! scope="col" width="120" | 3<br />
! scope="col" width="120" | 4<br />
! scope="col" width="120" | 5<br />
! scope="col" width="120" | 6<br />
|- align="center"<br />
|'''Sample'''<br />
|HyperLadder I<br />
|''L. opalescens''<br/> A1 primers <br/> 72.0°C<br />
|''L. opalescens''<br/> A1 primers <br/> 68.9°C<br />
|''L. opalescens''<br/> A1 primers <br/> 65.7°C<br />
|''L. opalescens''<br/> A1 primers <br/> 62.6°C<br />
|''L. opalescens''<br/> A1 primers <br/> 59.4°C<br />
|- align="center"<br />
!'''7'''<br />
!'''8'''<br />
!'''9'''<br />
!'''10'''<br />
!'''11'''<br />
!'''12'''<br />
!'''13'''<br />
|- align="center"<br />
|''L. opalescens''<br/> A1 primers <br/> 56.3°C<br />
|''L. opalescens''<br/> A1 primers <br/> 53.1°C<br />
|''L. opalescens''<br/> A1 primers <br/> 50.0°C<br />
|HyperLadder I<br />
|''L. opalescens''<br/> A2 primers <br/> 72.0°C<br />
|''L. opalescens''<br/> A2 primers <br/> 68.9°C<br />
|''L. opalescens''<br/> A2 primers <br/> 65.7°C<br />
|- align="center"<br />
!'''14'''<br />
!'''15'''<br />
!'''16'''<br />
!'''17'''<br />
!'''18'''<br />
!'''19'''<br />
!'''20'''<br />
|- align="center"<br />
|''L. opalescens''<br/> A2 primers <br/> 62.6°C<br />
|''L. opalsecens''<br/> A2 primers <br/> 59.4°C<br />
|''L. opalescens''<br/> A2 primers <br/> 56.3°C<br />
|''L. opalescens''<br/> A2 primers <br/> 53.1°C<br />
|''L. opalescens''<br/> A2 primers <br/> 50.0°C<br />
|HyperLadder I<br />
|''L. opalescens''<br/> genomic DNA<br />
|}<br />
Note: HyperLadder I is a set of molecular weight markers that allow for quantification and size determination within the 200bp - 10,000bp range.<br />
<br />
As we can see from the picture of the gel:<br />
*no bands are visible in the lanes loaded with the PCR products, and surprisingly neither primers nor template DNA can be detected as well<br />
*there is no trace of DNA in the last lane (numer 20) which was loaded with the supernatant obtained during the squid DNA extraction process <br />
<br />
[[File:cam_gelelectrophoresis_2107.jpg | center | thumb | 400px | A picture of the first gel with wells numbered from the left to the right. Only three lanes with HyperLadder are visible. ]]<br />
<br />
<br />
These observations suggest low efficiency of the extraction method. To check if this problem was unique to DNA from Loligo opalescens, we ran a second gel which included genomic DNA from the two ''Loligo vulgaris'' individuals as well as an array of PCR reactions conducted at different temperatures (65.7°C and 56.3°C) and with different sets of primers.<br />
<br />
{| border="1" style="text-align:center;"<br />
! scope="col" width="120" | Well<br />
! scope="col" width="120" | 1<br />
! scope="col" width="120" | 2<br />
! scope="col" width="120" | 3<br />
! scope="col" width="120" | 4<br />
! scope="col" width="120" | 5<br />
! scope="col" width="120" | 6<br />
|- align="center"<br />
|'''Sample'''<br />
|HyperLadder I<br />
|''L. vulgaris lf'' <br/> genomic DNA <br/> <br />
|''L. vulgaris ba'' <br/> genomic DNA <br/> <br />
|''L. opalescens'' <br/> B1 primers <br/> 65.7°C<br />
|''L. vulgaris lf'' <br/> A1 primers <br/> 65.7°C<br />
|''L. vulgaris lf'' <br/> A2 primers <br/> 65.7°C<br />
|- align="center"<br />
!'''7'''<br />
!'''8'''<br />
!'''9'''<br />
!'''10'''<br />
!'''11'''<br />
!'''12'''<br />
!'''13'''<br />
|- align="center"<br />
|''L. vulgaris lf''<br/> B1 primers <br/> 65.7°C<br />
|''L. vulgaris ba'' <br/> A1 primers <br/> 65.7°C<br />
|''L. vulgaris ba'' <br/> A2 primers <br/> 65.7°C<br />
|''L. vulgaris ba'' <br/> B1 primers <br/> 65.7°C<br />
|HyperLadder I<br />
|''L. opalescens''<br/> B1 primers <br/> 56.3°C<br />
|''L. vulgaris lf'' <br/> A1 primers <br/> 56.3°C<br />
|- align="center"<br />
!'''14'''<br />
!'''15'''<br />
!'''16'''<br />
!'''17'''<br />
!'''18'''<br />
!'''19'''<br />
!'''20'''<br />
|- align="center"<br />
|''L. vulgaris lf'' <br/> A2 primers <br/> 56.3°C<br />
|''L. vulgaris lf'' <br/> B1 primers <br/> 56.3°C<br />
|''L. vulgaris ba'' <br/> A1 primers <br/> 56.3°C<br />
|''L. vulgaris ba'' <br/> A2 primers <br/> 56.3°C<br />
|''L. vulgaris ba'' <br/> B1 primers <br/> 56.3°C<br />
|HyperLadder I<br />
|''L.opalescens''<br/> genomic DNA<br />
|}<br />
Note: ''L. vulgaris lf'' stands for the ''Loligo'' squid obtained from fishing bait suppliers, while ''L. vulgaris ba'' stands for the ''Loligo'' squid bought from the local culinary wholesaler. <br />
<br />
We could see a similar pattern in the second gel:<br />
*no products of PCR reactions, as well as primers and template DNA were visible,<br />
*no smear of genomic DNA from ''L. opalescens'' and ''L. vulgaris ba'' was detected,<br />
*however, we noticed a thin faint band of genomic DNA from ''L. vulgaris lf'', but the detectable amount of the template DNA did not improve the efficiency of corresponding PCR reactions.<br />
<br />
[[File:cam_gelelectrophoresisI_2107.jpg | center | thumb | 400px | A close-up of the second gel with the HyperLadder I on the leftmost lane and a faint band of ''L. vulgaris'' genomic DNA in the adjacent lane. ]]<br />
<br />
We concluded that the DNA extraction protocol applied in this experiment was not very efficient and that this was the main reason why we failed to obtain any amplified reflectin or reflectin-related genes.<br />
<br />
===References===<br />
#'''Anderson F.E. (2000)''' Phylogeny and historical biogeography of the loliginid squids (Mollusca: Cephalopoda) based on mitochondrial DNA sequence data. ''Molecular Phylogenetics and Evolution'', 15: 191-214.<br />
#'''Anderson F.E. (2000)''' Phylogenetic relationships among loliginid squids (Cephalopoda: Myopsida) based on analyses of multiple data sets. ''Zoological Journal of the Linnean Society'', 130: 603–633.<br />
#'''Crookes W.J., Ding L., Huang Q.L., Kimbell J.R., Horwitz J., McFall-Ngai M.J. (2004)''' Reflectins: the unusual proteins of squid reflective tissues. ''Science'', 303: 235–238.<br />
<br />
<br />
{{Template:Team:Cambridge/CAM_2011_EXPERIMENT_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/SocietyTeam:Cambridge/Society2011-09-22T01:30:52Z<p>Cat: /* Why talk to iGEMmers? */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==iGEMers on iGEM==<br />
<br />
The iGEM competition gathers hundreds of undergraduates together to explore the potential of synthetic biology. What happens to iGEM alumni after the Jamboree? Does the experience of such a unique summer project change the career paths of iGEM students? What becomes of all the innovation and hard-won expertise developed whilst building biobricks?<br />
<br />
To answer these questions, the 2011 Cambridge team conducted a census of iGEM alumni. [[Team:Cambridge/Society/Questionnaire | Take the questionnaire here]], [[Team:Cambridge/Society/report | read our discussion,]] or [[Team:Cambridge/Society/Questionnaire/Results | see some of our results]].<br />
<br />
Some previous iGEMers were generous enough to grant us interviews, allowing us to record their experiences of life after iGEM. As well as providing the quotes and case studies in our report, you can see [[Team:Cambridge/Society/interviews | excerpts from their interviews]]. ''(With thanks to Veronica Ranner for assistance in filming)''<br />
<br />
==Why talk to iGEMmers?==<br />
<br />
The experiences of those who do iGEM is logged in wikis and wetwork and BioBricks, but we think more emphasis should be placed on the long term influence of an intense summer of synthetic biology. <br />
<br />
After a talk on best practice for Human Practices projects by Andy Balmer at the [[Team:UEA-JIC_Norwich/UKConference | UK iGEM meetup]], we were inspired to focus on the underexplored areas of synthetic biology and its influence.<br />
<br />
As well as looking at the big picture of synthetic biology and the challenges and opportunities it can bring to society, we would like to encourage more teams to reflect on their own thoughts and feelings. The growing field of synthetic biology will be shaped by the people who do synthetic biology, so we hope to see a greater consideration of what makes people want to join the field. <br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Society/reportTeam:Cambridge/Society/report2011-09-22T01:21:28Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
We wanted to do a worthwhile investigation into how iGEM affects its participants. We know that surveys are fraught with difficulty if you intend to make concrete conclusions, but we saw contacting past iGEMers as an essential first step in understanding the impact of iGEM on its alumni. We composed a brief online [[Team:Cambridge/Society/Questionnaire | questionnaire]] in order to take a cross section of the population of iGEM alumni and select victims...erm, brains to pick further about the impact of iGEM. Due to the busy nature of our project we chose people who we could access here in Cambridge (Shuna Gould and Justin Pahara), but we tried to broaden our investigation by also recording a Skype conversation with Kim de Mora in Scotland, and a Google chat with an iGEM alumnus in the USA who requested not to be quoted directly in the write up.<br />
<br />
In short our questionnaire was aimed at finding our what people get into iGEM, how their experiences differ, and (more importantly for us) what are they doing now and has iGEM played a role in this. We also took the time to reflect on our own experience and compare this to what we found in our research.<br />
<br />
All of our interviews revealed that iGEM has profoundly influenced the careers people chose since iGEM, and in general iGEM seems to have a very positive impact in people's progression through these careers. For example; Shuna Gould mentioned that her PHD choice was swayed by her enjoyment of synthetic biology, her PHD supervisor was impressed with her training in synthetic biology, and she plans to implement specific techniques she first encountered in iGEM in her PHD (which is not strictly a synthetic biology project). Several of our team have had similar experiences in applications for future research opportunities: before we've even finished iGEM a couple of [[Team:Cambridge/Team/Students | us]] have had incredibly positive reactions to the mention of our synthetic biology training.<br />
<br />
Justin Pahara's career has been even more influenced by iGEM than Shuna. Since being part of iGEM 2007 and advising in 2008 he has moved to Cambridge to study a PHD in biotechnology and is now part of several start up companies, one which is heavily involved in working with synthetic biologists to improve their work through the use of software and IT. He says iGEM was a vital head start that opened his mind and has pushed him ahead of some of his colleagues in biotechnology.<br />
<br />
Kim de Mora certainly inspired us in his Skype interview, where he stressed the 'buzz' of the iGEM jamboree, told his own tale of iGEM swaying his research interests and helping him secure placements science. Many of our team have also commented how much iGEM has changed our future plans: Katy is considering a move into Biophysics, Matt wants to see if his third year university project can incorporate synthetic biology techniques to speed up construct production and modification, Catriona also wants to pursue synthetic biology further and Haydn (an engineer by training) is now taking "An Introduction to Molecular Bioengineering" as a module next year having worked with his future lecturers this summer.<br />
<br />
<br />
iGEM is unique in the amount of freedom given to undergrads to choose and organise their own research project. Shuna Gould made the point that teams also have to manage publicity and pitching their research to people with differing levels of specialist knowledge - valuable skills which are rarely developed in "normal" summer placement schemes.<br />
<br />
Our experience of iGEM has been overwhelmingly positive - despite the late nights editing the wiki and the occasional loss of direction, being part of such a unique project has been invaluable to all of us.<br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Society/reportTeam:Cambridge/Society/report2011-09-22T01:16:02Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
We wanted to do a worthwhile investigation into how iGEM affects its participants. We know that surveys are fraught with difficulty if you intend to make concrete conclusions, but we saw contacting past iGEMers as an essential first step in understanding the impact of iGEM on its alumni. We composed a brief online [[Team:Cambridge/Society/Questionnaire | questionnaire]] in order to take a cross section of the population of iGEM alumni and select victims...erm, brains to pick further about the impact of iGEM. Due to the busy nature of our project we chose people who we could access here in Cambridge (Shuna Gould and Justin Pahara), but we tried to broaden our investigation by also recording a Skype conversation with Kim de Mora in Scotland, and a Google chat with an iGEM alumnus in the USA who requested not to be quoted directly in the write up.<br />
<br />
In short our questionnaire was aimed at finding our what people get into iGEM, how their experiences differ, and (more importantly for us) what are they doing now and has iGEM played a role in this. We also took the time to reflect on our own experience and compare this to what we found in our research.<br />
<br />
All of our interviews revealed that iGEM has profoundly influenced the careers people chose since iGEM, and in general iGEM seems to have a very positive impact in people's progression through these careers. For example; Shuna Gould mentioned that her PHD choice was swayed by her enjoyment of synthetic biology, her PHD supervisor was impressed with her training in synthetic biology, and she plans to implement specific techniques she first encountered in iGEM in her PHD (which is not strictly a synthetic biology project). Several of our team have had similar experiences in applications for future research opportunities: before we've even finished iGEM a couple of [[Team:Cambridge/Team/Students | us]] have had incredibly positive reactions to the mention of our synthetic biology training.<br />
<br />
Justin Pahara's career has been even more influenced by iGEM than Shuna. Since being part of iGEM 2007 and advising in 2008 he has moved to Cambridge to study a PHD in biotechnology and is now part of several start up companies, one which is heavily involved in working with synthetic biologists to improve their work through the use of software and IT. He says iGEM was a vital head start that opened his mind and has pushed him ahead of some of his colleagues in biotechnology.<br />
<br />
Kim de Mora certainly inspired us in his Skype interview, where he stressed the 'buzz' of the iGEM jamboree, told his own tale of iGEM swaying his research interests and helping him secure placements science. Many of our team have also commented how much iGEM has changed our future plans: Katy is considering a move into Biophysics, Matt wants to see if his third year university project can incorporate synthetic biology techniques to speed up construct production and modification, Catriona also wants to pursue synthetic biology further and Haydn (an engineer by training) is now taking "An Introduction to Molecular Bioengineering" as a module next year having worked with his future lecturers this summer.<br />
<br />
<br />
iGEM is unique in the amount of freedom given to undergrads to choose and organise their own research project. Shuna Gould made the point that teams also have to manage publicity and pitching their research to people with differing levels of specialist knowledge - valuable skills which are rarely developed in "normal" summer placement schemes.<br />
<br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-22T00:16:22Z<p>Cat: /* Reflectin A1 GFP fusion */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==System diagrams==<br />
<br />
Our parts were designed for the two main branches of our labwork - [[Team:Cambridge/Project/In_Vivo | ''in vivo'' expression and export]] to try and achieve structural colour, and [[Team:Cambridge/Project/In_Vitro | overexpression for purification]] and ''in vitro'' studies of our recombinant reflectins.<br />
<br />
===Overexpression for purification===<br />
[[File:Cam_Overexpression_Construct.png | center | 600px]]<br />
<br />
===Export===<br />
[[File:Cam_Export_Construct.png | center | 600px]]<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures with optical properties. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage.<br />
<br />
In order to isolate pure reflectin, we built and submitted [http://partsregistry.org/wiki/index.php?title=Part:BBa_K638202 Poly-His tagged Reflectin A1 generator]. In pure form reflectins may be used to create [[Team:Cambridge/Project/In_Vitro | vibrantly coloured thin films]] and other devices. We used a poly-His affinity column to purify reflectin from cells for this purpose.<br />
<br style='clear:both' /><br />
[[File:Cam Reflectin-GFP-inclusionbodies.jpg | thumb | 175px | left | ''E. coli'' transformed with our pBAD-ReflectinA1-GFP construct]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638301 Reflectin A1 GFP fusion]===<br />
This BioBrick was created in order for us to control for the expression of reflectin and its location in the cell. Using it, we were able to determine that reflectin forms [[Team:Cambridge/Project/In_Vitro#Over-Expression | inclusion bodies]] when expressed at a high level.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 Improved TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K233307 the TorA tag already in the registry] and should be cheaper to obtain from primer synthesis.<br />
<br style='clear:both' /><br />
We also submitted [http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP], which we used in our [[Team:Cambridge/Project/In_Vivo#Periplasmic_Export | periplasm export]] attempt.<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team can be found below.<br />
(We also added our [http://partsregistry.org/Part:BBa_I0500:Experience experience of part I0500] to the registry.)<br />
<html><br />
<link rel="stylesheet" type="text/css" href="/common/tablesorter/themes/groupparts/style.css" /><br />
</html><br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Society/reportTeam:Cambridge/Society/report2011-09-21T23:40:41Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
Why iGEM? What kind of backgrounds does it attract<br />
<br />
Advantages - freedom to research, inspiring, interdisciplinary <br />
<br />
iGEM is unique in the amount of freedom given to undergrads to choose and organise their own research project. Shuna Gould made the point that teams also have to manage publicity and pitching their research to people with differing levels of specialist knowledge - valuable skills which are rarely developed in "normal" summer placement schemes.<br />
<br />
Effect on future plans etc<br />
<br />
are we excited by synbio enough to do it?<br />
<br />
Staying involved?<br />
<br />
We wanted to do a worthwhile investigation into how iGEM affects its participants. We know that surveys are fraught with difficulty if you intend to make concrete conclusions, but we saw contacting past iGEMMERs an essential first step in assessing the impact of iGEM on its alumni. We composed a brief<br />
<br />
<br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Society/reportTeam:Cambridge/Society/report2011-09-21T22:46:35Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
Why iGEM? What kind of backgrounds does it attract<br />
<br />
Advantages - freedom to research, inspiring, interdisciplinary <br />
<br />
iGEM is unique in the amount of freedom given to undergrads to choose and organise their own research project. Shuna Gould made the point that teams also have to manage publicity and pitching their research to people with differing levels of specialist knowledge - valuable skills which are rarely developed in "normal" summer placement schemes.<br />
<br />
Effect on future plans etc<br />
<br />
are we excited by synbio enough to do it?<br />
<br />
Staying involved?<br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/SocietyTeam:Cambridge/Society2011-09-21T22:33:40Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==iGEMers on iGEM==<br />
<br />
The iGEM competition gathers hundreds of undergraduates together to explore the potential of synthetic biology. What happens to iGEM alumni after the Jamboree? Does the experience of such a unique summer project change the career paths of iGEM students? What becomes of all the innovation and hard-won expertise developed whilst building biobricks?<br />
<br />
To answer these questions, the 2011 Cambridge team conducted a census of iGEM alumni. [[Team:Cambridge/Society/Questionnaire | Take the questionnaire here]], [[Team:Cambridge/Society/report | read our discussion,]] or [[Team:Cambridge/Society/Questionnaire/Results | see some of our results]].<br />
<br />
Some previous iGEMers were generous enough to grant us interviews, allowing us to record their experiences of life after iGEM. As well as providing the quotes and case studies in our report, you can see [[Team:Cambridge/Society/interviews | excerpts from their interviews]]. ''(With thanks to Veronica Ranner for assistance in filming)''<br />
<br />
=Why talk to iGEMmers?=<br />
<br />
The experiences of those who do iGEM is logged in wikis and wetwork and BioBricks, but we think more emphasis should be placed on the long term influence of an intense summer of synthetic biology. As well as looking at the big picture of synthetic biology and the challenges and opportunities it can bring to society, we would like to encourage more teams to reflect on their own thoughts and feelings. The growing field of synthetic biology will be shaped by the people who do synthetic biology, so we hope to see a greater consideration of what makes people want to join the field. <br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/SocietyTeam:Cambridge/Society2011-09-21T22:32:10Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==iGEMers on iGEM==<br />
<br />
The iGEM competition gathers hundreds of undergraduates together to explore the potential of synthetic biology. What happens to iGEM alumni after the Jamboree? Does the experience of such a unique summer project change the career paths of iGEM students? What becomes of all the innovation and hard-won expertise developed whilst building biobricks?<br />
<br />
To answer these questions, the 2011 Cambridge team conducted a census of iGEM alumni. [[Team:Cambridge/Society/Questionnaire | Take the questionnaire here]], [[Team:Cambridge/Society/report | read our discussion,]] or [[Team:Cambridge/Society/Questionnaire/Results | see some of our results]].<br />
<br />
Some previous iGEMers were generous enough to grant us interviews, allowing us to record their experiences of life after iGEM. As well as providing the quotes and case studies in our report, you can see [[Team/Cambridge/Society/interviews | excerpts from their interviews]]. ''(With thanks to Veronica Ranner for assistance in filming)''<br />
<br />
=Why talk to iGEMmers?=<br />
<br />
The experiences of those who do iGEM is logged in wikis and wetwork and BioBricks, but we think more emphasis should be placed on the long term influence of an intense summer of synthetic biology. As well as looking at the big picture of synthetic biology and the challenges and opportunities it can bring to society, we would like to encourage more teams to reflect on their own thoughts and feelings. The growing field of synthetic biology will be shaped by the people who do synthetic biology, so we hope to see a greater consideration of what makes people want to join the field. <br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/SocietyTeam:Cambridge/Society2011-09-21T22:20:11Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==iGEMers on iGEM==<br />
<br />
The iGEM competition gathers hundreds of undergraduates together to explore the potential of synthetic biology. What happens to iGEM alumni after the Jamboree? Does the experience of such a unique summer project change the career paths of iGEM students? What becomes of all the innovation and hard-won expertise developed whilst building biobricks?<br />
<br />
To answer these questions, the 2011 Cambridge team conducted a census of iGEM alumni. [[Team:Cambridge/Society/Questionnaire | Take the questionnaire here]], [[Team:Cambridge/Society/Report | read our discussion,]] or [[Team:Cambridge/Society/Questionnaire/Results | see some of our results]].<br />
<br />
Some previous iGEMers were generous enough to grant us interviews, allowing us to record their experiences of life after iGEM. As well as providing the quotes and case studies in our report, you can see [[Team/Cambridge/Society/interviews | excerpts from their interviews]]. ''(With thanks to Veronica Ranner for assistance in filming)''<br />
<br />
=Why talk to iGEMmers?=<br />
<br />
The experiences of those who do iGEM is logged in wikis and wetwork and BioBricks, but we think more emphasis should be placed on the long term influence of an intense summer of synthetic biology. As well as looking at the big picture of synthetic biology and the challenges and opportunities it can bring to society, we would like to encourage more teams to reflect on their own thoughts and feelings. The growing field of synthetic biology will be shaped by the people who do synthetic biology, so we hope to see a greater consideration of what makes people want to join the field. <br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/desc/society/reportTeam:Cambridge/desc/society/report2011-09-21T22:13:35Z<p>Cat: </p>
<hr />
<div><p><br />
Is there life after iGEM? We conducted a <a href='/Team:Cambridge/Society/Questionnaire/Results'>brief poll</a> of past iGEMers and <a href='/Team:Cambridge/Society/interviews'>interviewed a few alumni</a> to find out how they feel about synthetic biology now, in order to better reflect upon our own experiences.<br />
</p></div>Cathttp://2011.igem.org/Team:Cambridge/desc/society/reportTeam:Cambridge/desc/society/report2011-09-21T22:12:52Z<p>Cat: </p>
<hr />
<div><p><br />
Is there life after iGEM? We conducted a <a href='/Team:Cambridge/Society/Questionnaire/Results'>brief poll</a> of past iGEMers, and <a href='/Team:Cambridge/Society/interviews'>interviewed a few alumni</a> to find out how they feel about synthetic biology now, in order to better reflect upon our own experiences.<br />
</p></div>Cathttp://2011.igem.org/Team:Cambridge/Project/In_VitroTeam:Cambridge/Project/In Vitro2011-09-21T21:38:25Z<p>Cat: /* Thin Films */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
=Isolating Reflectin and Making Thin Films=<br />
Previous in vitro investigations of reflectin showed that it was possible to use a method called [[Team:Cambridge/Protocols/Flow_coating | flow coating]] to deposit a thin layer of reflectin onto a silicon substrate which would then demonstrate structural colour.<br />
<br />
We wanted to investigate using a different coating method – [[Team:Cambridge/Protocols/Spin_Coating | spin coating]] – and to try out different methods for [[Team:Cambridge/Protocols/Protein_Purification | protein purification]]. A polyhis-tagged variant of the protein was overexpressed and purified using a polyhis affinity column. Thin films were made by precipitating with either [[Team:Cambridge/Protocols/Acetone_Precipitation_of_Proteins | acetone]] or [[Team:Cambridge/Protocols/Ethanol_Precipitation_of_Proteins | ethanol]] and resuspending in HFIP before either spin coating or flow coating onto a silicon wafer.<br />
<br />
We also investigated an [[Team:Cambridge/Protocols/Inclusion_Body_Prep | inclusion body prep]], as the protein we found that the protein formed inclusion bodies upon over-expression.<br />
<br />
By varying our purification and coating methods, we were able to create vibrant thin films.<br />
<br />
[[File:CAM_invitro_flow.png| centre | 300px]]<br />
<br />
==Polyhis-Tagging Reflectin==<br />
<br />
The reflectin genes were polyhis-tagged by incorporating the sequence of part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K128005 BBa_K128005] into the primers used to clone the gene. Polyhis tagging was chosen because it allows proteins to be purified with relatively simple apparatus, an affinity column, to which a metal containing resin is added traps the protein when cell lysate is added.<br />
<br />
==Over-Expression==<br />
<br />
Reflectin was placed on a high copy plasmid ([http://partsregistry.org/Part:pSB1A2 PSB1A2]) under an arabinose inducible promoter ([http://partsregistry.org/Part:BBa_I0500 pBad]) in order to express reflectin at a high level. As well as a negative control, we used an sfGFP-reflectin fusion as a control for reflecitn production.<br />
<br />
In the non-GFP cells we found that there were brightly lit points at the ends of nearly every cell, which were not present in the negative control. In the GFP fusion cells, these spots glowed strongly, while the rest of the cell was relatively dark. This indicated that reflectin produces inclusion bodies when expressed at high level.<br />
<br />
[[File:Cam Reflectin-GFP-inclusionbodies.jpg | thumb| 400px| center| ''E. coli'' transformed with our pBAD-ReflectinA1-GFP construct, induced by adding 1mM arabinose, demonstrating localisation of the protein.]]<br />
<br />
==Extraction & Purification==<br />
We investigated two principal methods for extracting the protein from the transformed <i>E. coli</i> - we lysed the cells and ran the lysate through a polyhis affinity column and we tried a proprietary inclusion body prep.<br />
<br />
===[[Team:Cambridge/Protocols/Protein_Purification | Polyhis-Tag Affinity Column]]===<br />
The polyhis affinity column allowed us to extract our tagged protein from the lysate without too much difficulty. In order to increase the purity of our sample we tried using an [[Team:Cambridge/Protocols/Acetone_Precipitation_of_Proteins | acetone]] or [[Team:Cambridge/Protocols/Ethanol_Precipitation_of_Protein | ethanol]] precipitation step to remove chemicals retained in the elution buffer and to concentrate the protein for downstream processing.<br />
<br />
Having done this, we ran our sample on an SDS Page protein gel, to verify that we had in fact purified reflectin. We were expecting to see a thick band of reflectin at around 43 kDa, which – as the image shows – was the case.<br />
<br />
[[File:CAM_SDS_POLYHIS.png | center | thumb | 500px | The result of the Polyhis affinity column SDS Page gel]]<br />
<br />
===[[Team:Cambridge/Protocols/Inclusion_Body_Prep | Inclusion Body Prep]]===<br />
<br />
The Norgen Proteospin inclusion body prep kit was experimented with as a commercial alternative to the previous purification method. The kit was simple to use, but required the use of an ultracentrifuge capable of spinning up to 25ml of fluid at 27,000g. It was found that the spinning forces specified in the protocol for these steps weren't capable of moving fluid through the necessary columns and higher forces were needed. <br />
<br />
On analysing the end-product of the purification with SDS-PAGE approximately 20 discrete bands were observed from 10-200 kda with 2 particularly bold bands, both of which are too short for reflectin. However, a reasonably thick band of protein was present at 43kDA (the correct length for reflctin).<br />
<br />
[[File:CAM_SDS_INCLUSION_BODY.png | center | thumb | 450px | The result of the inclusion body SDS Page gel]]<br />
<br />
As the gel shows, the resulting protein is nowhere near as pure as the result of the previous method as the inclusion bodies do not contain only reflectin. However, greater purity might have been achieved by using the result of the inclusion body prep as the first stage of the polyhis affinity protocol, as it would reduce the amount of non-polyhis protein going in to the column. This was not tested in practice, however, as our protein appeared to be pure enough for processing, and we did not have the time to experiment further.<br />
<br />
<div id="Thin Films"></div><br />
==[[Team:Cambridge/Experiments/Thin_Films | Thin Films]]==<br />
In order to demonstrate structural colour, thin films were created by re-suspending the purified protein in HFIP and [[Team:Cambridge/Protocols/Spin_Coating | spin coating]] or [[Team:Cambridge/Protocols/Flow_coating | flow coating]] the re-suspended protein onto a silicon substrate. This gave us brightly coloured thin films which responded to water vapour in the air by changing colour. We performed controls by making HFIP films and films with Bovine Serum Albumin (BSA, a generic protein) neither of which showed any structural colour.<br />
<br />
<center><br />
<gallery widths=150px caption='An initial thin film (left), alongside its controls'><br />
File:Cam Reflectin Thin Film2.jpg | ''Reflectin HFIP solution showed iridescence after coating''<br />
File:Cam HFIP only control thinfilm.jpg | ''HFIP (solvent) only control does not exhibit iridescence''<br />
File:BSAcontrolfilm1.jpg | ''Bovine Serum Albumin makes a dull, striated thin film''<br />
</gallery><br />
</center><br />
<br />
Our initial films were not uniform in colour and would form crystals when allowed to dry out. We improved this by increasing the purity of our protein as well as refining our coating technique.<br />
<br />
We first tried to reduce the urea content of our sample, in the belief that this would reduce the tendency to crystallise, however, this did not work as our sample became too dilute. Almost by accident, we tried using a [[Team:Cambridge/Experiments/Reflectin_Thin_Films_IV | lower volume of protein]] and found - to our great surprise - that the quality of the thin films produced increased greatly, and the crystallisation was greatly reduced.<br />
<br />
The reflectin thin films were dynamic - they responded to water vapour in the air. The video below shows the effect of breathing several times on a thin film - the colour change is quite dramatic.<br />
<br />
<center><br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/X3pexBYWOrY" frameborder="0" allowfullscreen></iframe></html><br />
</center><br />
<br />
Flushed with success, we then pushed on to make [[Team:Cambridge/Experiments/Reflectin_Thin_Films_VI | multi layers]] of Reflectin, hoping that this would again increase the brightness and uniformity of the thin films. The beauty of the results was astonishing.<br />
<br />
<center><br />
<gallery caption='A sample of the microscope images taken of multi-layers.' widths=140px><br />
File:Cam Multilayer drop 1.jpg <br />
File:Cam Crazy multilayer single AP 2k spin2nd.jpg<br />
File:Cam crazy multilayer2.jpg<br />
File:Cam_crazy_multilayer3.jpg<br />
</gallery><br />
</center><br />
<br />
<br />
===Future Work===<br />
As well as further improving our thin film's colour, we also wanted to improve their long-term stability, as well as investigate controlling their colour electrically.<br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:CambridgeTeam:Cambridge2011-09-21T20:06:57Z<p>Cat: </p>
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<div class='cam-home-box cam-home-left cam-home-abstract'><br />
<div class='cam-home-box-title'><br />
<p>Abstract</p><br />
</div><br />
<div class='cam-home-box-content'><br />
<p>We investigated the properties of a novel yet under-researched group of proteins called reflectins.</p><br />
<p>Reflectins are interesting and unusual because of their self-organising properties, which cause them to produce dynamic structural colour.</p><br />
<p>We expressed codon-optimised reflectin in E. coli, and investigated the proteins' in vivo and in vitro effects.</p><br />
</div><br />
</div><br />
</a><br />
<br />
<a href='/Team:Cambridge/Project/Background' class='cam-color-exempt'><br />
<div class='cam-home-box cam-home-right cam-home-colour'><br />
<div class='cam-home-box-title'><br />
<p>Structural Colour</p><br />
</div><br />
<div class='cam-home-box-content'><br />
<p>Nature’s colours don’t just come from pigments, but from structure too.</p><br />
<p>Cephalopods camouflage themselves using intracellular structures made from reflectins.</p><br />
<p>These are the only known proteinaceous structures to exhibit structural colour. They are inspiring a new class of responsive optical materials.</p><br />
</div><br />
</div><br />
</a><br />
<br />
<div style='clear:both;'></div><br />
</div><br />
<br />
<br />
<div class='cam-home-photos cam-home-row'><br />
<div class='cam-home-photo' style='margin-right:6px;'><br />
<a href='/Team:Cambridge/Project/In_Vitro'><br />
<img src='/wiki/images/6/60/CAM_home_image1.jpg' width='238px' height='140px' alt='Image 1' title='In-Vitro Thin Films of Reflectin'></img><br />
</a><br />
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<div class='cam-home-photo' style='margin-right:6px;'><br />
<a href='/Team:Cambridge/Team'><br />
<img src='/wiki/images/3/37/CAM_home_image2.jpg' width='238px' height='140px' alt='Image 2' title='The Team'></img><br />
</a><br />
</div><br />
<div class='cam-home-photo'><br />
<a href='/Team:Cambridge/Project/Microscopy'><br />
<img src='/wiki/images/b/b1/CAM_home_image3.jpg' width='238px' height='140px' alt='Image 3' title='Microscopy'></img><br />
</a><br />
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<br />
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<div class='cam-home-box cam-home-left cam-home-survey'><br />
<div class='cam-home-box-title'><br />
<p>Impact of iGEM</p><br />
</div><br />
<div class='cam-home-box-content'><br />
<p>We wanted to know what effect the iGEM competition has had on its past contestants.</p><br />
<p>We were also interested in what past participants thought of iGEM and how it shaped their view of synthetic biology.</p><br />
<p>To this end we got in touch with a number of iGEM alumni and asked them to share their experience.</p><br />
</div><br />
</div><br />
</a><br />
<br />
<a href='/Team:Cambridge/Project/Gibthon' class='cam-color-exempt'><br />
<div class='cam-home-box cam-home-right cam-home-tools'><br />
<div class='cam-home-box-title'><br />
<p>Tools</p><br />
</div><br />
<div class='cam-home-box-content'><br />
<p>We used Gibson Assembly exclusively for construct assembly during the competition, allowing us to assemble plasmids faster and with greater ease than would be possible using standard techniques.</p><br />
<p>We contributed to a collection of software tools called <i>Gibthon</i> (initiated by Cambridge iGEM 2010), which aids the design of primers for Gibson assembly.</p><br />
</div><br />
</div><br />
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{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-21T20:04:09Z<p>Cat: /* Reflectin A1 with poly-His tag */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==System diagrams==<br />
<br />
Our parts were designed for the two main branches of our labwork - [[Team:Cambridge/Project/In_Vivo | ''in vivo'' expression and export]] to try and achieve structural colour, and [[Team:Cambridge/Project/In_Vitro | overexpression for purification]] and ''in vitro'' studies of our recombinant reflectins.<br />
<br />
===Overexpression for purification===<br />
[[File:Cam_Overexpression_Construct.png | 600px]]<br />
===Export===<br />
[[File:Cam_Export_Construct.png | 600px]]<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures with optical properties. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638202 Poly-His tagged Reflectin A1 generator]===<br />
In pure form reflectins may be used to create [[Team:Cambridge/Project/In_Vitro | vibrantly coloured thin films]] and other devices. We used a poly-His affinity column to purify reflectin from cells for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 Improved TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K233307 the TorA tag already in the registry] and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team can be found below.<br />
(We also added our [http://partsregistry.org/Part:BBa_I0500:Experience experience of part I0500] to the registry.)<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:CambridgeTeam:Cambridge2011-09-21T19:58:33Z<p>Cat: typos</p>
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<div class='cam-home-box cam-home-left cam-home-abstract'><br />
<div class='cam-home-box-title'><br />
<p>Abstract</p><br />
</div><br />
<div class='cam-home-box-content'><br />
<p>We investigated the properties of a novel yet under-researched group of proteins called reflectins.</p><br />
<p>Reflectins are interesting and unusual because of their self-organising properties, which cause them to produce dynamic structural colour.</p><br />
<p>We expressed codon-optimised reflectin in E. coli, and investigated the protein's in vivo and in vitro effects.</p><br />
</div><br />
</div><br />
</a><br />
<br />
<a href='/Team:Cambridge/Project/Background' class='cam-color-exempt'><br />
<div class='cam-home-box cam-home-right cam-home-colour'><br />
<div class='cam-home-box-title'><br />
<p>Structural Colour</p><br />
</div><br />
<div class='cam-home-box-content'><br />
<p>Nature’s colours don’t just come from pigments, but from structure too.</p><br />
<p>Cephalopods camouflage themselves using intracellular structures made from reflectins.</p><br />
<p>These are the only known proteinaceous structures to exhibit structural colour. They are inspiring a new class of responsive optical materials.</p><br />
</div><br />
</div><br />
</a><br />
<br />
<div style='clear:both;'></div><br />
</div><br />
<br />
<br />
<div class='cam-home-photos cam-home-row'><br />
<div class='cam-home-photo' style='margin-right:6px;'><br />
<a href='/Team:Cambridge/Project/In_Vitro'><br />
<img src='/wiki/images/6/60/CAM_home_image1.jpg' width='238px' height='140px' alt='Image 1' title='In-Vitro Thin Films of Reflectin'></img><br />
</a><br />
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<div class='cam-home-photo' style='margin-right:6px;'><br />
<a href='/Team:Cambridge/Team'><br />
<img src='/wiki/images/3/37/CAM_home_image2.jpg' width='238px' height='140px' alt='Image 2' title='The Team'></img><br />
</a><br />
</div><br />
<div class='cam-home-photo'><br />
<a href='/Team:Cambridge/Project/Microscopy'><br />
<img src='/wiki/images/b/b1/CAM_home_image3.jpg' width='238px' height='140px' alt='Image 3' title='Microscopy'></img><br />
</a><br />
</div><br />
<div style='clear:both;'></div><br />
</div><br />
<br />
<br />
<div class='cam-home-row'><br />
<a href='/Team:Cambridge/Society' class='cam-color-exempt'><br />
<div class='cam-home-box cam-home-left cam-home-survey'><br />
<div class='cam-home-box-title'><br />
<p>Impact of iGEM</p><br />
</div><br />
<div class='cam-home-box-content'><br />
<p>We wanted to know what effect the iGEM competition has had on its past contestants.</p><br />
<p>We were also interested in what past participants thought of iGEM and how it shaped their view of synthetic biology.</p><br />
<p>To this end we got in touch with a number of iGEM alumni and asked them to share their experience.</p><br />
</div><br />
</div><br />
</a><br />
<br />
<a href='/Team:Cambridge/Project/Gibthon' class='cam-color-exempt'><br />
<div class='cam-home-box cam-home-right cam-home-tools'><br />
<div class='cam-home-box-title'><br />
<p>Tools</p><br />
</div><br />
<div class='cam-home-box-content'><br />
<p>We used Gibson Assembly exclusively for construct assembly during the competition, allowing us to assemble plasmids faster and with greater ease than would be possible using standard techniques.</p><br />
<p>We contributed to a collection of software tools called <i>Gibthon</i> (initiated by Cambridge iGEM 2010), which aids the design of primers for Gibson assembly.</p><br />
</div><br />
</div><br />
</a><br />
<div style='clear:both;'></div><br />
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{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/TeamTeam:Cambridge/Team2011-09-21T17:53:19Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
Our core team was made of 9 Cambridge University [[Team:Cambridge/Team/Students | undergraduates]] from a wide range of courses. There were 4 biologists, three engineers, one physicist and one mathematician. While every member of the group broadened their knowledge, our different areas of expertise allowed us to simultaneously approach problems from many angles. '''Unless otherwise stated, all work done on the wiki was carried out by us.'''<br />
<br />
We were also aided by a student from the Royal College of Art (RCA) who was able to provide us with a completely different perspective on our work. Her advice and creative input was invaluable to our project.<br />
<br />
We were advised by several [[Team:Cambridge/Team/Academics | academics]] (both from within the university and externally), both professors and PhD students, who provided technical assistance and insight. They all did a fantastic job of providing positive feedback, without guiding the direction of our project.<br />
<br />
'''Work carried out primarily by these advisors is clearly labelled as such. In particular we are grateful to Paul Grant for assistance operating the confocal and for his advice during [[Team:Cambridge/Project/Microscopy| our microscopy]]'''.<br />
<br />
With the team's budget especially tight this year due to the increased cost of the competition, our [[Team:Cambridge/Team/Sponsors | sponsors]] were invaluable - they contributed the reagents, kits and cash which enabled us to take part.<br />
<br />
[[File:Team_photo.png|center|frame|From left to right: Haydn, Jonathan, Marta, Heather, Katy, Cat, Matt, Joe and Felix]]<br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-21T16:25:59Z<p>Cat: /* ...and the rest */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==System diagrams==<br />
<br />
Our parts were designed for the two main branches of our labwork - [[Team:Cambridge/Project/In_Vivo | ''in vivo'' expression and export]] to try and achieve structural colour, and [[Team:Cambridge/Project/In_Vitro | overexpression for purification]] and ''in vitro'' studies of our recombinant reflectins.<br />
<br />
===Overexpression for purification===<br />
[[File:Cam_Overexpression_Construct.png | 600px]]<br />
===Export===<br />
[[File:Cam_Export_Construct.png | 600px]]<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures with optical properties. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638201 Reflectin A1 with poly-His tag]===<br />
In pure form reflectins may be used to create [[Team:Cambridge/Project/In_Vitro | vibrantly coloured thin films]] and other devices. We used a poly-His affinity column to purify reflectin from cells for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 Improved TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K233307 the TorA tag already in the registry] and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team can be found below.<br />
(We also added our [http://partsregistry.org/Part:BBa_I0500:Experience experience of part I0500] to the registry.)<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-21T16:24:09Z<p>Cat: /* ...and the rest */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==System diagrams==<br />
<br />
Our parts were designed for the two main branches of our labwork - [[Team:Cambridge/Project/In_Vivo | ''in vivo'' expression and export]] to try and achieve structural colour, and [[Team:Cambridge/Project/In_Vitro | overexpression for purification]] and ''in vitro'' studies of our recombinant reflectins.<br />
<br />
===Overexpression for purification===<br />
[[File:Cam_Overexpression_Construct.png | 600px]]<br />
===Export===<br />
[[File:Cam_Export_Construct.png | 600px]]<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures with optical properties. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638201 Reflectin A1 with poly-His tag]===<br />
In pure form reflectins may be used to create [[Team:Cambridge/Project/In_Vitro | vibrantly coloured thin films]] and other devices. We used a poly-His affinity column to purify reflectin from cells for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 Improved TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K233307 the TorA tag already in the registry] and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team:<br />
(We also added our [http://partsregistry.org/Part:BBa_I0500:Experience experience of part I0500] to the registry.)<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Team/AcademicsTeam:Cambridge/Team/Academics2011-09-21T12:46:34Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
== '''Advisers''' ==<br />
==== Jim Haseloff ====<br />
----<br />
[[File:Team:Cambridge/Team/cam_jimh.jpg | right | thumb | 200px | Jim Haseloff]]<br />
http://www.plantsci.cam.ac.uk/Haseloff/<br />
<br style="clear: both" /><br />
==== Jim Ajioka ====<br />
----<br />
[[File:Team:Cambridge/Team/cam_jima.jpg | right | thumb | 200px | Jim Ajioka]]<br />
http://www.path.cam.ac.uk/research/investigators/ajioka/<br />
<br style="clear: both" /><br />
<br />
==== Gos Micklem ====<br />
----<br />
[[File:Team:Cambridge/Team/cam_gos.jpg | right | thumb | 200px | Gos Micklem]]<br />
http://www.gen.cam.ac.uk/research/micklem.html <br />
<br style="clear: both" /><br />
==== Fernan Federici====<br />
----<br />
[[File:Team:Cambridge/Team/cam_fernan.jpg | right | thumb | 200px | Fernan Federici]]<br />
http://www.plantsci.cam.ac.uk/Haseloff/people/people.html<br />
<br style="clear: both" /><br />
==== Paul Grant====<br />
----<br />
[[File:Team:Cambridge/Team/cam_paul.jpg | right | thumb | 200px | Paul Grant]]<br />
http://www.plantsci.cam.ac.uk/Haseloff/people/people.html<br />
<br style="clear: both" /><br />
==== PJ Steiner====<br />
----<br />
[[File:Team:Cambridge/Team/cam_pj.jpg | right | thumb | 200px | PJ Steiner]]<br />
http://www.plantsci.cam.ac.uk/Haseloff/people/people.html<br />
<br style="clear: both" /><br />
==== James Brown====<br />
----<br />
[[File:Team:Cambridge/Team/cam_jamesb.jpg | right | thumb | 200px | James Brown]]<br />
http://www.plantsci.cam.ac.uk/Haseloff/people/people.html<br />
<br style="clear: both" /><br />
=='''RCA Student'''==<br />
==== Veronica Ranner ====<br />
----<br />
[[File:Cam_Veronica_Ranner.jpg | right | thumb | 200px | Veronica]]<br />
Veronica recently graduated from the Design Interactions course at the Royal College of Arts. She has contributed a wealth of expertise in the design, marketing, creative thought processes and refinement of ideas, film-making and sketching. We're very grateful to have her on the team!<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Team/StudentsTeam:Cambridge/Team/Students2011-09-21T12:46:09Z<p>Cat: /* RCA Student */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
== '''Cambridge Students''' ==<br />
==== Marta Bozek ====<br />
----<br />
[[File:CAM_Marta.JPG | right | thumb | 200px | Marta]]<br />
I finished the second year of Natural Sciences at Cambridge University and I plan to specialize in Genetics from October, being particularly interested in different methods of regulating gene expression. The iGEM experience was especially valuable for me because it allowed me to get a glimpse into the world of real science and showed how fascinating the work in the lab can be! During the project I was mainly involved in the wet work, especially enjoying sealing gel tanks, vortexing and laborious pipetting. Apart from biology, I'm interested in modern art and underground Polish music scene.<br />
<br />
<br style='clear: both;' /><br />
<br />
==== Xin Gao / Heather ====<br />
----<br />
[[File:Cam_Heather.jpg | right | thumb | 200px | Heather]]<br />
I just finished my second year engineering course, still haven't quite decided which area of engineering to specialize in. In the past one and half week, I enjoyed roaming the unfamiliar territory of synthetic biology despite the steep learning curve.<br />
<br />
<br style='clear: both;' /><br />
==== Joe Harvey ====<br />
----<br />
[[File:joe.jpg | right | thumb | 200px | Joe]]<br />
Soon-to-be third year maths student intent on wheedling his way into the world of biology.<br />
<br />
<br style='clear: both;' /><br />
<br />
==== Matthew Jones ====<br />
----<br />
[[File:Cam_Matt_J.jpeg | right | thumb | 200px | Matt]]<br />
Biological Natural Sciences - I've just finished my second year of Natural Sciences here at Cambridge University. So far I have studied modules in Cell Biology, Evolution and Behaviour, Plant Physiology, Animal Physiology, Neurobiology and Developmental Biology. iGEM has so far introduced me to lots of Biology that is outside of the courses I have studied in the last couple of years and I have really enjoyed stepping outside of my comfort zone and building a working 8x8 LED matrix my first Arduino project and getting stuck into some computer programming with the Microsoft Research team. I have high hopes for our team this summer.<br />
<br />
<br style='clear: both;' /><br />
==== Haydn King ====<br />
----<br />
[[File:Haydn.jpg | right | thumb | 200px | Haydn]]<br />
I have just finished second year Engineering at the University, and am likely to be studying Information and Computer Engineering next year. Having no biological training since GCSE, I am very interested to learn more about the subject during the course of this project. I am particularly interested in the parallels which can be drawn between computation 'in silico' and that which can be done within a living cell.<br />
<br />
I come from a small village called Brechfa near Carmarthen in south west Wales, and am a fluent Welsh speaker.<br />
<br />
<br style='clear: both;' /><br />
==== Catriona McMurran ====<br />
----<br />
[[File:Cat.jpg | right |thumb | 200px| Cat]]<br />
Soon to be a third year Biochemist, currently learning more than anyone needs to know about<br />
what a squid looks like on the inside. Thanks to iGEM I have become mildly obsessed with the number of followers on the team's Twitter feed, and have read so much about reflectins I can pull references out of my ears. Having studied mostly pure biology in my degree so far, I'm finding the engineering-based approach of synthetic biology really interesting. <br />
<br style='clear: both;' /><br />
<br />
==== Jonathan Very ====<br />
----<br />
[[File:Cam_Jonathan.jpg | right | thumb | 200px | Jonathan]]<br />
I'm a Natural Scientist who's specialised in the most abstruse of all the Biological subjects: Biochemistry (I like to think of it as being like the 'particle physics' of life). My role in the group is to document the peculiar and often amusing things that go on our lab (see the blog), track down ex-iGEMers and charm them into taking our questionnaire (see the 'society' page) and occasionally put what little biological skill I've gleaned from my two years' study into some labwork. When not doing such things I can be found playing and performing Renaissance lute pieces, and writing fiction.<br />
<br />
<br style='clear: both;' /><br />
<br />
==== Katy Wei ====<br />
----<br />
[[File:CambridgeKaty.jpg | right |thumb | 200px| Katy]]<br />
I'm a physicist who has not encountered biology since GCSE times, and I joined iGEM fearing that I wouldn't contribute any useful knowledge to the group - to my pleasant surprise, however, the physics of optics proved extremely important in our project. IGEM was a great experience for me to get familiar with biological research, and I really enjoyed getting my hands dirty in the wetwork too. Impressively, the field seems to me even more mysterious and open-ended than current research in theoretical physics, which I was previously looking to specalise in. I now confess to being a complete convert, and am looking forward to working in biophysics in the future! <br />
<br />
<br style='clear: both;' /><br />
<br />
==== Felix Zhou ====<br />
----<br />
[[File:FELIX.jpg | right | thumb | 200px | Felix]]<br />
An excitable engineer who 'boos' <br />
<br />
<br style='clear: both;' /><br />
<br />
<br />
<br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Project/BackgroundTeam:Cambridge/Project/Background2011-09-21T10:47:19Z<p>Cat: /* Reflectins as Novel Polymers */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==Reflectins in Cephalopods <br/><i style='margin-left:25px;font-size:70%;'>or</i><br/> How to Disappear Completely==<br />
<br />
Reflectins are a family of proteins which self assemble into a multilayered structure in association with membranes. This multilayer interacts with light to give colours in cephalopods due to [[#Interference | thin film interference.]]<br />
<br />
The beautiful optical properties which reflectins make possible are used for a multitude of purposes within nature. Manipulation of reflectance allows squids to communicate through polarised light whilst altering the refractive index of their skin to match that of the water column allows cephalopods to hide from their predators, a living invisibility cloak. <br />
<br />
Reflectin was first identified as such in the Hawaiian bobtail squid [http://en.wikipedia.org/wiki/Euprymna_scolopes ''Euprymna scolopes''] as the protein responsible for a reflective layer in the " light organ". This layer reflects light emitted by symbiotic bacteria to be reflected downwards away from the squid for camouflage, like a [http://en.wikipedia.org/wiki/Headlamp#Reflector_lamps car headlamp]. By controlling the light emitted from the light organ, Hawaiian bobtail squid reduce their shadow against the brighter sky, concealing them from predators below.<br />
<br />
<br />
Whilst the iridescent effect is static in ''E.scolopes'', the Longfin inshore squid [http://en.wikipedia.org/wiki/Longfin_Inshore_Squid ''Loligo pealei''] demonstrates dynamic iridescence, controlled by the nervous system<sup>[[#Izumi|[5]]]</sup>. Dynamic iridescence is controlled by the release of the neurotransmitter acetylcholine. Acetylcholine detection by reflectin containing cells is believed to activate a [http://en.wikipedia.org/wiki/Kinase protein kinase], an enzyme which adds negatively charged phosphate groups to the positively charged protein. Within the squid reflectin is structured hierarchially - the structure of the reflectin protein itself, the complex platelets which form, and then the final multi-layer 'Bragg stack' of platelets which is contained within reflective cells called iridophores<sup>[[#Morse|[3]]]</sup>. The negatively charged phosphate groups alters the spacing between the protein layers and therefore modifies the colour by changing the attraction/repulsion between the reflectin platelets.(Read more about structural colour below.)<br />
<br />
<center><br />
<gallery><br />
File:CAM_LOLIGO_IRIDOCYTES.jpg | EM of reflectin ultrastructure (dark bands) in cells from ''Loligo opalescens''<sup>[[#Octopus|[6]]]</sup><br />
File:CAM_OCTOPUS_IRIDOCYTES.jpg | EM of reflectin ultrastructure (dark bands) in reflector cells from ''Octopus dofleini''<sup>[[#Octopus|[6]]]</sup><br />
File:CAM_CLAM.jpg | Structural colour in the mantle of a giant clam creates beautiful blue-green hues<br />
</gallery><br />
</center><br />
<br />
Iridophores are not merely confined to cephalopods but are suspected to be present in other members of the mollusc phylum e.g. giant clams<sup>[[#Clams|[4]]]</sup>where they are responsible for the stunning iridescent colours of the mantle, and they may play a role in protecting against harmful UV and maximising capture of sunlight for the photosynthetic symbionts.<br />
<br />
<div id="Interference"></div><br />
<br />
=Light and interference - The physics behind structural colour=<br />
[[File:Cam_ThinFilmInterference.jpg | thumb | Diagram showing how light reflects from a thin film]]<br />
Structural colour is a common occurrence in nature - butterfly wings, fish scales and even [http://en.wikipedia.org/wiki/Tapetum_lucidum cats's eyes] contain light reflecting components rather than pigments. It occurs due to the phenomenon of [http://en.wikipedia.org/wiki/Thin-film_interference '''thin film interference'''].<br />
<br />
The difference in [http://en.wikipedia.org/wiki/Refractive_index refractive index] between the alternating layers leads to reflection (bouncing off) and transmission of light. This process is repeated at each layer. When the two reflected light waves meet, they sum together but are no longer in sync (due to differences in the optical paths they have taken) leading to the phenomenon known as interference. [[File:Cam-2D_interference.png | thumb | Examples of 2D interference patterns]] Some wavelengths will destructively interfere more than others. The result is that not all colours are reflected with the same intensity, creating rainbow-like effects like those of oil droplets on the surface of water or the surface of shimmering soap bubbles. <br />
<br />
The observed colour changes with viewing angle, angle of incidence of light and alterations in the spacing of the layers or refractive indices which alters the mechanism of interference. In fact, if you use layers of materials with matching refractive indices, total transparency and hence invisibility of the material can be achieved.<br />
<br />
This principle of interference is believed to be the primary mechanism by which colour is controlled in the skin of squid. Experimental observations of changes in the thickness and spacing between reflectin layers ''in-vivo'' has been observed following post translational modification.<br />
<br />
=Reflectins as Novel Proteins=<br />
<br />
Reflectins unique properties partly stem from their unique amino acid composition - residues normally common in proteins (alanine, isoleucine, leucine and lysine) are nowhere to be found in any reflectins identified thus far. Meanwhile typically rare residues (arginine, methionine, tryptophan and tyrosine) make up almost 57% of the protein! The family of reflectin proteins also share a repeated domain which could also possess some unique optical characteristics.<br />
<br />
[[File:Cam-beta_barrel.png | thumb |A typical example of a beta-barrel structure]]<br />
The 3D protein structure of reflectin has not yet been documented. It may be natively unstructured, Weiss ''et al'' hypothesised that it may form a [http://en.wikipedia.org/wiki/Beta_barrel beta barrel] like structure when interacting with membranes, as recombinant reflectin-like proteins associate strongly with artificial membrane structures after cell-free expression.<br />
<br />
Kramer ''et al'' and Tao and DeMartini ''et al'' have demonstrated the remarkable capability of reflectin proteins to self assemble in vitro to create complex structures. Recombinant reflectin, refolded ''in vitro'', can be carefully spread along a silicon slide to make thin films with intense structural colours which showed iridescence. Measurement of the refractive index revealed reflectin possessing the highest refractive index of any natural known protein. In addition Kramer ''et al'' demonstrated the ability of reflectin to form a diffraction grating when ionic solvents used to dissolve it, were diffused away in a water bath. <br />
<br />
[[File:CAM_THIN_FILM.jpg | thumb | Thin films cast from different concentrations of reflectin<sup>[[#Kramer|[1]]]</sup>]]<br />
<br />
=='''References'''==<br />
<div id="Kramer"></div>[http://www.nature.com/nmat/journal/v6/n7/abs/nmat1930.html] Kramer ''et al.'' '''The self-organizing properties of squid reflectin protein''' Nature Materials 533-538 VOL6 JULY 2007<br />
<div id="Crookes"></div>[http://www.sciencemag.org/content/303/5655/235.short] Crookes ''et al.'' '''Reflectins: The Unusual Proteins of Squid Reflective Tissues''' SCIENCE 235-238 VOL303 9 JANUARY 2004<br />
<div id="Morse"></div>[http://www.sciencedirect.com/science/article/pii/S0142961209011442] Morse ''et al.'' '''The role of protein assembly in dynamically tunable bio-optical tissues''' Biomaterials 793-801 VOL31 FEBRUARY 2010<br />
<div id="Clams"></div>[http://www.publish.csiro.au/paper/ZO9920319.html] Griffiths ''et al''. '''Iridophores in the mantle of giant clams.''' Australian Journal of Zoology (1992) Volume: 40, Issue: 3 Pages: 319-326<br />
<div id="Izumi"></div>[http://www.ncbi.nlm.nih.gov/pubmed/19776150] Izumi ''et al''. '''Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid.''' J. R. Soc. Interface 6 March 2010 vol. 7 no. 44 549-560 <br />
<div id="Octopus"></div>[http://www.springerlink.com/content/bba14b73ad35f495/]Brocco ''et al''. '''Reflector cells in the skin of ''Octopus dofleini''''' Cell and Tissue Research, Volume 205, Number 2, 167-186, 1980<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-20T22:54:28Z<p>Cat: /* System diagrams */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==System diagrams==<br />
<br />
Our parts were designed for the two main branches of our labwork - [[Team:Cambridge/Project/In_Vivo | ''in vivo'' expression and export]] to try and achieve structural colour, and [[Team:Cambridge/Project/In_Vitro | overexpression for purification]] and ''in vitro'' studies of our recombinant reflectins.<br />
<br />
===Overexpression for purification===<br />
[[File:Cam_Overexpression_Construct.png | 600px]]<br />
===Export===<br />
[[File:Cam_Export_Construct.png | 600px]]<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures with optical properties. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage, and see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 our registry page] for information about our BioBrick submission.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638201 Reflectin A1 with poly-His tag]===<br />
In pure form reflectins may be used to create [[Team:Cambridge/Project/In_Vitro | vibrantly coloured thin films]] and other devices. We used a poly-His affinity column to purify reflectin from cells for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 Improved TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K233307 the TorA tag already in the registry] and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team:<br />
<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/File:Cam_Overexpression_Construct.pngFile:Cam Overexpression Construct.png2011-09-20T22:48:26Z<p>Cat: </p>
<hr />
<div></div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-20T22:48:02Z<p>Cat: /* System diagrams */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==System diagrams==<br />
===Overexpression for purification===<br />
[[File:Cam_Overexpression_Construct.png | 600px]]<br />
===Export===<br />
[[File:Cam_Export_Construct.png | 600px]]<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures with optical properties. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage, and see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 our registry page] for information about our BioBrick submission.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638201 Reflectin A1 with poly-His tag]===<br />
In pure form reflectins may be used to create [[Team:Cambridge/Project/In_Vitro | vibrantly coloured thin films]] and other devices. We used a poly-His affinity column to purify reflectin from cells for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 Improved TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K233307 the TorA tag already in the registry] and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team:<br />
<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-20T22:32:36Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
==System diagrams==<br />
===Overexpression for purification===<br />
===Export===<br />
[[File:Cam_Export_Construct.png | 700px]]<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures with optical properties. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage, and see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 our registry page] for information about our BioBrick submission.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638201 Reflectin A1 with poly-His tag]===<br />
In pure form reflectins may be used to create [[Team:Cambridge/Project/In_Vitro | vibrantly coloured thin films]] and other devices. We used a poly-His affinity column to purify reflectin from cells for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 Improved TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K233307 the TorA tag already in the registry] and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team:<br />
<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/File:Cam_Export_Construct.pngFile:Cam Export Construct.png2011-09-20T22:31:59Z<p>Cat: </p>
<hr />
<div></div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-20T20:21:22Z<p>Cat: /* Reflectin A1 */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures with optical properties. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage, and see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 our registry page] for information about our BioBrick submission.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638201 Reflectin A1 with poly-His tag]===<br />
In pure form reflectins may be used to create [[Team:Cambridge/Project/In_Vitro | vibrantly coloured thin films]] and other devices. We used a poly-His affinity column to purify reflectin from cells for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 Improved TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K233307 the TorA tag already in the registry] and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team:<br />
<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-20T20:20:40Z<p>Cat: /* TorA tag */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage, and see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 our registry page] for information about our BioBrick submission.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638201 Reflectin A1 with poly-His tag]===<br />
In pure form reflectins may be used to create [[Team:Cambridge/Project/In_Vitro | vibrantly coloured thin films]] and other devices. We used a poly-His affinity column to purify reflectin from cells for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 Improved TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K233307 the TorA tag already in the registry] and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team:<br />
<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-20T20:08:54Z<p>Cat: /* TorA tag */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage, and see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 our registry page] for information about our BioBrick submission.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638201 Reflectin A1 with poly-His tag]===<br />
In pure form reflectins may be used to create [[Team:Cambridge/Project/In_Vitro | vibrantly coloured thin films]] and other devices. We used a poly-His affinity column to purify reflectin from cells for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from [http://partsregistry.org/wiki/index.php?title=Part:BBa_K233307 the TorA tag already in the registry] and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team:<br />
<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-20T20:05:55Z<p>Cat: /* Reflectin A1 with poly-His tag */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage, and see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 our registry page] for information about our BioBrick submission.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638201 Reflectin A1 with poly-His tag]===<br />
In pure form reflectins may be used to create [[Team:Cambridge/Project/In_Vitro | vibrantly coloured thin films]] and other devices. We used a poly-His affinity column to purify reflectin from cells for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from the TorA tag already in the registry and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team:<br />
<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-20T20:03:42Z<p>Cat: /* Reflectin A1 */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures. We worked with Reflectin A1 from Loligo pealeii. See our [[Team:Cambridge/Project/Background | background page]] for more information about reflectins and their role in cephalopod camouflage, and see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 our registry page] for information about our BioBrick submission.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638201 Reflectin A1 with poly-His tag]===<br />
In pure form reflectins may be used to create vibrantly coloured thin films and other devices. We used a poly-His affinity column to purify reflectin for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from the TorA tag already in the registry and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team:<br />
<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/PartsTeam:Cambridge/Parts2011-09-20T20:02:18Z<p>Cat: /* TorA tag */</p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<br />
==Featured Parts==<br />
<br />
[[Image:Cam_Multilayer_drop_1.jpg|400px|right|thumb| A thin film composed of Reflectin A1 from Loligo pealeii, purified using a poly-His affinity column]]<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638001 Reflectin A1]===<br />
Reflectins are a family of proteins which help give cephalopods their amazing camouflage abilities and which can self-assemble into a variety of multilayered structures. We worked with Reflectin A1 from Loligo pealeii. See our Background page for more information about reflectins and their role in cephalopod camouflage, and see our registry page for information about our BioBrick submission.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638201 Reflectin A1 with poly-His tag]===<br />
In pure form reflectins may be used to create vibrantly coloured thin films and other devices. We used a poly-His affinity column to purify reflectin for this purpose.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638402 TorA tag]===<br />
We submitted a variant of the TorA leader sequence export tag. This TorA leader sequence variant has had been successfully used to export GFP to the periplasm of E.coli as described [http://www.ncbi.nlm.nih.gov/pubmed/11123687 here]. Our tag differs slightly from the TorA tag already in the registry and should be cheaper to obtain from primer synthesis.<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638401 Reflectin A1 with N-terminal TorA tag]===<br />
<br />
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K638403 Reflectin A1 with N-terminal TorA tag and C-terminal sfGFP]===<br />
<br />
===...and the rest===<br />
<br />
A complete list of parts submitted to the Registry by this year's Cambridge team:<br />
<br />
<groupparts>iGEM011 Cambridge</groupparts><br />
<br />
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/desc/overview/homeTeam:Cambridge/desc/overview/home2011-09-20T19:59:18Z<p>Cat: </p>
<hr />
<div><p>Welcome to the Cambridge 2011 iGEM team's homepage!</p><br />
<p>From here, you can find out more about our <a href='/Team:Cambridge/Project'>project</a>, see the <a href='/Team:Cambridge/Parts'>parts</a> we submitted, browse the <a href='/Team:Cambridge/Experiments'>experiments</a> we performed to characterise them and read up on the <a href='/Team:Cambridge/Team'>team</a>.</p><br />
<p>You can also take a look at what we've done to help the wider <a href='/Team:Cambridge/Project/Gibthon'> SynBio community</a>, as well as read our <a href='/Team:Cambridge/Society'>report</a> on what effect iGEM has on its participants.</p></div>Cathttp://2011.igem.org/Team:Cambridge/desc/overview/partsTeam:Cambridge/desc/overview/parts2011-09-20T18:08:46Z<p>Cat: Created page with "<p>Our best characterised biobricks are highlighted here, along with diagrams of how our reflectin producing systems work.</p> <p>This page also links to the relevant registry p..."</p>
<hr />
<div><p>Our best characterised biobricks are highlighted here, along with diagrams of how our reflectin producing systems work.</p><br />
<br />
<p>This page also links to the relevant registry pages for all our parts.</p></div>Cathttp://2011.igem.org/Team:Cambridge/BlogTeam:Cambridge/Blog2011-09-20T17:23:45Z<p>Cat: Redirected page to Team:Cambridge/Blog/Week 12</p>
<hr />
<div>#REDIRECT [[Team:Cambridge/Blog/Week_12]]<br />
{{Template:Team:Cambridge/BLOG_HEAD}}<br />
<br />
{{Template:Team:Cambridge/BLOG_FOOT}}</div>Cathttp://2011.igem.org/Team:Cambridge/Society/Questionnaire/ResultsTeam:Cambridge/Society/Questionnaire/Results2011-09-20T17:18:01Z<p>Cat: </p>
<hr />
<div>{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}<br />
<div><br />
<br />
<p><font size="3" face="Helvetica">The iGEM census</font> <br><br />
</p><br />
<br />
<p><font size="3" face="Helvetica"><u>Preface</u></font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">When a new scientific field is just <br />
budding, outreach organisations and events are key to bringing in new <br />
minds and catalysing expansion. Synthetic biology is no different, and <br />
the iGEM (international Genetically Engineered Machine) competition <br />
initiated by Tom Knight and Randy Witberg is arguably the biggest outreach <br />
effort that has ever been launched in this area. The annual competition <br />
encourages students to dedicate a single summer vacation to learning <br />
and applying the basic principles of synthetic biology to create a genetically <br />
engineered organism that can be ‘useful’ to society. iGEM has grown <br />
exponentially since its humble beginnings as an IAP at MIT in 2003 to <br />
become an international community with thousands of participants from <br />
all over the world. Many iGEM teams from around the world have conducted <br />
their own outreach events, introducing synthetic biology to an even <br />
wider audience including children who have not even left primary school. </font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">One question strikes us, however <br />
- just how effective has iGEM been as means to engage more scientists <br />
to work in synthetic biology? What has its effect been on its participants, <br />
and the field as a whole? By making some advances to answer this question, <br />
valuable insight can be made into what similar outreach programmes can <br />
work to achieve, and what they can do to improve their effectiveness.</font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">To this end, a short online questionnaire <br />
was designed and distributed by the Cambridge iGEM team in 2011, with <br />
the aim of creating a ‘census’ of the students who have taken part <br />
in iGEM teams over the past 8 years of the competition’s history. <br />
With over 60 respondents, we have amassed a sufficient amount of data <br />
to make some valid analyses; although we do urge caution against drawing <br />
any broad conclusions about the competition, since our sample is only <br />
a statistically insignificant “”% of the population size. However, <br />
on an individual scale, we do believe that we have a lot of interesting <br />
tales to tell. </font> <br></p><br />
<p><font size="3" face="Helvetica">We would like to thank the many iGEM <br />
team supervisors from around the globe who have helped us to get in <br />
touch with these alumni, and without which this study would not have <br />
been possible.</font> <br> <br></p><br />
<br />
<p><font size="3" face="Helvetica"><u>Notes</u></font></p><br />
<p><font size="3" face="Helvetica">The respondents were not required <br />
to complete the survey, and so not all applicants responded to every <br />
question - as a result, the total number of respondents varies for each <br />
question.</font></p><br />
<p><font size="3" face="Helvetica">We assume familiarity with the iGEM <br />
competition in the remainder of this report. If necessary, you can find <br />
out more about iGEM on their website, at <a href="http://ung.igem.org" target="_blank">ung.igem.org</a>.</font></p><br />
<p><font size="3" face="Helvetica">On our graphs, we refrain from using <br />
percentages and deal with raw figures instead due to our small sample <br />
size. In this way, we wish to make it evident when our data may be more <br />
anecdotal than statistically significant.</font> <br> <br><br />
</p><br />
<p><font size="3" face="Helvetica"><u>Contents</u></font> <br><br />
</p><br />
<br />
<p><font size="3" face="Helvetica">The student sample (Graphs 1,2, map <br />
of team locations) </font> <br></p><br />
<p><font size="3" face="Helvetica">The iGEM projects (Graph 3)</font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">The impact of iGEM on career paths <br />
(Graphs 4,5,6,7, map of new locations)</font> <br></p><br />
<p><font size="3" face="Helvetica">Is iGEM self-sustaining? (Graph 8)</font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">Conclusions</font> <br></p><br />
<br />
<p><font size="3" face="Helvetica">Appendix 1 - The Questionnaire</font></p><br />
<p><font size="3" face="Helvetica">Appendix 2 - Additional Resources <br />
(published papers, videos)</font> <br></p><br />
<p><font size="3" face="Helvetica"><u>The student sample</u></font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">The target population of the iGEM <br />
outreach initiative was sampled for this study - this consisted of the <br />
‘alumni’ who initially took part in the iGEM competition as a student <br />
team member from 2003-2010 inclusive. The iGEM alumni who participated <br />
in our research were linked to the survey (hosted on <a href="http://www.esurveyspro.com" target="_blank">www.esurveyspro.com</a>) <br />
via various routes, including direct email contact as well as Twitter, <br />
Facebook and our iGEM wiki page. </font></p><br />
<p><font size="3" face="Helvetica">(Graph 1)</font></p><br />
<p><font size="3" face="Helvetica">We attempted to focus on alumni who <br />
participated in the earliest years of the competition, since their careers <br />
would be more likely to have progressed further after taking part in <br />
iGEM than more recent participants. Unfortunately, it was difficult <br />
to get in contact with students from 2003 and 2004, since it was only <br />
in 2005 that iGEM existed formally in its own right, as opposed to being <br />
an option within the internal IAP (independent activities period) programme <br />
at MIT.</font> <br></p><br />
<br />
<p><font size="3" face="Helvetica">(Graph 2)</font></p><br />
<p><font size="3" face="Helvetica">The vast majority of the respondents <br />
had specialised in biological sciences (although this spanned very different <br />
fields including microbiology, biochemistry, immunology and pharmacology) <br />
by the time they participated in iGEM, indicating that they were already <br />
set on a career in biology before taking part. Significantly, however, <br />
one third of respondents were not specialised in biology, with their <br />
backgrounds ranging from physics and engineering to computer science, <br />
mathematics and medicine. These students came from various years of <br />
the competition, and were present from the very beginning of the competition <br />
in 2003 through to the most recent years. iGEM seems to have been quite <br />
successful in attracting participants from various backgrounds, which <br />
is crucial in the development of such an interdisciplinary field.</font></p><br />
<p><font size="3" face="Helvetica">(Map 1 - team locations)</font></p><br />
<p><font size="3" face="Helvetica">The respondents came from teams across <br />
North America and Europe - although an effort was made to collect data <br />
from universities in other continents, we were not successful. This <br />
is partially due to the fact that the vast majority of iGEM teams were <br />
either North American or European up until 2009, when there was a large <br />
expansion in Asia. However, we are aware that this still introduces <br />
a bias to our results that needs to be bourne in mind.</font> <br><br />
</p><br />
<p><font size="3" face="Helvetica"><u>The iGEM Projects</u></font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">The significance of the actual projects <br />
that are undertaken by iGEM teams is a key part of the contribution <br />
that the iGEM community brings to the field of synthetic biology, together <br />
with building the online and physical library of standard DNA parts <br />
and of course inspiring students to participate in the new science. <br />
A criticism that has been made of the competition is that since the <br />
students have such little time to work on their projects, they either <br />
end up with ‘gimmicky’ machines or are not able to make significant <br />
headway in achieving the ambitious goals they set themselves at the <br />
outset. Evidently, more work must be done beyond the summer in order <br />
to develop projects further, but has this been the case in the past? </font></p><br />
<p><font size="3" face="Helvetica">(Graph 3)</font></p><br />
<br />
<p><font size="3" face="Helvetica">Graph 3 shows that over two thirds <br />
of the projects that respondents to the survey took part in were continued <br />
beyond the jamboree, though the extent of the work done varied significantly <br />
between projects. Some of the projects were only slightly augmented, <br />
with some further BioBrick modification, sometimes finishing off projects <br />
that weren’t completed in time for the jamboree. This work was sometimes <br />
done by the original team members, but also often carried on by their <br />
iGEM mentors or supervisors, who were often professors or post-Doctoral <br />
students in the lab they were working at. More recently, the judging <br />
criteria of the iGEM competition has encouraged new teams to further <br />
characterise the BioBrick parts submitted to the Registry by previous <br />
iGEM teams. This is important, since many parts that have been submitted <br />
have little or no documentation so future teams can do a lot to improve <br />
the usefulness of the BioBricks available. Some teams, such as Slovenia, <br />
build upon the work they do year on year, and have been extremely successful <br />
in the competition (winning 3 times in the past 5 years in Slovenia’s <br />
case). Teams such as Cambridge 2009 have also had their BioBricks requested <br />
from other scientists, who have subsequently used them in their own <br />
research. </font> <br></p><br />
<p><font size="3" face="Helvetica">Impressively, several projects were <br />
also continued by some members of the team as part of their Master’s <br />
or PhD thesis. Some papers were also published by the students which <br />
were printed in acclaimed journals such as Nature and Cell (team UTAustin <br />
2004,2005,2006) and presentations made at conferences such as BioSynBio. <br />
This is indicative of the scientific importance of the work done. Some <br />
titles of the publications can be found in Appendix 2.</font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">Through our research, we have found <br />
that some notable work has been produced following on from projects <br />
undertaken as entries in the iGEM competition. In this way, iGEM has <br />
allowed students to give a direct contribution to research in synthetic <br />
biology, very soon after participating. The students are often inspired <br />
to produce very ambitious biological machines, often aiming to solve <br />
a well-known global problem, such as facilitating the breakdown of hazardous <br />
chemicals (e.g. crude oil, TU Delft 2010), sustainable production of <br />
biomaterials (e.g. biocellulose, Imperial College London 2009). As a <br />
result, there is often much media coverage of iGEM projects (e.g. Bioluminescence <br />
by Cambridge 2010 was featured in New Scientist magazine), raising the <br />
public profile of synthetic biology in a positive light. In this sense, <br />
it can thus be argued that iGEM projects have done much to both publicise <br />
synthetic biology as well as contribute significantly to the science.</font> <br><br />
<br></p><br />
<p><font size="3" face="Helvetica"><u>The Impact of iGEM on Career Paths</u></font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">We asked iGEM alumni whether or not <br />
they personally believed that iGEM affected their subsequent career <br />
paths. The results are shown in Graph 4.</font></p><br />
<br />
<p><font size="3" face="Helvetica">(Graph 4)</font></p><br />
<p><font size="3" face="Helvetica">Over 90% of respondents thought that <br />
iGEM did have an effect, and over 50% thought that the effect was significant. <br />
The reasons for this varied widely; the most popular included:</font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">Research experience (often a decisive <br />
factor in whether or not the students subsequently pursued a career <br />
in academia)</font></p><br />
<p><font size="3" face="Helvetica">Developed lab skills (useful in their <br />
later career, and influential on their CV)</font></p><br />
<p><font size="3" face="Helvetica">Networking opportunities (many students <br />
are currently working as PhD students with their iGEM mentor as their <br />
supervisors, or in other iGEM labs which they got in contact with during <br />
the jamboree)</font></p><br />
<p><font size="3" face="Helvetica">Many students were inspired to work <br />
in synthetic biology later in their careers (see Graph 6 below)</font></p><br />
<p><font size="3" face="Helvetica">Students became more open-minded <br />
to working outside their specialist fields and with people from different <br />
disciplines (almost every respondent was new to synthetic biology when <br />
taking part)</font> <br></p><br />
<br />
<p><font size="3" face="Helvetica">(Graph 5)</font></p><br />
<p><font size="3" face="Helvetica">Graph 5 also shows that 75% of respondents <br />
were performing research in academia at the time of completing the survey <br />
- participating in iGEM was commonly said to be a valuable experience <br />
that helped students to choose such a career path. Conversely, a couple <br />
of alumni also stated that their experience in iGEM helped them to decide <i><br />
not </i>to go into research. </font> <br></p><br />
<p><font size="3" face="Helvetica">In addition, three alumni responding <br />
to our survey were co-founders of biotech startup companies (including <br />
Ginkgo Bioworks, </font><a href="http://www.diybio.com" target="_blank"><font color="#000099" size="3" face="Helvetica"><u>www.diybio.org</u></font></a><font size="3" face="Helvetica"> and Synbiota), all of which were related <br />
to synthetic biology, and cited participation in the iGEM competition <br />
as being crucial to their motivation for doing so.</font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">We also asked more specifically about <br />
whether or not taking part in iGEM actually inspired further participation <br />
in synthetic biology, which is key to judging the success of the outreach <br />
aspect of iGEM. </font></p><br />
<br />
<p><font size="3" face="Helvetica">(Graph 6)</font></p><br />
<p><font size="3" face="Helvetica">Almost half of our respondents did <br />
place their work within the field of synthetic biology, which is a very <br />
promising statistic given that all but one were new to the field prior <br />
to participating in the competition. In addition, several non-biologists <br />
(e.g. engineers) switched into biological research in areas such as <br />
systems biology and bioinformatics which they did not term ‘synthetic <br />
biology’, but are arguably very closely related. Biology students <br />
now working in other biological fields have also stated that the lab <br />
skills learned during iGEM have been extremely useful in their current <br />
research.</font> <br> <br></p><br />
<p><font size="3" face="Helvetica">A couple of notes to bear in mind <br />
- several of the respondents who stated that they were working in synthetic <br />
biology were studying PhDs in the field, and some are considering moving <br />
away from the field afterwards. It must also be considered that our <br />
sample could be quite biased, since iGEM alumni who enjoyed participating <br />
in iGEM may have been more likely to find out about and complete our <br />
survey. Nevertheless, the effect of participating in iGEM for these <br />
alumni is significant, and may be considered by the organisers of iGEM <br />
as a big success.</font> <br></p><br />
<p><font size="3" face="Helvetica">In order to shed more light on what <br />
particular aspects of participating in iGEM caused these effects, we <br />
also looked at where the individual students made their principle contribution <br />
to their projects.</font></p><br />
<p><font size="3" face="Helvetica">(Graph 7)</font></p><br />
<p><font size="3" face="Helvetica">The vast majority of respondents <br />
contributed principally to biological experimentation (or ‘wet-work’, <br />
as it is commonly termed) in their iGEM project - which is perhaps unsurprising <br />
given the practical nature of the competition. Almost all biologists <br />
focused on this side of the project, while students who focused on the <br />
other aspects of the project were all non-biologists, with the exception <br />
of a single biologist who worked on software design. However, approximately <br />
half of the non-biologists also named their largest contribution to <br />
the project to be biological experimentation. This suggests that, at <br />
least in some teams, new students have been introduced to biology through <br />
the iGEM programme. This has certainly been the case in our personal <br />
experience, as more than half of the Cambridge 2011 iGEM team were non-biologists, <br />
yet we all participated eagerly in wet-work. Engineers became specialists <br />
in making electrophoresis gels and Gibson assembly, while a physicist <br />
became heavily involved in protein extraction. As a consequence, several <br />
members of our team are now keen to participate in research with a strong <br />
biological bent in the future.</font> <br></p><br />
<p><font size="3" face="Helvetica">Several other teams, however, seem <br />
to restrict students to focus on aspects of the project that play only <br />
to their strengths, so biology students would devote all their time <br />
to the wet-work while computer scientists (for example) would only work <br />
on modelling and software development. From our research, this restriction <br />
does not seem to have affected the influence that iGEM has on the students. <br />
For example, we have spoken to an engineering student who worked solely <br />
on modelling in her iGEM project, but subsequently (after some networking <br />
at the jamboree) took up a PhD in synthetic biology at another iGEM <br />
lab. We possess little more than anecdotal evidence, however, so more <br />
data needs to be gathered in order to draw any confident conclusions.</font> <br><br />
<br />
</p><br />
<p><font size="3" face="Helvetica"><u>Is iGEM Self-Sustaining?</u></font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">In order for an organisation to be <br />
sustainable over an extended period of time, it is crucial that there <br />
exists a continuing influx of people willing to participate in running <br />
it. We looked to find out whether or not iGEM alumni chose to be such <br />
people, since this would show that the participants enjoyed the experience <br />
to such an extent that they would willingly take part again, and perhaps <br />
believed in the effectiveness of the outreach programme. If a sufficient <br />
proportion of alumni were to contribute further to the iGEM community, <br />
it would also make the community self-sustaining, in that solely alumni <br />
of the programme itself would be required to keep the organisation running, <br />
with no other recruitment necessary (although also possible, and likely <br />
to be very welcome!).</font> <br></p><br />
<p><font size="3" face="Helvetica">(Graph 8)</font> <br></p><br />
<p><font size="3" face="Helvetica">Graph 8 shows that almost half of <br />
the respondents have participated in iGEM beyond being a student team <br />
member. The extent of involvement varied widely, from passively following <br />
the progress of new iGEM teams (and communicating with them via social <br />
networking sites such as Facebook and Twitter) to working at iGEM HQ <br />
and becoming a member of the judging panel at the jamboree. Every level <br />
of administration seemed to have been covered, though the most popular <br />
means of participation was supervising/advising new teams. If this statistic <br />
were representative of the entire population of iGEM alumni, which looks <br />
to increase by several thousand students each year, then it would seem <br />
that iGEM is successfully self-sustaining. However, once again caution <br />
must be made against such sweeping statements due to the small size <br />
of our sample.</font> <br></p><br />
<p><font size="3" face="Helvetica"><u>Conclusions</u></font> <br><br />
<br />
</p><br />
<p><font size="3" face="Helvetica">At the outset of our study, we sought <br />
to find out how successful iGEM has been as an outreach programme attracting <br />
students to the field of synthetic biology, and deduce what lessons <br />
could be learned in order to improve such efforts in the future. Though <br />
our research has been by no means complete, we do believe that we have <br />
garnered sufficient data to draw some tentative conclusions.</font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">Generally, the iGEM alumni who responded <br />
to our questionnaire have indeed been influenced by their experiences <br />
in the competition. Almost half went on to work in synthetic biology <br />
in their future careers, and several more worked in closely related <br />
fields. This included many students who were not even studying a biological <br />
subject as an undergraduate. </font> <br></p><br />
<p><font size="3" face="Helvetica">Many of the projects were also continued <br />
well beyond the jamboree, with some findings published in highly-regarded <br />
scientific journals. This reflects the significance of the scientific <br />
findings that have been found or inspired within the context of iGEM <br />
projects, and shows that participants can not only learn about the field, <br />
but also contribute actively in the science. </font> <br></p><br />
<p><font size="3" face="Helvetica">These alumni also often went on to <br />
contribute further in the iGEM community, contributing to the sustainability <br />
of the organisation in the long-term.</font> <br></p><br />
<p><font size="3" face="Helvetica">There are some aspects of iGEM that <br />
we believe have been key to its success - </font></p><br />
<p><font size="3" face="Helvetica">-The focus on creating machines that <br />
solve problems or create innovations with applications in the real world <br />
seems to be really inspirational to students, creating a really memorable <br />
experience.</font></p><br />
<br />
<p><font size="3" face="Helvetica">-The freedom given to undergraduate <br />
students to participate in ‘PhD-style’ research gives them a valuable <br />
opportunity to find out whether or not they wish to work in academia, <br />
and also gives the students a chance to pursue the areas which interest <br />
them most within synthetic biology. This is arguably the best way to <br />
allow students to enjoy the experience as much as possible.</font></p><br />
<p><font size="3" face="Helvetica">-The encouragement to create novel <br />
projects pushes students to the edge of current research, allowing students <br />
to feel a real sense of pride for their achievements, and inspires work <br />
that is genuinely significant in the wider scientific community.</font></p><br />
<p><font size="3" face="Helvetica">-The target for outreach, undergraduate <br />
students, seems to be pitched to the right level. Within interdisciplinary <br />
teams, the students have a wide knowledge base, and are capable of both <br />
learning and understanding the challenging material without having to <br />
‘dumb-down’ the science. However, the students are also at a stage <br />
in their career that is early enough for them to later specialise straightforwardly <br />
in synthetic biology before leaving education (for example, by undertaking <br />
a PhD in the subject).</font></p><br />
<p><font size="3" face="Helvetica">-The jamboree has been cited by many <br />
alumni as the best aspect of the competition - it is an international <br />
scientific conference on a very large scale, and the enthusiasm of the <br />
thousands of students present make it quite unique. It has been cited <br />
by some alumni as the one key event that really made them want to return <br />
in the future, and networking opportunities are well-utilised.</font> <br><br />
<br></p><br />
<p><font size="3" face="Helvetica">In addition, however, we believe <br />
some improvements could be made to really maximise the success of iGEM <br />
as an outreach programme for synthetic biology:</font> <br></p><br />
<p><font size="3" face="Helvetica">-The importance of iGEM projects <br />
in the wider scientific community should be publicised by the organisation, <br />
with papers and conferences in which references have been made to iGEM <br />
projects highlighted. This is likely to inspire several other teams <br />
that have been successful in the iGEM competition to prepare similar <br />
publications, which could also be successful.</font></p><br />
<p><font size="3" face="Helvetica">-The opportunities for further involvement <br />
by iGEM alumni in other aspects of the iGEM community should be made <br />
clear, and alumni should be encouraged to participate. This is likely <br />
to be popular, since almost all the students appeared to have enjoyed <br />
their first experience.</font></p><br />
<br />
<p><font size="3" face="Helvetica">-Students from all backgrounds should <br />
be able to participate in all aspects of the project - although restricting <br />
students to their specific areas of expertise did not seem to have too <br />
much effect on their appreciation of the science, in our personal experience <br />
we believe it to be a lot more enjoyable when all the students take <br />
the opportunity to step out of their comfort zone and explore new fields <br />
- bringing a new sense of perspective that we value highly.</font></p><br />
<p><font size="3" face="Helvetica">-Funding has been said to be an issue <br />
facing many teams, that have hampered efforts to both expand projects <br />
beyond the jamboree and also to start up new teams. If iGEM could perhaps <br />
lower the cost of participation, and/or offer special grants for particularly <br />
promising applications, this could help to over come the problem.</font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">Despite leaving much room for improvement, <br />
within a few weeks of research we have managed to identify some key <br />
learning points that could be extremely useful for both the iGEM administrators <br />
and others hoping to lead similar outreach programmes. The most accurate <br />
and efficient way for organisers to find their strengths and weaknesses <br />
is straight from the participants themselves. We would like to encourage <br />
such practice to optimise outreach, in order to create enjoyable programmes <br />
that are maximally effective.</font> <br></p><br />
<p><font size="3" face="Helvetica"><u>Appendix 1</u></font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">Screenshots of the full questionnaire <br />
(and link to the site?)</font> <br></p><br />
<p><font size="3" face="Helvetica"><u>Appendix 2</u></font> <br><br />
<br />
</p><br />
<p><font size="3" face="Helvetica"><b>Papers published as a result of <br />
extended iGEM projects:</b></font> <br></p><br />
<p><font size="3" face="Helvetica">Levskaya et al. Nature 2005 -<i> <br />
Synthetic Biology: engineering Escherichia coli to see light</i></font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">Tabor et al. Cell 2009 - <i>A synthetic <br />
genetic edge detection program</i></font> <br></p><br />
<p><font size="3" face="Helvetica">Joshi et al. Desalination 2009 - <i><br />
Novel approaches to biosensors for detection of arsenic in drinking <br />
water</i> (Presented at the Water and Sanitation in International Development <br />
and Disaster Relief (WSIDDR) International Workshop Edinburgh, Scotland, <br />
UK, 28-30 May 2008.</font> <br></p><br />
<br />
<p><font size="3" face="Helvetica">Mora et al. Analytical &amp; Bioanalytical <br />
Chemistry 2011 - <i>A pH-based biosensor for detection of arsenic in <br />
drinking water</i></font> <br></p><br />
<p><font size="3" face="Helvetica"><b>Additional links:</b></font> <br><br />
</p><br />
<p><font size="3" face="Helvetica">An interview with an iGEM alumnus <br />
who co-founded <a href="http://diybio.org" target="_blank">diybio.org</a> has also been posted to YouTube by user kkleiner, <br />
with the title “Singularity Hub Interviews Mac Cowell of <a href="http://diybio.org" target="_blank">diybio.org</a> <br />
(diy bio)”. This interview was not conducted by the Cambridge iGEM <br />
team.</font> <br></p><br />
<br />
<p><font size="3" face="Helvetica">A blog set up by an iGEM alumnus <br />
who is “blogging about iGEM from the spectator’s viewpoint”: </font><a href="http://bloggingaboutigem.wordpress.com" target="_blank"><font color="#000099" size="3" face="Helvetica"><u>http://bloggingaboutigem.<WBR>wordpress.com</u></font></a><font size="3" face="Helvetica">/</font></p><br />
<br />
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==iGEMers on iGEM==<br />
<br />
The iGEM competition gathers hundreds of undergraduates together to explore the potential of synthetic biology. What happens to iGEM alumni after the Jamboree? Does the experience of such a unique summer project change the career paths of iGEM students? What becomes of all the innovation and hard-won expertise developed whilst building biobricks?<br />
<br />
To answer these questions, the 2011 Cambridge team conducted a census of iGEM alumni. [[Team:Cambridge/Society/Questionnaire | Take the questionnaire here]], [[Team:Cambridge/Society/Questionnaire/Results | see some of our results]] or [[Team:Cambridge/Society/Report | read our discussion about what we found out.]]<br />
<br />
Some previous iGEMers were generous enough to grant us interviews, allowing us to record their experiences of life after iGEM. As well as providing the quotes and case studies in our report, you can see [[Team/Cambridge/Society/interviews | excerpts from their interviews]]. ''(With thanks to Veronica Ranner for assistance in filming and editing)''<br />
<br />
=Why talk to iGEMmers?=<br />
<br />
The experiences of those who do iGEM is logged in wikis and wetwork and BioBricks, but we think more emphasis should be placed on the long term influence of an intense summer of synthetic biology. As well as looking at the big picture of synthetic biology and the challenges and opportunities it can bring to society, we would like to encourage more teams to reflect on their own thoughts and feelings. The growing field of synthetic biology will be shaped by the people who do synthetic biology, so we hope to see a greater consideration of what makes people want to join the field. <br />
<br />
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<br />
==Gibson Assembly==<br />
<br />
This year, we at the Cambridge team used [http://en.wikipedia.org/wiki/Gibson_assembly Gibson Assembly] exclusively for fabrication of our [[Team:Cambridge/Experiments/Plasmid_Constructs | plasmids]]. Gibson assembly was pioneered in iGEM by the 2010 Cambridge team, who submitted an [http://www.cambridgeigem.org/RFC57.pdf RFC] (Request For Comments) to the [http://biobricks.org/ BioBricks Foundation].<br />
<br />
European teams at this year's iGEM competition have an unprecedented short time in which to complete their projects. We found that Gibson allowed us to construct our plasmids quickly and easily, saving us a lot of time.<br />
<br />
See our protocol for Gibson Assembly [[/Team:Cambridge/Protocols/Gibson_Assembly | here]].<br />
<br />
==Gibthon==<br />
[[File:CAM_gibthon_BillCollins.jpg | 200px | thumb | right | Bill Collins, seen here submerged under our laboratory pet Krysia, began writing Gibthon in 2010]]<br />
[http://www.gibthon.org Gibthon], initiated by Bill Collins of the Cambridge 2010 iGEM team, is a collection of web based tools to facilitate the design of primers for Gibson Assembly. Currently, primer design is a bit of a 'dark art' – one must spend a large amount of time manually copying and pasting sequences into various other tools in order to check for annealing temperature, mispriming and secondary structure.<br />
<br />
Gibthon's aim is to be an easy to use web based tool for primer design, reducing the chance of errors while designing primers. While some degree of common sense will always be required, Gibthon will reduce the chance of making mistakes while designing these primers.<br />
<br />
<br style='clear:both;' /><br />
===Our Contribution===<br />
Gibthon's source code is hosted on GitHub. You can see exactly what we have contributed this year by looking at my [https://github.com/haydnKing/Gibthon gibthon repository].<br />
<br />
The most important changes are summarised below.<br />
<br />
<html><br />
<table border='1' cellpadding='5' ><br />
<tr><th style='width:100px;'>Change</th><th>Before</th><th>After</th></tr><br />
<tr><br />
<td>Improvements to fragment display</td><br />
<td>Gibthon could only display fragments as raw text, making it difficult to decipher where features were on the strand.</td><br />
<td>Gibthon now displays fragments in a much more intuitive way, allowing the user to select parts of the sequence and easily copy from themm</td><br />
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<td>Fragment import improvements</td><br />
<td>Gibthon had basic support for: partsregistry.org import (via an ugly hack), NCBI/Entrez import (by accession number only) and genbank file upload (much of the genbank metadata was lost)</td><br />
<td>Gibthon can now directly import from the partsregistry by part name (more below), seamlessly converting biobrick features and metadata into genbank format. It can also search a number of NCBI/Entrez databases, display search results, and import the results. Gibthon can now handle multiple file upload and supports both genbank and fasta format. You can now manually import raw sequences into Gibthon</td><br />
</tr><br />
<tr><br />
<td>Metadata Editing</td><br />
<td>Once a fragment was imported, it was impossible for the user to edit any of the metadata (annotations) associated with the fragment</td><br />
<td>Users can now easily update the metadata associated with a fragment – this is particularly useful, an example annotation might be “location: fridge C, shelf 2”</td><br />
</tr><br />
<tr><br />
<td>Fragment deletion</td><td>Gibthon did not allow you to delete a fragment from your fragment library</td><td>You can now</td><br />
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Gibthon is an important tool not just for future iGEM teams, but for synthetic biology as a whole. Its open nature puts it in a great position to become <i>the</i> tool for construct assembly.<br />
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I am therefore intending to make further improvements after the wiki freeze, which will be:<br />
<br />
* A few fixes for the fragment display (gibthon's UI is going through some improvements at the moment, so things may be a bit lopsided...)<br />
* Great improvements to the Construct Designer.<br />
** The construct designer (as is) works well, but it is not particularly intuitive for people new to the field (as many igemmers are) so I plan on making it far more visual – this will also make it harder for people to make mistakes!<br />
** This will mean using the html5 canvas tag – People using IE6 will see a curt (but polite) explanation of why they should upgrade their browser!<br />
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=== Gibthon's Future ===<br />
We hope that gibthon will become a leading tool for construct design, both within iGEM and the wider synbio community. Gibthon will always be open source and free to use.<br />
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== The Future of Gibson Assembly ==<br />
We hope that more people in synbio will start using this revolutionary technique, it has certainly saved us a large amount of trouble and strife. While Gibson assembly does not require the prefix and suffix which are required for the BioBrick standard, it is perfectly straightforward to add and remove them using Gibson as required.<br />
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However, as the cost of synthesis comes ever lower, people will begin to move away from assembly – who wants to go to the hassle of building their construct if you can get the entire thing synthesised at little cost? When this begins to happen, physical DNA libraries will be less and less important, while tools to manipulate digital sequences of DNA will become vital.<br />
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==iGEMers on iGEM==<br />
<br />
The iGEM competition gathers hundreds of undergraduates together to explore the potential of synthetic biology. What happens to iGEM alumni after the Jamboree? Does the experience of such a unique summer project change the career paths of iGEM students? What becomes of all the innovation and hard-won expertise developed whilst building biobricks?<br />
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To answer these questions, the 2011 Cambridge team conducted a census of iGEM alumni. [[Team:Cambridge/Society/Questionnaire | Take the questionnaire here]], or [[Team:Cambridge/Society/Report | see our discussion of the results.]]<br />
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Some previous iGEMers were generous enough to grant us interviews, allowing us to record their experiences of life after iGEM. As well as providing the quotes and case studies in our report, you can see [[Team/Cambridge/Society/interviews | excerpts from their interviews]]. ''(With thanks to Veronica Ranner for assistance in filming and editing)''<br />
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=Why talk to iGEMmers?=<br />
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The experiences of those who do iGEM is logged in wikis and wetwork and BioBricks, but we think more emphasis should be placed on looking at the long term influence of an intense summer of synthetic biology. <br />
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