http://2010.igem.org/wiki/index.php?title=Special:Contributions/Porter&feed=atom&limit=50&target=Porter&year=&month=2010.igem.org - User contributions [en]2024-03-29T15:02:29ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Aberdeen_Scotland/AdvisorsTeam:Aberdeen Scotland/Advisors2010-10-27T21:34:34Z<p>Porter: </p>
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<h4>Dr. Oliver Ebenhoeh</h4><br />
<p>Oliver Ebenhoeh studied Mathematics and Physics at the University of Heidelberg (Germany), <br />
and did his PhD in Theoretical Biophysics at the Humboldt University, Berlin (Germany), <br />
where he continued to work as a postdoctoral researcher until 2006. He established his research group<br />
'Systems Biology and Mathematical Modelling' at the Max-Planck-Institute of Molecular Plant Physiology in Potsdam (Germany)<br />
in 2007. He moved to the University of Aberdeen in 2009 where he was appointed Reader in Systems Biology <br />
as a joint position of the Institute for Complex Systems and Mathematical Biology and the Institute of Medical Sciences.<br />
<br><br><br />
<a href="http://www.abdn.ac.uk/ims/staff/details.php?id=ebenhoeh">http://www.abdn.ac.uk/ims/staff/details.php?id=ebenhoeh</a></p><br />
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<h4>Dr. John B. Geddes</h4><br />
<p>Dr. Geddes is the Associate Dean for Faculty Affairs and Research and Professor<br />
of Mathematics at Olin College. He graduated in 1990 from Heriot-Watt University, Edinburgh<br />
with a B.Sc. in Physics and received his Ph.D. in Applied Mathematics in 1994 from the University<br />
of Arizona. Dr. Geddes has worked closely with the Institute for Complex Systems and Mathematical<br />
Biology at the University of Aberdeen on various projects including the mathmatics of micro-vascular<br />
blood flow.<br />
<br><br><br />
<a href="http://www.olin.edu/faculty_staff/bios/bio_jgeddes.html">http://www.olin.edu/faculty_staff/bios/bio_jgeddes.html</a></p><br />
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No picture of Alessandro is available.<br />
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<h4>Dr. Alessandro De Moura</h4><br />
<p>Dr Alessandro De Moura is a lecturer at the University of Aberdeen and a member of the Applied Dynamics Research Group within the Institute of Dynamics and Control.<br />
<br><br><br />
<a href="http://www.abdn.ac.uk/engineering/people/details.php?id=a.moura">http://www.abdn.ac.uk/engineering/people/details.php?id=a.moura</a></p><br />
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<td><img src="https://static.igem.org/mediawiki/2010/1/1f/Mamen.jpg"/></td><br />
<td><h4>Dr. Mamen Romano</h4><br />
<p>Field: Nonlinear dynamics and time series analysis, modelling of biological<br />
systems<br />
<br><br />
I studied Physics in Spain and Germany; then I did my PhD at the University of<br />
Potsdam (Germany) in 2004, and stayed there for two more years doing a<br />
PostDoc. I moved to Aberdeen at the end of 2006 and became Lecturer in 2007.<br />
<br><br><br />
<a href="http://www.abdn.ac.uk/ims/staff/details.php?id=m.romano">http://www.abdn.ac.uk/ims/staff/details.php?id=m.romano</a></p></td></tr><br />
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<h4>Dr. Yasushi Saka</h4><br />
<p>Yasushi was a graduate student in Mitsuhiro Yanagida’s laboratory in the Biophysics Department of Kyoto University, and his early postdoctoral work was with Professor Jim Smith and centred on the early Xenopus embryogenesis, especially the function of activin, an archetypical morphogen, and its downstream targets. <br />
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<a href="http://www.sulsa.ac.uk/saka">http://www.sulsa.ac.uk/saka</a></p><br />
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<td><h4>Prof. Duncan Shaw</h4><br />
<p>BSc, University of Bristol (Biochemistry), 2.1 Honours 1975.<br />
PhD, University of Wales Cardiff (Biochemistry) 1976-1980.<br />
Postdoctoral Research Fellow, University of Sheffield (Microbiology) 1980-82 (including 3 months at University of Pittsburgh, USA).<br />
Research Fellow/Lecturer/Senior Lecturer/Reader, University of Wales College of Medicine (Medical Genetics), 1982-1994.<br />
<br><br><br />
<a href="http://www.abdn.ac.uk/ims/staff/details.php?id=d.shaw">http://www.abdn.ac.uk/ims/staff/details.php?id=d.shaw</a></p></td></tr><br />
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<td><h4>Dr. Ian Stansfield</h4><br />
<p>Ian Stansfield graduated from the University of Sheffield with a BSc (Hons) Microbiology in 1986, and was awarded a PhD in 1990. His post-doctoral research was carried out with Professor Mick Tuite at the University of Kent from 1990 to 1996. This work focused on studies of protein synthesis in yeast, investigating how the accuracy of protein synthesis is maintained, and the mechanism of translation termination. In 1996, he moved to the University of Aberdeen to take up a Lectureship position. <br />
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<a href="http://www.abdn.ac.uk/ims/staff/details.php?id=i.stansfield">http://www.abdn.ac.uk/ims/staff/details.php?id=i.stansfield</a></p></td></tr><br />
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<td><img src="https://static.igem.org/mediawiki/2010/7/7d/Marco.jpg"/></td><br />
<td><h4>Dr. Marco Thiel</h4><br />
<p>2004 PhD in Nonlinear Dynamics (Mathematical Physics)<br />
<br><br />
2004-2006 Post-Doc, University of Potsdam (Germany)<br />
<br><br />
2006- RCUK Academic Research Fellow<br />
<br><br><br />
<a href="http://www.abdn.ac.uk/~wpe009/people/details.php?id=m.thiel">http://www.abdn.ac.uk/~wpe009/people/details.php?id=m.thiel</a></p><br />
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<h4>Dr. Ekkehard Ullner</h4><br />
<p>Ekkehard Ullner was appointed SULSA Lecturer in Systems Biology at the University of Aberdeen in 2009. In 2004, he was awarded a PhD from the University of Potsdam for the thesis “Noise-induced phenomena of signal transmission in excitable neural models”. He investigates genetic networks with cell-to-cell communications and deduced numerical simulations of the repressilator model with cell-to-cell communication mediated by an additional quorum-sensing module.<br />
<br><br><br />
<a href="http://www.abdn.ac.uk/ims/staff/details.php?id=e.ullner">http://www.abdn.ac.uk/ims/staff/details.php?id=e.ullner</a></p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Project_OverviewTeam:Aberdeen Scotland/Project Overview2010-10-27T20:43:34Z<p>Porter: </p>
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<h1>Project Overview</h1><br />
<h3>Introduction</h3><br />
<p> <br />
For this year’s iGEM competition the Aberdeen team has worked on developing a translationally controlled toggle switch embedded in yeast.<a href="#ref1"><sup style="font-size:10px">[1]</sup></a> Genetic toggle switches are a vital component for synthetic biology circuits , enabling functional control of biological functions. The majority of toggle switches used for iGEM are embedded in Escherichia coli and can only be controlled at the transcriptional level <a href="#ref2"><sup style="font-size:10px">[2]</sup></a><sup style="font-size:10px">,</sup><a href="#ref3"><sup style="font-size:10px">[3]</sup></a>. Our main goal was to create and model a novel gene circuit, wherein yeast cells can be switched between mutually exclusive fluorescent proteins under exposure to environmental factors. This switching behaviour would be regulated at the translational level, an innovation over previous systems that only demonstrated transcriptional regulation <a href="#ref4"><sup style="font-size:10px">[4]</sup></a><sup style="font-size:10px">,</sup><a href="#ref5"><sup style="font-size:10px">[5]</sup></a>.The novel genetic toggle switch operated by controlling gene expression at the translational level consisted of two gene expression constructs expressing an RNA-binding protein fused to either Green (GFP) or Cyan (CFP) fluorescent protein in the presence of appropriate inducer. When co-expressed in yeast, these translational fusions would be mutually inhibitory at the translational level, thereby forming a biological, ‘Toggle Switch’ system. <br />
</p><br />
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<h3>The AyeSwitch</h3><br />
<p>The toggle switch is shown by Fig 1 and was named the ‘AyeSwitch’. It is regulated by controlling the two constructs, GAL1p-[Npeptide-GFP] and CUP1p-[MS2-CFP], via inducible yeast promoters GAL1 or CUP1 in the presence or absence of galactose and Cu2+ ions respectively. <br />
</p><br />
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<p><br />
For example, in the presence of galactose only, GAL1 is induced and there is expression of N-peptide-GFP protein. The subsequent addition of Cu2+ then induces the transcription of mRNA coding for MS2 coat binding protein and CFP. In addition to this, the mRNA also codes for a Bbox stem loop sequence that can be bound by N-peptide. </p><br />
<p><br><br />
Ideally, there is initial inhibition of MS2-CFP translation by Npeptide-GFP binding to the Bbox stem loop. Evolution of time corresponds to the ratio of MS2-CFP mRNA to N-peptide-GFP protein increasing allowing some MS2-CFP to be produced until CFP ‘switches ON’ as it gains dominance over GFP.</p><br />
<p><br />
Additionally, N-peptide-GFP protein translation can also be inhibited by MS2-CFP via MS2 protein binding to the MS2 stem loops on the N-peptide-GFP mRNA. This may help the switching ON of CFP and also means GFP would face a similar situation if the inducer was changed from Cu2+ to galactose.</p><br />
<p><br><br />
However, additional variables may come into play affecting the outcomes described above. It is likely that the concentration of each inducer present, the translational rate and binding efficiency of stem loop binding proteins to mRNA stem loop and degradation rate of proteins can also affect the outcome. Reversing the order of inducer present may also affect the outcome. </p><br />
<br><br />
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<h3>Experimental Characterisation of the AyeSwitch</h3><br />
<p><br />
The experimental work addressed these issues by initially characterising the promoters in terms of their dose response and time response using constructs GAL1-[GFP] and CUP1-[GFP]. These experiments were then extended to characterise GAL1p-[Npeptide-GFP] and CUP1p-[MS2-CFP] which discovered that CUP1p-[MS2-CFP] did not function as expected.</p><br><br />
<p><br />
The experimental work diverged from this point to troubleshoot CUP1p-[MS2-CFP], investigating the translation inhibition of GAL1p-[Npeptide-GFP] by MS2 coat protein using construct MET17p - [MS2], Bio-brick construction and testing of Bio-brick E2050 mOrange.</p><br />
<br><br />
<br><br />
<h3>Modelling Characterisation of the Ayeswitch</h3><br />
<p><br />
Our team proposed a novel model to describe the functioning of the Aye-switch, based on ordinary differential equations (ODEs). The proposed system of ODEs was carefully and systematically studied both analytically and computationally. A bifurcation analysis was performed and the bistability of the system was investigated with respect to large variations in the parameters of the system. The deterministic simulations were compared with stochastic ones, using the Gillespie algorithm. The parameter space of the model was thoroughly investigated, using two different approaches: Monte-Carlo and directed evolution. These two approaches are very useful for a wide range of projects in synthetic biology. The theoretical predictions led to the proposition of optimised parameters for the Aye-switch that allow a very robust translational switch.</p><br />
<br><br />
<br><br />
<h3>Troubleshooting CUP1p-[MS2-CFP]</h3><br />
<p><br />
Troubleshooting of CUP1p-[MS2-CFP] was carried out through a series of gene cassette replacement experiments testing the promoter and CFP sequences for functionality. The conclusions to these experiments suggest that the Bbox Stem loop, usually located in the 3’untranslated region but is in the 5’ untranslated region of our construct may be preventing the expression of downstream proteins. It may also be that the fusion of MS2 to CFP results in inappropriate protein folding, inhibiting expression.</p> <br />
<br><br />
<br><br />
<h3>Verification of Translation Inhibition as a Regulatory Mechanism</h3><br />
<p><br />
It was shown that the translational inhibition of GAL1p-[Npeptide-GFP] by MS2 coat protein was possible, confirming that translational regulation is viable. Further work if time permitted would investigate if this inhibition could work in the context of a toggle switch.</p><br />
<br><br />
<br><br />
<h3>Bio-brick construction and testing </h3><br />
<p><br />
In parallel, Bio-bricks were constructed and submitted to the Registry of parts whilst testing of the Bio-brick E2050 mOrange using fluorimetry and FACS analysis lead to the conclusion that the mOrange sequence did not function within our GAL1p-[Npeptide-GFP] construct that was shown to be able to express GFP appropriately. </p><br />
<br><br><br />
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<h1>Attribution and Contributions</h1><br />
<h3>Biological circuit construction and testing </h3><br />
<p><br />
The students within the experimental section of the team were provided (by their host lab) with two yeast strains that had Gal1p-GFP and Cup1p-GFP integrated into the genome (see 'DNA constructs). They then used these constructs to analyse the properties of the CUP1 and GAL1 promoters. With some instructor oversight, the student team themselves then completely designed constructs Gal1p-(Npep-GFP) and Cup1p-(MS2-CFP), which were then synthesised by a synthetic DNA supply company. The students then tested these constructs, and further engineered them during the trouble-shooting phase of the project.<br><br />
All the experimental work described on the wiki, involving characterisation, testing and re-engineering of the bio-bricks, was carried out by the student members of the team. All the construction and sequencing of the four submitted bio-bricks was also carried out by members of the student team.<br />
<br><br><br />
<br />
<h3>Mathematical modelling of the AyeSwitch </h3><br />
<p><br />
The students within the theoretical section of the team carried out all the described modelling. Team activities were overseen by the Instructors, but all model coding and model analysis was performed by the students within the team.<br />
<br />
<br><br><br />
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<h3> References</h3><br><br />
<p><br />
<a name="ref1"></a><br />
<a href="http://www.nature.com/msb/journal/v2/n1/full/msb4100073.html"target="_blank"><b><sup style="font-size:10px">[1]</sup></b></a> Ernesto Andrianantoandro et al. Synthetic biology: new engineering rules for an emerging discipline Molecular Systems Biology 2:2006.0028</p><br><br />
<p><br />
<a name="ref2"></a><br />
<a href="http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html"target="_blank"><b><sup style="font-size:10px">[2]</sup></b></a> Timothy S. Gardner et al. Construction of a genetic toggle switch in Escherichia coli Nature 403, 339-342 (20 January 2000)</p><br><br />
<p><br />
<a name="ref3"></a><br />
<a href="http://www.cell.com/retrieve/pii/S0092867403003465"target="_blank"><b><sup style="font-size:10px">[3]</sup></b></a> Mariette R. Atkinson et al. Development of Genetic Circuitry Exhibiting Toggle Switch or Oscillatory Behavior in Escherichia coli Cell, Volume 113, Issue 5, 597-607, 30 May 2003 </p><br><br />
<p><br />
<a name="ref4"></a><br />
<a href="http://www.nature.com/emboj/journal/v17/n14/abs/7591108a.html"target="_blank"><b><sup style="font-size:10px">[4]</sup></b></a> Adam Platt and Richard J Reece The yeast galactose genetic switch is mediated by the formation of a Gal4p–Gal80p–Gal3p complex The EMBO Journal (1998) 17, 4086 - 4091 </p><br><br />
<p><br />
<a name="ref5"></a><br />
<a href="http://www.pnas.org/content/88/19/8597.abstract"target="_blank"><b><sup style="font-size:10px">[5]</sup></b></a> D W Griggs and M Johnston Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression PNAS October 1, 1991 vol. 88 no. 19 8597-8601</i></p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Biobrick_RelatedTeam:Aberdeen Scotland/Biobrick Related2010-10-27T20:38:59Z<p>Porter: </p>
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<h1>Lab Diary concerning Biobricks</h1><br><br />
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<h2>Preparation week</h2><br><br />
<ul><li>1.Preparation / rescue of iGEM DNA samples:<br><br />
<ul><br />
<li>i.BBa_E2050 – mOrange fluorescent protein, KanR.<br><br />
<li>ii.BBa_J63005 – ADH1 promoter, AmpR.<br><br />
<li>iii.BBa_E2030 – yEYFP (yellow fluorescent protein), KanR.<br><br />
<li>iv.BBa_I716101 with J04450 (plasmid), AmpR.<br><br />
<li>v.BBa_I716101 with P1010 (plasmid with CcdB), AmpR<br></ul><br />
<li>2.Transformation of rescued iGEM DNA samples into XL1 – Blue Subcloning – Grade Competent Cells.<br><br />
<li>3.Transfer of Transformants into fresh plates to prevent the depletion of nutrients.<br></ul><br />
<br><br />
<h2>Week 1 (14th – 18th of June)</h2><br><br />
<ul><li>1.Confirmation of the identity of Biobricks YFP (pSB2k3 + E2030)<br><br />
<li>2.Plasmid purification (Miniprep) of desired BioBrick constructs:<br><br />
<ul><li>i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)<br><br />
<li>ii.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
<li>iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul><br />
<li>3.BioBricks digestion – gel electrophoresis, to verify plasmid sizes, of:<br><br />
<ul><li>i.BBa_J63010 plasmid and BBa_J63005 (ADH1 promoter)<br><br />
<li>ii.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
<li>iii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul></ul><br />
<br><br />
<h2>Week 2 (21st – 25th of June)</h2><br><br />
<ul><li>1.Design of Primers for construction of Biobricks<br><br />
<ul><li>i.MS2 protein<br><br />
<li>ii.MS2 loops: 5’leader<br><br />
<li>iii.N peptide (1)<br><br />
<li>iv.N peptide (2)<br><br />
<li>v.Bbox – 5’leader<br></ul><br />
<li>2.Due to unclear results obtained in the prior digestion we repeated the digestion of BioBricks:<br><br />
<ul><li>i.BBa_I716101 plasmid and BBa_J04450 (RFP)<br><br />
<li>ii.BBa_I716101 plasmid and BBa_P1010 (CcdB – death gene)<br></ul></ul><br />
<br><br />
<h2>Week 3 (28th – 2nd July)</h2><br><br />
<ul><li>1.Bgl Brick Preparation:<br><br />
<ul><li>i.Digestion 1, of BglBrick 1, using EcoR1 and Xho1 – higher volume than protocol since concentration of plasmid was very low. <br><br />
<li>ii.Enzymes then heat inactivated<br><br />
<li>iii.Vector then alkaline phosphatase treated, with enzymes again heat inactived after treatment.<br></ul></ul><br />
<br><br />
<h2>Week 4 (5th – 9th of July)</h2><br><br />
<ul><li>1.Miniprep of BglBrick 1<br><br />
<li>2.Digestion of BglBrick using EcoR1 and Xho1 – due to low yield when previously digested.<br><br />
<li>3.PCR 1 reaction of inserts (to use for BglBrick cloning)<br><br />
<li>4.Transformation of BglBrick into one shot competent cells E.coli cells, to try and improve plasmid propagation – no improved propagation observed.<br></ul><br />
<br><br />
<h2>Week 5 (12th – 16th of July)</h2><br><br />
<ul><li>1.Further Bgl Brick preparation:<br><br />
<ul><li>i.Miniprep of BglBrick<br><br />
<li>ii.Digestion of BglBrick<br><br />
<li>iii.Enzyme heat inactivation<br><br />
<li>iv.Alkaline phosphatase treatment.<br></ul><br />
<li>2.Digestion 1 of PCR products using EcoR1 and Xho1<br><br />
<li>3.Ligation 1 of PCR products (inserts 1) and vector BglBrick<br><br />
<ul><li>Used Nanodrop to determine concentration, we have concluded the data obtained in this way is unreliable, which is why this procedure may not have been successful.<br></ul><br />
<li>4.Transformation 1, of cloned products into XL1 Blue subcloning – grade cells E.coli cells.<br><br />
<li>5.Transformation 2, of cloned products into XL1 Blue competent E.coli cells. Since transformation 1 gave poor efficiency, a more competent cell was tried.<br><br />
<li>6.PCR of E.coli colonies from BglBrick 1 – Did not work.<br><br />
<li>7.Design and order of mOrange primers for Bio-brick testing<br><br />
<li>8.Background reading for Bgl-Brick vector and Bgl-bricking<br><br />
<li>9.Mini-prep, digest and gel electrophoresis of Bgl-Brick vector and pRS415 vector <br></ul><br />
<br><br />
<h2>Week 6 (19th – 23rd of July)</h2><br><br />
<ul><li>1.More Bgl Brick preparation since previous involved Nanodrop concentration determination but was decided this analysis is unrealiable:<br><br />
<ul><li>i.Miniprep of BglBrick<br><br />
<li>ii.Digestion of BglBrick<br><br />
<li>iii.Enzyme heat inactivation<br><br />
<li>iv.Alkaline phosphatase treatment.<br></ul><br />
<li>2.Background reading and planning for PCR <br><br />
<li>3.Background reading and planning of alkaline phosphatase and ligation reaction for Bgl-bricking <br></ul><br />
<br><br />
<h2>Week 7 (26th – 30th of July)</h2><br><br />
<ul><li>1.PCR of E.coli colonies from BglBrick 2 – positive PCR.<br><br />
<li>2.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br><br />
<li>3.Transformation 3, of cloned products (inserts 1) into subcloning efficiency DH5α E.coli cells.<br><br />
<li>4.PCR of Transformation 3 colonies which were then also plated, to confirm required insert presence.<br><br />
<li>5.Second stage digest , (PvuII) of pRS415 and gel electrophoeris for Bgl-bricking <br><br />
<li>6.Master plate of Bgl-brick<br><br />
<li>7.PCR of mORange<br><br />
<li>8.Transformation of BY4741 ΔTrp with pRS415 and mOrange (homologous recombination)<br></ul><br />
<br><br />
<h2>Week 8 (2nd – 7th of August)</h2><br><br />
<ul><li>1.Confirmation of BglBricks produced:<br><br />
<ul><li>i.Plasmid purification (miniprep) of E.coli colonies from Transformation 3 colonies.<br><br />
<li>ii.Digestion of plasmid with<br><br />
<li>iii. Ecor1 and Xhol1<br><br />
<li>iv.Ecor1 – since insert was not being observed on the gel when cut with two enzymes.<br></ul><br />
<li>2.Further Bgl Brick preparation as we ran out:<br><br />
<ul><li>i.6 X Miniprep of BglBrick<br><br />
<li>ii.Combined all 6 from step i. To obtain a higher concentration of BglBrick using Qiagen PCR kit (check name of kit).<br><br />
<li>iii.Digestion of BglBrick using EcoR1 and Xho1<br><br />
<li>iv.Digestion had to be done twice as some uncut vector remained after the first cut.<br><br />
<li>v.Enzyme heat inactivation<br><br />
<li>vi.Alkaline phosphatase treatment.<br></ul><br />
<li>3.Tested BY4741 pRS415 mOrange transformants <br><br />
<li>4.Read through protocol for PCR colony screening <br></ul><br />
<br><br />
<h2>Week 9 (9th – 14th of August)</h2><br><br />
<ul><li>1.Digestion 1, of E.coli colonies from BglBrick 2 using EcoR1 and Xho1.<br><br />
<li>2.Digestion 2, of E.coli colonies from BglBrick 2 using EcoR1, since digest with two enzymes showed no insert band on the gel<br><br />
<li>3.Digestion 2, of PCR products using EcoR1 and Xho1<br><br />
<li>4.Ligation 2, of PCR products (inserts 1) and vector BglBrick<br><br />
<li>5.Transformation 2, of cloned products into XL1 Blue competent – grade E.coli cells.<br><br />
<li>6.PCR Colony screening of mOrange transformants<br><br />
<li>7.Bgl-bricking transformation experiment<br><br />
<li>8.Tested BY4741 pRS415 mOrange transformants using fluorometer<br><br />
<li>9.Mini-prep Bgl-brick vectors<br></ul><br />
<br><br />
<h2>Week 10 (15th – 21st of August)</h2><br><br />
<ul><li>1. Repeated: PCR of inserts chosen to submit into biobricks.<br><br />
<li>2. pSB1C3 plasmid preparation:<br />
<li>i.Digestion of linear plasmid with EcoR1 and Pst1<br />
<li>ii.Enzyme heat inactivation <br><br />
<li>3. Digestion of PCR products using EcoR1, Dpn1 and Pst1<br><br />
<li>4. Ligation 1 of PCR products and pSB1C3 plasmid. <br><br />
<li>5. Transformation 1, (1:1, plasmid:vector) of cloned products into XL1 Blue competent E.coli cells – grade cells E.coli cells.<br><br />
<li>6. Transformation 2, (1:3, plasmid:vector)of cloned products into XL1 Blue competent E.coli cells. <br><br />
<li>7. PCR of E.coli colonies<br></ul><br />
<br><br />
<h2>Week 11 (22nd – 29th of August)</h2><br><br />
<ul><li>1. Design and order of primers for Bio-brick testing <br><br />
<li>2. Sent Biobricks to iGEM registry of parts <br><br />
<li>3.FACS analysis of dose response of <br></ul><br />
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<h1>Parameter Space Analysis</h1><br />
<br />
<p>Recall that the equations of our system are as follows (see <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Equations">Equations</a> page for parameter definitions):</p><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/e/e4/Equations3.png"><br />
</center><br />
<br><br />
<p>We wrote a program which takes the equations for our system, calculates the nullclines and records whether or not bistability is achieved. Whether or not bistability is achieved depends strongly on the parameters λ<sub style="font-size:10px">i</sub>, K<sub style="font-size:10px">i</sub>, μ<sub style="font-size:10px">i</sub> and n<sub style="font-size:10px">i</sub>.</p> <br />
<br><br />
<p>The values for the parameters λ<sub style="font-size:10px">i</sub>, K<sub style="font-size:10px">i</sub> and μ<sub style="font-size:10px">i</sub> were taken from literature<a href="#ref1"><sup style="font-size:10px">[1]-[13]</sup></a> but have a large uncertainty attached to them. To take this uncertainty into account we selected each parameter value randomly from a range of two orders of magnitude around the literature value for each parameter. We knew that n<sub style="font-size:10px">2</sub> was between 1 and 3 and n<sub style="font-size:10px">4</sub>=1. This is due to the number of stem loops on the two DNA strands (see Fig 1 in <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Equations">Equations</a>). The strand associated with GFP has two stem loops. A protein can bind to one stem loop and this can then hinder the binding of a protein to the second stem loop (thus making n<sub style="font-size:10px">2</sub> less than 2 but more than 1) or encourage the binding of a protein to the second stem loop (thus making n<sub style="font-size:10px">2</sub> more than 2 but less than 3). The strand associated with the CFP has only one stem loop and thus n<sub style="font-size:10px">4</sub>=1. Please see <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Curve_Fitting">Determination of the Hill coefficient n<sub style="font-size:10px">2</sub></a> for an explanation on how we narrowed down the value for n<sub style="font-size:10px">2</sub>.<br />
<br><br />
<br><br />
We ran the program for various combinations of Hill coefficients (n<sub style="font-size:10px">i</sub>) between 1 and 5. Each time the program was run, the Hill coefficients remained constant but the other parameters varied. </p><br />
<br><br />
<p>Choosing the parameters in this way meant that each time the program was run we would sometimes achieve a combination of parameters that allowed bistability, and sometimes not. <br />
<br><br />
<br><br />
The program was run 100 times and the number of times bistability was achieved was output to the screen as a percentage. <br />
<br><br />
<br><br />
We then decided to run the program for various different parameter ranges. We cannot change individual parameters specifically, but in the lab it is possible to vary the order of magnitude of a parameter. We wanted to know, if we varied our parameters and Hill coefficients, what is the best percentage we can possibly get? These results are shown in scenarios 2-4.</p><br />
<br><br />
<h3>Scenario 1 – unmodified parameters </h3><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/2f/Table1a.png"><br />
</center><br />
<br><br />
<p>These parameters were taken from, or derived from, the information contained within various sources of literature<a href="#ref1"><sup style="font-size:10px">[1]-[13]</sup></a>. The parameters were converted from concentrations to numbers of molecules.<br />
<br><br />
<br><br />
We did not know what concentration of galactose and methionine was optimal, so we ran various tests and found the value for which the percentages were maximised. We found that the optimal percentages occurred if we have 100000 times more galactose than methionine. See figure 1a and 1b.</p><br />
<br><br />
<br><br />
<center><br />
a)<img src="https://static.igem.org/mediawiki/2010/d/d2/Table1.png"> <br />
<br><br />
<br><br />
b)<img src="https://static.igem.org/mediawiki/2010/9/9f/Original_parameters_colorplot.png"><br />
<p style="font-size:10px">Figure 1. a) The percentage of parameter combinations tested that will give us bistability for various combinations of Hill coefficients. n<sub style="font-size:5px">2</sub> is the Hill coefficient for the GFP rate-reaction equation and n<sub style="font-size:5px">4</sub> is the Hill coefficient for the CFP rate-reaction equation. b) Graphical representation of 1a. </p><br />
</center><br />
<br><br />
<br><br />
<p>Here we can see that if n<sub style="font-size:10px">4</sub>=1, then the best scenario we can hope for is that n<sub style="font-size:10px">2</sub>=5. In this situation, 3.29% of the parameter combinations tested will give bistability and the possibility of switching. However, with n<sub style="font-size:10px">2</sub> estimated to be between 1 and 3, we can say that at most 2.03% of the parameter combinations tested gave bistability. </p> <br />
<br><br />
<h3>Scenario 2 – modified parameters 1</h3><br />
<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/2c/Table2a.png"><br />
</center><br />
<br><br />
<br><br />
<p>Percentages are maximised if we have 100000 times more galactose than methionine. See figure 2a and 2b.</p><br />
<br><br />
<br><br />
<center><br />
a) <img src="https://static.igem.org/mediawiki/2010/d/dd/Table2b.png"><br />
<br><br />
<br><br />
b) <img src="https://static.igem.org/mediawiki/2010/3/3b/Mod1.png"><br />
<p style="font-size:10px">Figure 2. a) The percentage of parameter combinations tested that will give us bistability for various combinations of Hill coefficients. b) Graphical representation of 2a.</p><br />
</center><br />
<br><br />
<br><br />
<p>The best scenario is if we have n<sub style="font-size:10px">2</sub>=5 and n<sub style="font-size:10px">4</sub>=5.</p><br />
<br><br />
<h3>Scenario 3 – modified parameters 2</h3><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/7/76/Table3a.png"><br />
</center><br />
<br><br />
<br><br />
<p>Percentages are maximised if there is 100000 times more galactose than methionine. See figure 3a and 3b.</p><br />
<br><br />
<br><br />
<center><br />
a) <img src="https://static.igem.org/mediawiki/2010/2/29/Table3b.png"><br />
<br><br />
<br><br />
b) <img src="https://static.igem.org/mediawiki/2010/d/da/Mod2.png"><br />
<p style="font-size:10px">Figure 3. a) The percentage of parameter combinations tested that will give us bistability for various combinations of Hill coefficients. b) Graphical representation of 3a.</p><br />
</center><br />
<br><br />
<br><br />
<p>Here we find that the optimal scenario is n<sub style="font-size:10px">2</sub>=3 and n<sub style="font-size:10px">4</sub>=5. The trend which has developed in this table will be discussed at the end of scenario 4. </p><br />
<br><br />
<h3>Scenario 4 – modified parameters 3 </h3><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/0/00/Table4a.png"><br />
</center><br />
<br><br />
<br><br />
<p>Percentages are maximised if we have 10000000 times more galactose than methionine. See Figure 4a and 4b.</p><br />
<br><br />
<br><br />
<center><br />
a) <img src="https://static.igem.org/mediawiki/2010/2/22/Table4b.png"><br />
<br><br />
<br><br />
b) <img src="https://static.igem.org/mediawiki/2010/1/1b/Modified_parameters_colorplot.png"><br />
<p style="font-size:10px">Figure 4. a) The percentage of parameter combinations tested that will give us bistability for various combinations of Hill coefficients. b) Graphical representation of 4a.</p><br />
</center><br />
<br><br />
<br><br />
<p>An interesting trend is seen in scenarios 3 and 4. The percentages increase to a maximum at n<sub style="font-size:10px">2</sub>=2 and n<sub style="font-size:10px">4</sub>=5 (n<sub style="font-size:10px">2</sub>=3 and n<sub style="font-size:10px">4</sub>=5 in scenario 3) and then decrease, quite rapidly, towards Hill coefficients of n<sub style="font-size:10px">2</sub>=5 and n<sub style="font-size:10px">4</sub>=5. In the parameter space, there is a region where bistability is possible. For the smaller Hill coefficients, the randomly chosen parameters (as described above) would allow the nullclines to have a shape such that they would cross three or more times and bistability is achieved. The higher Hill coefficients, however, for the same range of parameters, would have nullclines which were shaped so that they did not cross three or more times – in parameter space, they fall outside the region of bistability. </p><br />
<br><br />
<h3>Conclusions</h3><br />
<br />
<p>The ideal scenario is to change the parameters to that in scenario 4 and have Hill coefficients of n<sub style="font-size:10px">2</sub>=2 and n<sub style="font-size:10px">4</sub>=5. Here 98.17% of the parameter combinations tested gave bistability. Contrary to our initial assumptions, depending on the range of parameters, higher Hill coefficients are not always better. The parameters will dictate the shape and position of the nullclines and whether or not bistability is possible. <br />
<br><br />
<br><br />
It is possible to determine the optimal values of the parameters for an ideal working of the switch. However, to change the parameters in the experimental construct so much would require much more time than we had available. </p><br />
<br><br />
<h3>References</h3><br />
<br><br />
<a name="ref1"></a><br />
<p><sup style="font-size:10px">[1]</sup> Hershey, J., Sonenberg, N., and Mathews, M. (2007), Origins and Principles of Translational Control. In Translational Control (pp.1-29). Cold Spring Harbor, NY: Cold Spring Harbor Press. </p><br />
<br><br />
<p><sup style="font-size:10px">[2]</sup> Edwards, A.M., Kane, C.M., Young, R.A., and Kornberg, R.D. (1991), Two Dissociable Subunits of Yeast RNA Polymerase II Stimulate the Initiation of Transcription at a Promoter in Vitro, <i>J. Biol. Chem.</i>, Vol. 266, No.1, pp.71-75 </p><br />
<br><br />
<p><sup style="font-size:10px">[3]</sup> Fürst, P., and Hamer, D. (1989), Cooperative activation of a eukaryotic transcription factor: Interaction between Cu(I) and yeast ACE1 protein, <i>Proc. Natl. Acad. Sci. USA</i>, Vol. 86, pp. 5267-5271 </p><br />
<br><br />
<p><sup style="font-size:10px">[4]</sup> Jensen, L.T., Posewitz, M.C., Srinivasan, C., and Winge, D.R. (1998), Mapping of the DNA binding domain of the copper-responsive transcription factor Mac1 from Saccharomyces cerevisiae, <i>J. Biol. Chem.</i>, Vol. 273, No. 37, pp.23805-23811 </p><br />
<br><br />
<p><sup style="font-size:10px">[5]</sup> Rodgers, K.K., and Coleman, J.E. (1994), DNA binding and bending by the transcription factors GAL4(62*) and GAL4(149*), <i>Protein Science</i>, Vol.3, No. 4, pp. 608-619 </p><br />
<br><br />
<p><sup style="font-size:10px">[6]</sup> Garcia-Martinez, J., Aranda, A., and Perez-Ortin, J.E. (2004), Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms, <i>Mol. Cell.</i>, Vol. 15, No. 2, pp.303-313 </p><br />
<br><br />
<p><sup style="font-size:10px">[7]</sup> Wang, Y., Liu, C.L., Storey, J.D., Tibshirani, R.J., Herschlag, D., and Brown, P.O. (2002), Precision and functional specificity in mRNA decay, <i>Proc. Natl. Acad. Sci. USA</i>, Vol. 99, No.9, pp.5860-5865 </p><br />
<br><br />
<p><sup style="font-size:10px">[8]</sup> Beyer, A. et al. (2004), Post-transcriptional sxpression regulation in the Yeast Saccharomyces Cerevisiae on a genomic scale, <i>Molecular and Cellular Proteomics</i>, Vol. 3, No. 11, pp.1083-1092 </p><br />
<br><br />
<p><sup style="font-size:10px">[9]</sup> Mateus, C. and Avery, S.V. (2000), Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry, <i>Yeast</i>, Vol.16, No.14, pp.1313-1323 </p><br />
<br><br />
<p><sup style="font-size:10px">[10]</sup> Carey, J., Cameron, V., de Haseth, P.L., and Uhlenbeck, O.C. (1983), Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site, <i>Biochemistry</i>, Vol.22, No.11, pp.2601-2610 </p><br />
<br><br />
<p><sup style="font-size:10px">[11]</sup> Talbot, S.J., Goodman, S., Bates, S.R., Fishwick, C.W., and Stockley, P.G. (1990), Use of synthetic oligoribonucleotides to probe RNA-protein interactions in the MS2 translational operator complex, <i>Nucleic Acids Res.</i>, Vol.18, No.12, pp.3521-3528 </p><br />
<br><br />
<p><sup style="font-size:10px">[12]</sup> Arava, Y., Wang, Y., Storey, J.D., Liu, C.L., Brown, P.O., and Herschlag, D. (2003), Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae, <i>Proc. Natl. Acad. Sci. USA</i>, Vol. 100, No.7, pp.3889-3894 </p><br />
<br><br />
<p><sup style="font-size:10px">[13]</sup> Chattopadhyay, S., Garcia-Mena, J., DeVito, J., Wolska, K., and Das, A. (1995), Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda, <i>Proc. Natl. Acad. Sci. USA</i>, Vol.92, No.9, pp.4061-4065 </p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Stochastic_ModelTeam:Aberdeen Scotland/Stochastic Model2010-10-27T20:34:33Z<p>Porter: </p>
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<h1>The Stochastic Model</h1><br />
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<p> Stochastic modelling can be used when modelling biochemical processes and intracellular dynamics <a href="#ref2"><sup style="font-size:10px">[2]</sup></a>. It is also widely used in many other areas such as physics, economics, geophysical systems and even music. </p> <br />
<br><br />
<h3>Overview of the Deterministic Model</h3><br />
<br />
<p>A first look at how our system behaved involved modelling it deterministically. The deterministic model is based on constructing a set of reaction-rate equations to describe the biochemical pathways being studied. These equations are non-linear ordinary differential equations (ODEs). The concentrations of chemical species are the variables and the parameters are the reaction rate constants. Solving the ODEs gives how the resulting concentrations of the chemical species change in time <a href="#ref2"><sup style="font-size:10px">[2]</sup></a><sup style="font-size:10px">,</sup><a href="#ref3"><sup style="font-size:10px">[3]</sup></a>. <br />
<br><br />
<br><br />
In a deterministic system, the time evolution is considered to be continuous and predictable. Strictly speaking, the time evolution of a chemically reacting system is not a continuous process because molecular population levels can only change by discrete integer amounts. In order to predict the molecular population levels at a future time by means of a deterministic approach, we have to take into account the precise positions and velocities of all the molecules in the system <a href="#ref1"><sup style="font-size:10px">[1]</sup></a>, which becomes practically impossible.<br />
<br><br />
<br><br />
In general, concentrations are only defined for large numbers of molecules. In this situation, when numbers change by one or two units in a reaction, these changes can be treated differentially. Also, when the number of molecules is large, any two reactions can take place at the same time. The system of ODEs thus represents a collection of reactions occurring simultaneously <a href="#ref3"><sup style="font-size:10px">[3]</sup></a>. <br />
<br><br />
<br><br />
The deterministic model has some disadvantages however. It is unable to describe the fluctuations in the molecular population levels, which can become very important if the numbers of the molecules involved in the reactions are very low <a href="#ref1"><sup style="font-size:10px">[1]</sup></a>.</p><br />
<br><br />
<h3>Introducing the Stochastic Model</h3><br />
<br />
<p> The stochastic model is a way of following the dynamics of individual molecules <a href="#ref1"><sup style="font-size:10px">[4]</sup></a>. In addition to the deterministic simulation, we also perform the stochastic simulation to see if the potential fluctuations due to the low numbers of molecules involved in our system play a major role. We assume that reactions cannot happen simultaneously and that they do not happen continuously throughout time. There is also now a probability attached to when a reaction will happen and which reaction will occur. <br />
<br><br />
<br><br />
We consider the time evolution of a chemical system to be a discrete process instead of a continuous, deterministic process. The stochastic model thinks of the time evolution as a random process with probabilities as variables <a href="#ref1"><sup style="font-size:10px">[1]</sup></a><sup style="font-size:10px">,</sup><a href="#ref2"><sup style="font-size:10px">[2]</sup></a>. The stochastic model involves the same decomposition of a pathway into elementary reactions. However, here we are looking at numbers of molecules instead of concentrations <a href="#ref2"><sup style="font-size:10px">[2]</sup></a>. The connection with molecular concentrations appears when, in the stochastic model, averages are taken over many cells. These averages satisfy the same equations as the concentrations. Thus the behaviour of concentrations can be interpreted as that of a population average, provided that fluctuations around the average are small <a href="#ref3"><sup style="font-size:10px">[3]</sup></a>.</p><br />
<br><br />
<h3>How to Model Systems Stochastically</h3><br />
<br />
<p>Modelling a system of differential equations stochastically can be done using the Gillespie Algorithm which was devised by Daniel T. Gillespie in 1977 <a href="#ref1"><sup style="font-size:10px">[1]</sup></a>. However, Gillespie also devised a variation on his method which is known as the tau-leaping method. This method results in faster simulations because it is less exact than the actual Gillespie Algorithm as it uses much larger time steps and therefore has less calculations to perform.<br />
<br><br />
<br><br />
Due to time constraints we modelled our system using the tau-leaping model. <br />
<br><br />
<br><br />
The tau-leaping method proceeds as follows: </p><br />
<br><br />
<OL><br />
<LI>Specify a time step, tau.<br />
<LI>Multiply each term of each differential equation by tau.<br />
<LI>Apply Poisson distribution function to each result from step 2.<br />
<LI>Generate a random number using the Poisson distribution created in step 3.<br />
<LI>Calculate the new number of molecules by adding or subtracting each random number from step 4, from the initial number of molecules. Adding if the change has a positive influence on the system, subtracting if it has a negative influence on the system. <br />
<LI>Repeat steps 1-5 for a specified number of reactions, N.<br />
</OL><br />
<br><br />
<p>This algorithm allowed us to view what our model should look like stochastically. The only problem is that we do not know exactly how many molecules we are starting with. However, this is a problem which affects the deterministic model as well as the stochastic model. In both models, the initial concentrations or number of molecules needs to be specified. Until we know how many molecules we begin with, this program will only give us an idea of how our system could evolve. We decided to start our simulations with only one molecule of each of the mRNAs and proteins and watch how the system evolved from there.</p><br />
<br><br />
<h3>Results</h3><br />
<br />
<p>The deterministic model describes the average change in concentration over time. Since the stochastic model deals with individual particles we would expect the stochastic model to follow the trend of the deterministic model but there would be fluctuations around the average value. If we were to take the average of these particles over time, it would look like the deterministic model. The results showed exactly what we expected. The stochastic model follows the trend set by the deterministic model but it fluctuates, sometimes wildly, around the average. <br />
<br />
One thing to note is that the deterministic model sometimes suggests that we will not see bistability. However, because of the fluctuations around the average in the stochastic model, one fluctuation could push us into the realm of bistability.<br />
<br />
In the pictures below the stochastic model is on the left and the deterministic model is on the right. One can clearly see the similarity in the overall shape and it is clear that the deterministic model is just the average of the stochastic model.<br />
<br><br />
<br><br />
<b>Scenario 1: GAL and METH present, winner GFP</b><br />
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<img src="https://static.igem.org/mediawiki/2010/3/34/Stoch_gal_meth.png" width="400" height="320"><br />
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<img src="https://static.igem.org/mediawiki/2010/a/af/Deter_meth_gal.png" width="400" height="320"><br />
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<br><br />
When both galactose and methionine are present, the methionine inhibits the production of the CFP and the galactose encourages the production of the GFP. With nothing to inhibit the production of GFP, the GFP dominates. <br />
<br><br />
<br><br />
<b>Scenario 2: no METH and GAL present, winner – either, depends on other parameters!</b><br />
<br><br />
<br><br />
<left><br />
<img src="https://static.igem.org/mediawiki/2010/b/b0/Stoch_gal_no_meth.jpg" width="400" height="320"><br />
</left><br />
<right><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/Deter_gal_no_meth.jpg" width="400" height="320"><br />
</right><br />
<br><br />
<br><br />
When no methionine is present, CFP can be produced. Also, when GAL is present GFP can be produced. When both proteins are produced, they will both inhibit each other. In this scenario, the dominant protein will depend on the parameters of the system. The modelling we have done suggests that the parameters which affect the system are the transcription/translation rates (λ values) and the binding coefficients (K values). As shown in the above graphs, the parameters of our system dictate that CFP will win. <br />
<br><br />
<br><br />
<b>Scenario 3: no METH and no GAL present, winner CFP</b><br />
<br><br />
<br><br />
<left><br />
<img src="https://static.igem.org/mediawiki/2010/b/b0/Stoch_gal_no_meth.jpg" width="400" height="320"><br />
</left><br />
<right><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/Deter_gal_no_meth.jpg" width="400" height="320"><br />
</right><br />
<br><br />
<br><br />
No methionine present leads to CFP being produced. No galactose present means that no GFP will be produced. With nothing to inhibit the CFP production, CFP will dominate. <br />
<br><br />
<br><br />
Also, a simulation was run with variable galactose and methionine over time. The aim of this was to model the switching behaviour of our system. The picture below shows the results of the stochastic simulation. What we see, is a clear switching behaviour, but only if we actively remove one protein before adding the other.<br />
<br><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/21/Stoch_switch.jpg" width="700" height="470"> <br />
</center><br />
<br><br />
<br><br />
<p>As mentioned previously, when GAL and no METH are present in the system (at 80000 iterations), the winning protein will depend heavily on the parameters of the system. Our experience has shown us that the values for the transcription/translation rates (λ values) and the dissociation constants (K values) strongly affect how the system behaves and thus determines what protein will dominate.</p></p><br />
<br><br />
<h3>Conclusions</h3><br />
<br />
<p>The stochastic model is a more accurate method of modelling a biochemical system where low numbers of molecules are involved. The stochastic model follows the actions of individual molecules whereas the ODEs based model describes how concentrations change continuously in time. The deterministic model is basically just the average of the stochastic model and does not represent any fluctuations due to the individual molecules present. </p><br />
<br><br />
<h3>References</h3><br />
<br><br />
<a name="ref1"></a><br />
<p><sup style="font-size:10px">[1]</sup> Gillespie, D.T. (1977), Exact Stochastic Simulation of Coupled Chemical Reactions, <i>The Journal of Physical Chemistry</i>, Vol. 81, No. 25.</p><br />
<br><br />
<a name="ref2"></a><br />
<p><sup style="font-size:10px">[2]</sup> Ullah, M., Schmidt, H., Cho, K.-H. and Wolkenhauer, O. (2006), Deterministic modelling and stochastic simulation of biochemical pathways using MATLAB, <i>IEE Proc.-Syst. Biol.</i>, Vol. 153, No. 2.</p><br />
<br><br />
<a name="ref3"></a><br />
<p><sup style="font-size:10px">[3]</sup> Hayot, F. (2008), Single Cell experiments and Gillespie’s algorithm. Retrieved from <a href="http://tsb.mssm.edu/summerschool/images/4/4d/HayotSlides.pdf">http://tsb.mssm.edu/summerschool/images/4/4d/HayotSlides.pdf</a> </p><br />
<br><br />
<a name="ref4"></a><br />
<p><sup style="font-size:10px">[4]</sup> Department of Computational & Applied Mathematics, Rice University, Modeling and Simulation of Reaction Networks. Retrieved from <a href="http://www.caam.rice.edu/~caam210/reac/lec.html">http://www.caam.rice.edu/~caam210/reac/lec.html</a> </p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Stochastic_ModelTeam:Aberdeen Scotland/Stochastic Model2010-10-27T20:32:07Z<p>Porter: </p>
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<h1>The Stochastic Model</h1><br />
<br />
<p> Stochastic modelling can be used when modelling biochemical processes and intracellular dynamics <a href="#ref2"><sup style="font-size:10px">[2]</sup></a>. It is also widely used in many other areas such as physics, economics, geophysical systems and even music. </p> <br />
<br><br />
<h3>Overview of the Deterministic Model</h3><br />
<br />
<p>A first look at how our system behaved involved modelling it deterministically. The deterministic model is based on constructing a set of reaction-rate equations to describe the biochemical pathways being studied. These equations are non-linear ordinary differential equations (ODEs). The concentrations of chemical species are the variables and the parameters are the reaction rate constants. Solving the ODEs gives how the resulting concentrations of the chemical species change in time <a href="#ref2"><sup style="font-size:10px">[2]</sup></a><sup style="font-size:10px">,</sup><a href="#ref3"><sup style="font-size:10px">[3]</sup></a>. <br />
<br><br />
<br><br />
In a deterministic system, the time evolution is considered to be continuous and predictable. Strictly speaking, the time evolution of a chemically reacting system is not a continuous process because molecular population levels can only change by discrete integer amounts. In order to predict the molecular population levels at a future time by means of a deterministic approach, we have to take into account the precise positions and velocities of all the molecules in the system <a href="#ref1"><sup style="font-size:10px">[1]</sup></a>, which becomes practically impossible.<br />
<br><br />
<br><br />
In general, concentrations are only defined for large numbers of molecules. In this situation, when numbers change by one or two units in a reaction, these changes can be treated differentially. Also, when the number of molecules is large, any two reactions can take place at the same time. The system of ODEs thus represents a collection of reactions occurring simultaneously <a href="#ref3"><sup style="font-size:10px">[3]</sup></a>. <br />
<br><br />
<br><br />
The deterministic model has some disadvantages however. It is unable to describe the fluctuations in the molecular population levels, which can become very important if the numbers of the molecules involved in the reactions are very low <a href="#ref1"><sup style="font-size:10px">[1]</sup></a>.</p><br />
<br><br />
<h3>Introducing the Stochastic Model</h3><br />
<br />
<p> The stochastic model is a way of following the dynamics of individual molecules <a href="#ref1"><sup style="font-size:10px">[4]</sup></a>. In addition to the deterministic simulation, we also perform the stochastic simulation to see if the potential fluctuations due to the low numbers of molecules involved in our system play a major role. We assume that reactions cannot happen simultaneously and that they do not happen continuously throughout time. There is also now a probability attached to when a reaction will happen and which reaction will occur. <br />
<br><br />
<br><br />
We consider the time evolution of a chemical system to be a discrete process instead of a continuous, deterministic process. The stochastic model thinks of the time evolution as a random process with probabilities as variables <a href="#ref1"><sup style="font-size:10px">[1]</sup></a><sup style="font-size:10px">,</sup><a href="#ref2"><sup style="font-size:10px">[2]</sup></a>. The stochastic model involves the same decomposition of a pathway into elementary reactions. However, here we are looking at numbers of molecules instead of concentrations <a href="#ref2"><sup style="font-size:10px">[2]</sup></a>. The connection with molecular concentrations appears when, in the stochastic model, averages are taken over many cells. These averages satisfy the same equations as the concentrations. Thus the behaviour of concentrations can be interpreted as that of a population average, provided that fluctuations around the average are small <a href="#ref3"><sup style="font-size:10px">[3]</sup></a>.</p><br />
<br><br />
<h3>How to Model Systems Stochastically</h3><br />
<br />
<p>Modelling a system of differential equations stochastically can be done using the Gillespie Algorithm which was devised by Daniel T. Gillespie in 1977 <a href="#ref1"><sup style="font-size:10px">[1]</sup></a>. However, Gillespie also devised a variation on his method which is known as the tau-leaping method. This method results in faster simulations because it is less exact than the actual Gillespie Algorithm as it uses much larger time steps and therefore has less calculations to perform.<br />
<br><br />
<br><br />
Due to time constraints we modelled our system using the tau-leaping model. <br />
<br><br />
<br><br />
The tau-leaping method proceeds as follows: </p><br />
<br><br />
<OL><br />
<LI>Specify a time step, tau.<br />
<LI>Multiply each term of each differential equation by tau.<br />
<LI>Apply Poisson distribution function to each result from step 2.<br />
<LI>Generate a random number using the Poisson distribution created in step 3.<br />
<LI>Calculate the new number of molecules by adding or subtracting each random number from step 4, from the initial number of molecules. Adding if the change has a positive influence on the system, subtracting if it has a negative influence on the system. <br />
<LI>Repeat steps 1-5 for a specified number of reactions, N.<br />
</OL><br />
<br><br />
<p>This algorithm allowed us to view what our model should look like stochastically. The only problem is that we do not know exactly how many molecules we are starting with. However, this is a problem which affects the deterministic model as well as the stochastic model. In both models, the initial concentrations or number of molecules needs to be specified. Until we know how many molecules we begin with, this program will only give us an idea of how our system could evolve. We decided to start our simulations with only one molecule of each of the mRNAs and proteins and watch how the system evolved from there.</p><br />
<br><br />
<h3>Results</h3><br />
<br />
<p>The deterministic model describes the average change in concentration over time. Since the stochastic model deals with individual particles we would expect the stochastic model to follow the trend of the deterministic model but there would be fluctuations around the average value. If we were to take the average of these particles over time, it would look like the deterministic model. The results showed exactly what we expected. The stochastic model follows the trend set by the deterministic model but it fluctuates, sometimes wildly, around the average. <br />
<br />
One thing to note is that the deterministic model sometimes suggests that we will not see bistability. However, because of the fluctuations around the average in the stochastic model, one fluctuation could push us into the realm of bistability.<br />
<br />
In the pictures below the stochastic model is on the left and the deterministic model is on the right. One can clearly see the similarity in the overall shape and it is clear that the deterministic model is just the average of the stochastic model.<br />
<br><br />
<br><br />
<b>Scenario 1: GAL and METH present, winner GFP</b><br />
<br><br />
<br><br />
<left><br />
<img src="https://static.igem.org/mediawiki/2010/3/34/Stoch_gal_meth.png" width="400" height="320"><br />
</left><br />
<right><br />
<img src="https://static.igem.org/mediawiki/2010/a/af/Deter_meth_gal.png" width="400" height="320"><br />
</right><br />
<br><br />
<br><br />
When both galactose and methionine are present, the methionine inhibits the production of the CFP and the galactose encourages the production of the GFP. With nothing to inhibit the production of GFP, the GFP dominates. <br />
<br><br />
<br><br />
<b>Scenario 2: no METH and GAL present, winner – either, depends on other parameters!</b><br />
<br><br />
<br><br />
<left><br />
<img src="https://static.igem.org/mediawiki/2010/b/b0/Stoch_gal_no_meth.jpg" width="400" height="320"><br />
</left><br />
<right><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/Deter_gal_no_meth.jpg" width="400" height="320"><br />
</right><br />
<br><br />
<br><br />
When no methionine is present, CFP can be produced. Also, when GAL is present GFP can be produced. When both proteins are produced, they will both inhibit each other. In this scenario, the dominant protein will depend on the parameters of the system. The modelling we have done suggests that the parameters which affect the system are the transcription/translation rates (λ values) and the binding coefficients (K values). As shown in the above graphs, the parameters of our system dictate that CFP will win. <br />
<br><br />
<br><br />
<b>Scenario 3: no METH and no GAL present, winner CFP</b><br />
<br><br />
<br><br />
<left><br />
<img src="https://static.igem.org/mediawiki/2010/b/b0/Stoch_gal_no_meth.jpg" width="400" height="320"><br />
</left><br />
<right><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/Deter_gal_no_meth.jpg" width="400" height="320"><br />
</right><br />
<br><br />
<br><br />
No methionine present leads to CFP being produced. No galactose present means that no GFP will be produced. With nothing to inhibit the CFP production, CFP will dominate. <br />
<br><br />
<br><br />
Also, a simulation was run with variable galactose and methionine over time. The aim of this was to model the switching behaviour of our system. The picture below shows the results of the stochastic simulation. What we see, is a clear switching behaviour, but only if we actively remove one protein before adding the other.<br />
<br><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/21/Stoch_switch.jpg" width="700" height="470"> <br />
</center><br />
<br><br />
<br><br />
<p>As mentioned previously, when GAL and no METH are present in the system (at 80000 iterations), the winning protein will depend heavily on the parameters of the system. Our experience has shown us that the values for the transcription/translation rates (λ values) and the dissociation constants (K values) strongly affect how the system behaves and thus determines what protein will dominate.</p></p><br />
<br><br />
<h3>Conclusions</h3><br />
<br />
<p>The stochastic model is a more accurate method of modelling a biochemical system where low numbers of molecules are involved. The stochastic model follows the actions of individual molecules whereas the ODEs based model describes how concentrations change continuously in time. The deterministic model is basically just the average of the stochastic model and does not represent any fluctuations due to the individual molecules present. </p><br />
<br><br />
<h3>References</h3><br />
<br><br />
<a name="ref1"></a><br />
<p><sup style="font-size:10px">[1]</sup> Gillespie, D.T. (1977), Exact Stochastic Simulation of Coupled Chemical Reactions, <i>The Journal of Physical Chemistry</i>, Vol. 81, No. 25.</p><br />
<br><br />
<a name="ref2"></a><br />
<p><sup style="font-size:10px">[2]</sup> Ullah, M., Schmidt, H., Cho, K.-H. and Wolkenhauer, O. (2006), Deterministic modelling and stochastic simulation of biochemical pathways using MATLAB, <i>IEE Proc.-Syst. Biol.</i>, Vol. 153, No. 2.</p><br />
<br><br />
<a name="ref3"></a><br />
<p><sup style="font-size:10px">[3]</sup> Hayot, F. (2008), Single Cell experiments and Gillespie’s algorithm. Retrieved from <a href="http://tsb.mssm.edu/summerschool/images/4/4d/HayotSlides.pdf">http://tsb.mssm.edu/summerschool/images/4/4d/HayotSlides.pdf</a> </p><br />
<br><br />
<a name="ref4"></a><br />
<p><sup style="font-size:10px">[4]</sup> Department of Computational & Applied Mathematics, Rice University, Modeling and Simulation of Reaction Networks. Retrieved from <a href="http://www.caam.rice.edu/~caam210/reac/lec.html">http://www.caam.rice.edu/~caam210/reac/lec.html</a> </p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Fixed_PointsTeam:Aberdeen Scotland/Fixed Points2010-10-27T20:31:14Z<p>Porter: </p>
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<h1>Fixed Points</h1><br />
<p>Fixed points are the points where an equation’s rate of change, or its slope, is zero. There are three main types of equilibrium points: stable, unstable, and saddle-node points. A stable equilibrium is a value which the function converges towards, whereas an unstable equilibrium is a value the function will diverge from. A saddle-node is where the function both converges to and diverges from, the equilibrium, depending on the direction from which we approach the point (S. Strogatz, Nonlinear dynamics and Chaos).</p><br />
<br><br />
<h3>Why are fixed points important?</h3><br />
<p>Finding fixed points is the first step in analyzing the stability of a system. In particular, we are interested in the nature of our system’s bistability and how it changes with a variation of the parameters, i.e., its bifurcation analysis. This analysis can be conveyed to the biologists to minimize experimental guessing.<br />
</p><br />
<br><br />
<h3>How are fixed points calculated?</h3><br />
<p>Fixed points are determined by setting all differential equations in a system equal to zero and solving for the variable being analyzed. The direct method of calculating equilibrium points is to find the roots of the system of equations using built-in root-finding functions such as fzero in MATLAB, and similar functions in Maple and C. The indirect method of calculating the equilibrium points is to plot and find the intersections of these nullclines, which represent equilibrium points.</p><br />
<br />
<br />
<h3>Results</h3><br />
<p>We were able to find the fixed points of the system analytically (for small Hill coefficients) and computationally. These points were used for bifurcation analysis, and for analyzing the probability that our system would exhibit bistable behavior.</p><br />
<br><br />
<br><br />
<br />
<h1>Nullclines</h1><br />
<p>In a system of differential equations, the nullclines are the solution curves for which all of the differential equations are equal to zero (Wikipedia).</p><br />
<br><br />
<h3>Why are nullclines important?</h3><br />
<p>The intersections of the nullclines give the equilibrium points of the system of differential equations. From graphs of the nullclines, it is possible to infer whether or not a system will be bistable. If the nullclines only intersect in one place the system is not bistable, since there is one single equilibrium point. If there are more than two intersections, the middle equilibrium point is often an unstable saddle point.</p><br />
<br><br />
<h3>How are the nullclines calculated?</h3><br />
<p>Just as in calculating the fixed points, we set the governing differential equations of the system equal to zero and plot the curves generated.</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/e/e4/Equations3.png"><br />
</center><br />
<br><br />
<h3>Simulations involved</h3><br />
<br />
<br />
<br />
<table><br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2010/1/18/Nullclines_diagram1.png"/></p><br />
</td><br />
<td><br />
<p><b>Figure 1.</b>This figure is a plot of the nullclines of the differential equations for CFP and GFP, where we solve them for CFP as a function of GFP. In this plot, both Hill coefficients are two, ie. n2 and n4 are both 2. The nullclines cross over at three fixed points, where the middle is a saddle-node fixed point. This is an ‘ideal’ bistability plot.<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2010/5/53/Nullclines_diagram2.png"></p><br />
</td><br />
<td><br />
<p><b>Figure 2.</b>This figure is a plot, again, of the nullclines of the differential equations for CFP and GFP. In this plot, both Hill coefficients are one. Note that the nullclines only cross once, resulting in only one fixed point and hence no possibility of bistability. This result allowed us to tell the biologists that the toggle switch would definitely not work if both of the Hill coefficients were one.<br />
</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2010/d/da/Nullclines_diagram3.png"/></p><br />
</td><br />
<td><br />
<p><b>Figure 3.</b> Figure 3: This figure is of CFP as a function of GFP for the two nullclines when the initial galactose and copper concentrations are changed. From this we can see how the fixed points change with different initial galactose and methionine concentrations. The blue line represents the GFP = CFP line. The further from this line equilibrium points are, the harder it will be to switch between stable states.</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<h3>Results</h3><br />
<p>We focused our efforts on plotting the nullclines of the system for a range of Hill coefficient combinations in order to get a general idea of which combinations would most likely produce robust bistability. We found that all combinations gave bistability, except when both Hill coefficients were one. The optimal Hill coefficient combination occurred when both Hill coefficients were two. We passed this information onto the biology team, letting them know that if we wanted the system to successfully switch, we could not have both Hill coefficients with a value of one.<br />
</p><br />
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<h1>Equations</h1><br />
<br />
<p>Here we define the equations and parameters that describe the novel genetic toggle switch that works at the translational level. The switch allows mutually exclusive expression of either green fluorescent protein (GFP) or cyan fluorescent protein (CFP). The synthetic biological circuit is represented in Fig 1.</p> <br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/ff/Toggle_switch.jpg"><br />
<br><br />
<br><br />
<p style="font-size:10px">Figure 1: Translation of DNA to mRNA.</p><br />
</center><br />
<br><br />
<br />
<p>We can regulate the system when we add galactose or methionine. Galactose will bind to the GAL promoter and activate the transcription of M1, allowing the system to express GFP. If we remove methionine from the system instead of adding galactose, it will bind to the MET1 promoter, the transcription of M2 will be activated, leading to the expression of CFP.</p><br />
<br><br />
<p>From Fig 1 it can be seen that there is mutual inhibition of the translation of the two mRNAs. That is because the translated proteins can bind to the corresponding stem loop structures on the opposing construct.</p><br />
<br><br />
<p>For our initial conditions, we began with more GFP than CFP and thus the production of CFP was inhibited. When methionine was added removed from the system, the rate of CFP production will increase and decrease for GFP. Eventually, we will see more CFP than GFP so the system will have switched. Once we have more CFP than GFP, galactose can then be added to switch back to an expression of GFP. </p><br />
<br><br />
<p>The N-Peptide and GFP strand has two MS2-Stem loops as we discovered that one single loop would not inhibit the production of CFP enough to achieve our switch.</p><br />
<br />
<br><br />
<h3>Equation 1</h3><br />
<br><br />
<div align="center"><br />
<table><br />
<tr><br />
<td><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/9/90/Dm1.jpg"></div><br />
</td><br />
<td><br />
<div align="right"><p><b>(1)</b></p></div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<p>This is the equation for the rate of change of the mRNA that is transcribed from the galactose promoter. The three terms represent production, degradation, and dilution respectively.</p><br />
<br><br />
<p>[GAL] represents the concentration of galactose that is added to the system. When galactose is added it binds to the promoter and activates the transcription of M1.<br />
<br><br />
<br><br />
[M1] is the concentration of mRNA that translates the N-peptide and GFP.</p><br />
<br><br />
<div align="center"><br />
<table><br />
<tr><br />
<td><br />
<div align="right"><b><p>Parameter<p></b></div><br />
</td><br />
<td><br />
<p><b>Description</b></p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>λ<sub style="font-size:10px">1</sub>:<p></b></div><br />
</td><br />
<td><br />
<p>Constant representing rate of transcription of the DNA that encodes for the production of N peptide and GFP</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>μ<sub style="font-size:10px">1</sub>:</p></b></div><br />
</td><br />
<td><br />
<p>Constant representing rate of degradation of mRNA</p><br />
</td><br />
</tr><br />
<tr><br />
<td> <br />
<div align="right"><b><p>n<sub style="font-size:10px">1</sub>:<p></b></div><br />
</td><br />
<td><br />
<p>Hill coefficient for the association between the galactose and the GAL promoter<p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>K<sub style="font-size:10px">1</sub>:</p></b></div><br />
</td><br />
<td><br />
<p>Dissociation constant for the GAL promoter</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>T:</p></b></div><br />
</td><br />
<td><br />
<p>Time constant representing rate of cellular division</p><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br />
<h3>Equation 2</h3><br />
<br><br />
<br />
<div align="center"><br />
<table><br />
<tr><br />
<td><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/7/7e/DGFP.jpg"></div><br />
</td><br />
<td><br />
<div align="right"><p><b>(2)</b></p></div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<p>This is the equation for the rate of change of protein that is translated from the mRNA for GFP. The three terms represent production, degradation, and dilution respectively.</p><br />
<br><br />
<p>[M1] is the concentration of mRNA that translates the N-peptide GFP.<br />
<br><br />
<br><br />
[GFP] represents the concentration of N-peptide and GFP.<br />
<br><br />
<br><br />
[CFP] represents the concentration of the MS2-protein and CFP.</p><br />
<br><br />
<div align="center"><br />
<table><br />
<tr><br />
<td><br />
<div align="right"><b><p>Parameter:<p></b></div><br />
</td><br />
<td><br />
<p><b>Description</b></p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>λ<sub style="font-size:10px">2</sub>:</p></b></div><br />
</td><br />
<td><br />
<p>Constant representing rate of translation of the mRNA that encodes for the production of N-peptide and GFP</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>μ<sub style="font-size:10px">2</sub>:</p></b></div><br />
</td><br />
<td><br />
<p>Constant representing rate of degradation of the GFP</p><br />
</td><br />
</tr><br />
<tr><br />
<td> <br />
<div align="right"><b><p>n<sub style="font-size:10px">2</sub>:<p></b></div><br />
</td><br />
<td><br />
<p>Hill coefficient of the CFP/MS2 stem loop association<p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>K<sub style="font-size:10px">2</sub>:</p></b></div><br />
</td><br />
<td><br />
<p>Dissociation constant for the MS2-CFP protein to MS2 loop</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>T:</p></b></div><br />
</td><br />
<td><br />
<p>Time constant representing rate of cellular division</p><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
<h3>Equation 3</h3><br />
<br><br />
<div align="center"><br />
<table><br />
<tr><br />
<td><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/2/23/Dm2.jpg"></div><br />
</td><br />
<td><br />
<div align="right"><p><b>(3)</b></p></div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br />
<p>This is the equation for the rate of change of the mRNA that is transcribed from the copper promoter. The three terms represent production, degradation, and dilution respectively.</p><br />
<br><br />
<p>[Cu<sup style="font-size:10px">2+</sup>] is the concentration of the copper added to the system that binds to the CUP1 promoter and activates the transcription of M2.<br />
<br><br />
<br><br />
[M2] represents the concentration of mRNA that translates the MS2-protein and CFP. </p><br />
<br><br />
<div align="center"><br />
<table><br />
<tr><br />
<td><br />
<div align="right"><b><p>Parameter</p></b></div><br />
</td><br />
<td><br />
<p><b>Description</b></p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>λ<sub style="font-size:10px">3</sup>:<p></b></div><br />
</td><br />
<td><br />
<p>Constant representing rate of transcription of the DNA that encodes for the production of the MS2-protein and CFP</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>μ<sub style="font-size:10px">3</sub>:</p></b></div><br />
</td><br />
<td><br />
<p>Constant representing rate of degradation of mRNA</p><br />
</td><br />
</tr><br />
<tr><br />
<td> <br />
<div align="right"><b><p>n<sub style="font-size:10px">3</sub>:<p></b></div><br />
</td><br />
<td><br />
<p>Hill coefficient of the association between copper and the CUP1 promoter<p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>K<sub style="font-size:10px">3</sub>:</p></b></div><br />
</td><br />
<td><br />
<p>Dissociation constant for Copper promoter</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>T:</p></b></div><br />
</td><br />
<td><br />
<p>Time constant representing rate of cellular division</p><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
<br />
<h3>Equation 4</h3><br />
<br><br />
<div align="center"><br />
<table><br />
<tr><br />
<td><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2010/d/d4/DCFP.jpg"></div><br />
</td><br />
<td><br />
<div align="right"><p><b>(4)</b></p></div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br />
<p>This is the equation for the rate of change of protein that is translated from the mRNA for CFP. The three terms represent production, degradation, and dilution respectively.</p><br />
<br><br />
<p>[M2] is the concentration of mRNA that translates to MS2-protein and CFP.<br />
<br><br />
<br><br />
[GFP] represents the concentration of the N-peptide and GFP.<br />
<br><br />
<br><br />
[CFP] represents the concentration of the MS2-protein and CFP.</p><br />
<br><br />
<div align="center"><br />
<table><br />
<tr><br />
<td><br />
<div align="right"><b><p>Parameters<p></b></div><br />
</td><br />
<td><br />
<p><b>Description</b></p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>λ<sub style="font-size:10px">4</sub>:</p></b></div><br />
</td><br />
<td><br />
<p>Constant representing rate of translation of the mRNA that encodes for the production of MS2-protein and CFP</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>μ<sub style="font-size:10px">4</sub>:</p></b></div><br />
</td><br />
<td><br />
<p>Constant representing rate of degradation of the CFP</p><br />
</td><br />
</tr><br />
<tr><br />
<td> <br />
<div align="right"><b><p>n<sub style="font-size:10px">4</sub>:<p></b></div><br />
</td><br />
<td><br />
<p>Hill coefficient of the GFP/Bbox stem loop association<p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>K<sub style="font-size:10px">4</sub>:</p></b></div><br />
</td><br />
<td><br />
<p>Dissociation constant for the N-Pep-GFP protein to the Bbox-stem loop</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><b><p>T:</p></b></div><br />
</td><br />
<td><br />
<p>time constant representing rate of cellular division</p><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<h1>Parameter Study</h1><br />
<p>The parameter values were first estimated based on the literature <a href="#ref1"><sup style="font-size:10px">[1]</sup></a> and after the first estimation, a possible range of variation for each parameter was assigned, also based on literature. Then, we studied the bistability of the model depending on the parameter values that were varied in the above mentioned ranges. For more information, see <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Probability">Parameter Space Analysis</a> and <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Evolution">Directed Evolution</a>.</p><br />
<br><br />
<h1>Modification of the construct</h1><br />
<br />
<p>Some experimental difficulties were encountered with the copper construct which led to the use of a methionine promoter to substitute it. Methionine acts as an inhibitor of the promoter, so that equation 3 had to be substituted by the following equation:<p><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/f2/Meth.png"><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2010/7/75/MET_toggle_switch.png"><br />
</center><br />
<br><br />
<p>The behaviour of the switch can then be summarise in the following table:</p><br />
<br><br />
<div align="center"><br />
<table><br />
<tr><br />
<td><br />
<div align="right"><b><p>What is present in the system</p></b></div><br />
</td><br />
<td><br />
<p><b>Protein(s) produced</b></p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><p>Galactose and Methionine</p></div><br />
</td><br />
<td><br />
<p>GFP</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><p>Galactose only</p></div><br />
</td><br />
<td><br />
<p>GFP, CFP (doses dependent)</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><p>Methionine only</p></div><br />
</td><br />
<td><br />
<p>No GFP or CFP</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<div align="right"><p>No Galactose and no Methionine</p></div><br />
</td><br />
<td><br />
<p>CFP</p><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br />
<h1>References</h1><br />
<a name="ref1"></a><br />
<p><sup style="font-size:10px">[1]</sup> Beyer A, Hollunder J, Nasheuer HP, Wilhelm T. (2004), Post-transcriptional expression regulation in the yeast Saccharomyces cerevisiae on a genomic scale, <i>Mol Cell Proteomics.</i>, Vol. 3, No.11, pp. 1083-1092.</p><br />
<br><br />
<p><sup style="font-size:10px">[2]</sup> Alon, U. (2006), An Introduction to Systems Biology: Design Principles of Biological Circuits, Chapman and Hall. </p><br />
<br />
<br />
<br />
<br><br><br />
<hr><br />
<table class="nav"><br />
<tr><br />
<td><br />
<a href="https://2010.igem.org/Team:Aberdeen_Scotland/Modeling"><img src="https://static.igem.org/mediawiki/2010/8/8e/Left_arrow.png">&nbsp;&nbsp;Return to the Modelling Summary</a><br />
</td><br />
<td align="right"><br />
<a href="https://2010.igem.org/Team:Aberdeen_Scotland/Fixed_Points">Continue to Nullclines and Fixed Points&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2010/3/36/Right_arrow.png"></a><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
</html><br />
{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/ModelingTeam:Aberdeen Scotland/Modeling2010-10-27T20:23:34Z<p>Porter: </p>
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{{:Team:Aberdeen_Scotland/Title}}<br />
<html><br />
<h1>Introduction to the Modelling of the ayeSwitch</h1><br />
<p>This page is an introduction to the different equations and techniques that we used to design and predict the behaviour of the ayeSwitch.<br />
<br><br />
</p> <br />
<br><br />
<a href="https://2010.igem.org/Team:Aberdeen_Scotland/Equations"><h3><font color="blue">Equations</font></h3></a><br />
<p>In this section we describe the process of developing a basic mathematical model for the ayeSwitch based on the promotion and inhibition behaviour necessary for mutual repression. We developed a set of four differential equations, one to model each of the two mRNAs and two proteins that are the active components of our system.</p><br />
<br><br />
<h3><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Fixed_Points"><font color="blue">Nullclines and Fixed Points</font></a></h3><br />
<p>In this section we describe how we used fixed point analysis to predict the equilibrium state(s) of the ayeSwitch system for different parameters. Three or more equilibrium points will give us bistability and the possibility of switching. </p><br />
<br><br />
<h3><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Bifurcation"><font color="blue">Bifurcation and Stability</font></a></h3><br />
<p>Bifurcation analysis allows us to track the number and relative position of the equilibrium points of our system for different parameters. This section describes in detail how bifurcation analysis can help us determine the optimal range for our parameters. </p><br />
<br><br />
<h3><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Stochastic_Model"><font color="blue">Stochastic Model</font></a></h3><br />
<p>There are two ways to model our system - deterministically and stochastically. Both methods have their advantages and disadvantages depending on the system in question. In our system, it was more beneficial to model the system stochastically due to the low numbers of molecules involved. This section describes in detail both methods, their advantages and disadvantages and how they are used to model our system.</p><br />
<br><br />
<h3><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Probability"><font color="blue">Parameter Space Analysis</font></a></h3><br />
<p>This section describes in detail how we analysed the parameter space for our system. The results of this will show when bistability is possible and when it is not. Using this information, we can determine the optimal parameter ranges for our system. </p><br />
<br><br />
<h3><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Curve_Fitting"><font color="blue">Determination of the Hill coefficient n<sub style="font-size:10px">1</sub></font></a></h3><br />
<p>The Hill coefficient relating to the CFP/MS2 stem loop association is assumed to be around 2 due to the number of stem loops present. This section details the method we used to calculate this value more accurately, and what the result means in terms of the parameter space analysis.</p><br />
<h3><a href="https://2010.igem.org/Team:Aberdeen_Scotland/Evolution"><font color="blue">Directed Evolution</font></a></h3><br />
<p>Here we describe one of the possible ways to improve our switch which we would have been able to attempt given more time. The results show an optimized version of our original system. </p><br />
<br />
<br><br><br />
<hr><br />
<table class="nav"><br />
<tr><br />
<td><br />
<a href="https://2010.igem.org/Team:Aberdeen_Scotland/Parts"><img src="https://static.igem.org/mediawiki/2010/8/8e/Left_arrow.png">&nbsp;&nbsp;Return to Parts Submitted to the Registry</a><br />
</td><br />
<td align="right"><br />
<a href="https://2010.igem.org/Team:Aberdeen_Scotland/Equations">Continue to Equations&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2010/3/36/Right_arrow.png"></a><br />
</td><br />
</tr><br />
</table><br />
<br />
</html><br />
{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Project_OverviewTeam:Aberdeen Scotland/Project Overview2010-10-27T19:34:40Z<p>Porter: </p>
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<h1>Project Overview</h1><br />
<h3>Introduction</h3><br />
<p> <br />
For this year’s iGEM competition the Aberdeen team has worked on developing a translationally controlled toggle switch embedded in yeast.<a href="#ref1"><sup style="font-size:10px">[1]</sup></a> Genetic toggle switches are a vital component for synthetic biology circuits , enabling functional control of biological functions. The majority of toggle switches used for iGEM are embedded in Escherichia coli and can only be controlled at the transcriptional level <a href="#ref2"><sup style="font-size:10px">[2]</sup></a><sup style="font-size:10px">,</sup><a href="#ref3"><sup style="font-size:10px">[3]</sup></a>. Our main goal was to create and model a novel gene circuit, wherein yeast cells can be switched between mutually exclusive fluorescent proteins under exposure to environmental factors. This switching behaviour would be regulated at the translational level, an innovation over previous systems that only demonstrated transcriptional regulation <a href="#ref4"><sup style="font-size:10px">[4]</sup></a><sup style="font-size:10px">,</sup><a href="#ref5"><sup style="font-size:10px">[5]</sup></a>.The novel genetic toggle switch operated by controlling gene expression at the translational level consisted of two gene expression constructs expressing an RNA-binding protein fused to either Green (GFP) or Cyan (CFP) fluorescent protein in the presence of appropriate inducer. When co-expressed in yeast, these translational fusions would be mutually inhibitory at the translational level, thereby forming a biological, ‘Toggle Switch’ system. <br />
</p><br />
<br><br />
<hr><br />
<h3>The AyeSwitch</h3><br />
<p>The toggle switch is shown by Fig 1 and was named the ‘AyeSwitch’. It is regulated by controlling the two constructs, GAL1p-[Npeptide-GFP] and CUP1p-[MS2-CFP], via inducible yeast promoters GAL1 or CUP1 in the presence or absence of galactose and Cu2+ ions respectively. <br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/ff/Toggle_switch.jpg"><br />
</center><br />
<br><br />
<p><br />
For example, in the presence of galactose only, GAL1 is induced and there is expression of N-peptide-GFP protein. The subsequent addition of Cu2+ then induces the transcription of mRNA coding for MS2 coat binding protein and CFP. In addition to this, the mRNA also codes for a Bbox stem loop sequence that can be bound by N-peptide. </p><br />
<p><br><br />
Ideally, there is initial inhibition of MS2-CFP translation by Npeptide-GFP binding to the Bbox stem loop. Evolution of time corresponds to the ratio of MS2-CFP mRNA to N-peptide-GFP protein increasing allowing some MS2-CFP to be produced until CFP ‘switches ON’ as it gains dominance over GFP.</p><br />
<p><br />
Additionally, N-peptide-GFP protein translation can also be inhibited by MS2-CFP via MS2 protein binding to the MS2 stem loops on the N-peptide-GFP mRNA. This may help the switching ON of CFP and also means GFP would face a similar situation if the inducer was changed from Cu2+ to galactose.</p><br />
<p><br><br />
However, additional variables may come into play affecting the outcomes described above. It is likely that the concentration of each inducer present, the translational rate and binding efficiency of stem loop binding proteins to mRNA stem loop and degradation rate of proteins can also affect the outcome. Reversing the order of inducer present may also affect the outcome. </p><br />
<br><br />
<hr><br />
<h3>Experimental Characterisation of the AyeSwitch</h3><br />
<p><br />
The experimental work addressed these issues by initially characterising the promoters in terms of their dose response and time response using constructs GAL1-[GFP] and CUP1-[GFP]. These experiments were then extended to characterise GAL1p-[Npeptide-GFP] and CUP1p-[MS2-CFP] which discovered that CUP1p-[MS2-CFP] did not function as expected.</p><br><br />
<p><br />
The experimental work diverged from this point to troubleshoot CUP1p-[MS2-CFP], investigating the translation inhibition of GAL1p-[Npeptide-GFP] by MS2 coat protein using construct MET17p - [MS2], Bio-brick construction and testing of Bio-brick E2050 mOrange.</p><br />
<br><br />
<hr><br />
<h3>Modelling Characterisation of the Ayeswitch</h3><br />
<p><br />
Our team proposed a novel model to describe the functioning of the Aye-switch, based on ordinary differential equations (ODEs). The proposed system of ODEs was carefully and systematically studied both analytically and computationally. A bifurcation analysis was performed and the bistability of the system was investigated with respect to large variations in the parameters of the system. The deterministic simulations were compared with stochastic ones, using the Gillespie algorithm. The parameter space of the model was thoroughly investigated, using two different approaches: Monte-Carlo and directed evolution. These two approaches are very useful for a wide range of projects in synthetic biology. The theoretical predictions led to the proposition of optimised parameters for the Aye-switch that allow a very robust translational switch.</p><br />
<br><br />
<hr><br />
<h3>Troubleshooting CUP1p-[MS2-CFP]</h3><br />
<p><br />
Troubleshooting of CUP1p-[MS2-CFP] was carried out through a series of gene cassette replacement experiments testing the promoter and CFP sequences for functionality. The conclusions to these experiments suggest that the Bbox Stem loop, usually located in the 3’untranslated region but is in the 5’ untranslated region of our construct may be preventing the expression of downstream proteins. It may also be that the fusion of MS2 to CFP results in inappropriate protein folding, inhibiting expression.</p> <br />
<br><br />
<hr><br />
<h3>Verification of Translation Inhibition as a Regulatory Mechanism</h3><br />
<p><br />
It was shown that the translational inhibition of GAL1p-[Npeptide-GFP] by MS2 coat protein was possible, confirming that translational regulation is viable. Further work if time permitted would investigate if this inhibition could work in the context of a toggle switch.</p><br />
<br><br />
<hr><br />
<h3>Bio-brick construction and testing </h3><br />
<p><br />
In parallel, Bio-bricks were constructed and submitted to the Registry of parts whilst testing of the Bio-brick E2050 mOrange using fluorimetry and FACS analysis lead to the conclusion that the mOrange sequence did not function within our GAL1p-[Npeptide-GFP] construct that was shown to be able to express GFP appropriately. </p><br />
<br><br><br />
<hr><br />
<br />
<br />
<h1>Attribution and Contributions</h1><br />
<h3>Biological circuit construction and testing </h3><br />
<p><br />
The students within the experimental section of the team were provided (by their host lab) with two yeast strains that had Gal1p-GFP and Cup1p-GFP integrated into the genome (see 'DNA constructs). They then used these constructs to analyse the properties of the CUP1 and GAL1 promoters. With some instructor oversight, the student team themselves then completely designed constructs Gal1p-(Npep-GFP) and Cup1p-(MS2-CFP), which were then synthesised by a synthetic DNA supply company. The students then tested these constructs, and further engineered them during the trouble-shooting phase of the project.<br><br />
All the experimental work described on the wiki, involving characterisation, testing and re-engineering of the bio-bricks, was carried out by the student members of the team. All the construction and sequencing of the four submitted bio-bricks was also carried out by members of the student team.<br />
<br><br><br />
<br />
<h3>Mathematical modelling of the AyeSwitch </h3><br />
<p><br />
The students within the theoretical section of the team carried out all the described modelling. Team activities were overseen by the Instructors, but all model coding and model analysis was performed by the students within the team.<br />
<br />
<br><br><br />
<hr><br />
<h3> References</h3><br><br />
<p><br />
<a name="ref1"></a><br />
<a href="http://www.nature.com/msb/journal/v2/n1/full/msb4100073.html"target="_blank"><b><sup style="font-size:10px">[1]</sup></b></a> Ernesto Andrianantoandro et al. Synthetic biology: new engineering rules for an emerging discipline Molecular Systems Biology 2:2006.0028</p><br><br />
<p><br />
<a name="ref2"></a><br />
<a href="http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html"target="_blank"><b><sup style="font-size:10px">[2]</sup></b></a> Timothy S. Gardner et al. Construction of a genetic toggle switch in Escherichia coli Nature 403, 339-342 (20 January 2000)</p><br><br />
<p><br />
<a name="ref3"></a><br />
<a href="http://www.cell.com/retrieve/pii/S0092867403003465"target="_blank"><b><sup style="font-size:10px">[3]</sup></b></a> Mariette R. Atkinson et al. Development of Genetic Circuitry Exhibiting Toggle Switch or Oscillatory Behavior in Escherichia coli Cell, Volume 113, Issue 5, 597-607, 30 May 2003 </p><br><br />
<p><br />
<a name="ref4"></a><br />
<a href="http://www.nature.com/emboj/journal/v17/n14/abs/7591108a.html"target="_blank"><b><sup style="font-size:10px">[4]</sup></b></a> Adam Platt and Richard J Reece The yeast galactose genetic switch is mediated by the formation of a Gal4p–Gal80p–Gal3p complex The EMBO Journal (1998) 17, 4086 - 4091 </p><br><br />
<p><br />
<a name="ref5"></a><br />
<a href="http://www.pnas.org/content/88/19/8597.abstract"target="_blank"><b><sup style="font-size:10px">[5]</sup></b></a> D W Griggs and M Johnston Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression PNAS October 1, 1991 vol. 88 no. 19 8597-8601</i></p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/FACS_analysis_of_fluorescent_proteinsFACS analysis of fluorescent proteins2010-10-27T19:31:31Z<p>Porter: </p>
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<br />
<h1>'''Flow cytometry Analysis of fluorescence Proteins'''</h1><br />
<br />
<p style="font-size:10pt">The flow cytometer used by our team was the Becton Dickinson 'LSRII '</p><br />
<br />
Please note that as a technique, flow cytometry was used in many of our experiments although this is frequently referred to in our wiki text as FACS (Fluorescent activated cell sorting) analysis . However, we stress that in fact no cell sorting was performed in our experiments. <br />
<br />
[[Image:Adbn_FACS.jpg|center|300 px]]<br />
<br />
<br />
<p style="font-size:12pt">'''Flow cytometry and Its Advantages'''</p><br />
Flow cytometry (FCM) is a technique used for counting and examining individual microscopic particles such as cells on the basis of their fluorescence. One of its unique features is that it measures fluorescence per cell or particle, contrasting with spectrophotometry which measures absorption and transmission of wavelengths as a bulk volume of the sample.<br />
<br />
<br />
<p style="font-size:12pt">'''How Flow Cytometry Works'''</p> <br />
The sample is injected into the center of the sheath stream of flow cytometer in a liquid state; therefore the particles are distributed randomly. The fluidics system is then responsible for separating out the particles into an ordered stream of single particles.<br />
<br />
<br />
<br />
[[Image:FACS_FC.jpg|center|450 px]]<br />
<br />
<br />
<br />
After hydrodynamic focusing, the cells or particles of interest pass through the laser beam therefore intercepting and scattering the light which excites the fluorochromes to a higher energy state. The energy is then released as a photon of light with spectral properties unique to specific fluorochromes. Light scattered in the forward direction (as shown in the below diagram) is collected by a lens which is in line with the beam known as the forward scatter channel (FSC). The FSC intensity gives the particles size and can give information used to distinguish between cellular debris and living cells. The side scatter channel (SSC) is perpendicular to the beam and provides information about the granular content within a particle. Both FSC and SSC are unique for each particle and a combination of the two may be used to differentiate between different cell types in a heterogeneous sample. <br />
<br />
[[Image:Fsc.ssc.JPG|center|600 px]]<br />
<br />
<p style="font-size:12pt">'''Cell Sorting'''</p><br />
Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. The rate of flow sorting at 10 000 cells/second provides a method for sorting a heterogeneous mixture of biological cells into separate storage containers. It is based upon the specific light scattering and fluorescent characteristics of each cell. It is an extremely useful scientific instrument, as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.<br />
<br />
<br />
<p style="font-size:12pt">'''How Cell Sorting Works'''</p><br />
After the cells have passed through the laser beam and the detectors, a vibrating mechanism causes the stream of cells to break into individual droplets. An electrical charge is placed at the point where the stream breaks into droplets immediately prior to the fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. The droplets then travel through a strong electrostatic field and are deflected based on their charge into waiting sample tubes. The number of cells and level of fluorescence in each tube is then known.<br />
<br />
<br />
<p style="font-size:12pt">'''Variables Considered for Our Project When Using the Flow Cytometry.''' </p><br />
During our experiment our choice of fluorochrome was restricted by the possibility of spectral overlap. When two or more fluorochromes are used during a single experiment there is a chance that their emission profiles will coincide, making measurement of the true fluorescence emitted by each particle very difficult. Therefore careful consideration of the excitation and emission wavelengths of the Green Fluorescent Protein and the Cyan Fluorescent Protein was carried out prior to the experiment to ensure there was no overlap.<br />
<br />
<br />
<p style="font-size:12pt">'''Data Received From Flow Cytometry and Analysis '''</p> <br />
The graph shown below is an example of a single-parameter histogram obtained from the FACS during our experiment. These graphs display a single measurement parameter; the relative fluorescence (as shown above) or light scatter intensity on the x-axis and the number of events (cell count) on the y-axis. This graph is very useful for evaluating the total number of cells in a sample that have the physical properties selected for or which express the marker of interest (as is the case with our project). The graph involves flow analysis on a mixed population of cells (some expressing GFP and some are not) this results in several peaks on the histogram. In order to identify the positive dataset, a positive and a negative control is used for positive identification of the peak corresponding to the cells which were and which were not expressing GFP.<br />
<br />
<br />
[[Image:Ka1.JPG|center|450 px]]<br />
<br />
<br />
Below is an example of a density plot taken during one of our experiments. In this plot, the particle counts are shown by dot density. Each cell recorded i.e. one of the dots shown above, is referred to as an event. The green colour represents larger number of events and the red one even more. The different colours are used to create a three-dimensional feel.<br />
<br />
<br />
[[Image:Ka2.JPG|center|450 px]]<br />
<br />
<br />
In preparation of the Flow cytometry analysis we;<br />
<br />
1. Washed and resuspended samples in PBS at a density of 10<sup style="font-size:10px">5</sup>-10<sup style="font-size:10px">7</sup> cells/ml.<br><br />
2. Less than 1 ml was required for analysis and cells were stored on ice until analysed then vortexed before analysed.<br />
<br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/FACS_analysis_of_fluorescent_proteinsFACS analysis of fluorescent proteins2010-10-27T19:31:04Z<p>Porter: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<br />
<h1>'''Flow cytometry Analysis of fluorescence Proteins'''</h1><br />
<br />
<p style="font-size:10pt">The flow cytometer used by our team was the Becton Dickinson 'LSRII '</p><br />
<br />
Please note that as a technique, flow cytometry was used in many of our experiments although this is frequently referred to in our wiki text as FACS (Fluorescent activated cell sorting) analysis . However, we stress that in fact no cell sorting was performed in our experiments. <br />
<br />
[[Image:Adbn_FACS.jpg|center|300 px]]<br />
<br />
<br />
<p style="font-size:12pt">'''Flow cytometry and Its Advantages'''</p><br />
Flow cytometry (FCM) is a technique used for counting and examining individual microscopic particles such as cells on the basis of their fluorescence. One of its unique features is that it measures fluorescence per cell or particle, contrasting with spectrophotometry which measures absorption and transmission of wavelengths as a bulk volume of the sample.<br />
<br />
<br />
<p style="font-size:12pt">'''How Flow Cytometry Works'''</p> <br />
The sample is injected into the center of the sheath stream of flow cytometer in a liquid state; therefore the particles are distributed randomly. The fluidics system is then responsible for separating out the particles into an ordered stream of single particles.<br />
<br />
<br />
<br />
[[Image:FACS_FC.jpg|center|450 px]]<br />
<br />
<br />
<br />
After hydrodynamic focusing, the cells or particles of interest pass through the laser beam therefore intercepting and scattering the light which excites the fluorochromes to a higher energy state. The energy is then released as a photon of light with spectral properties unique to specific fluorochromes. Light scattered in the forward direction (as shown in the below diagram) is collected by a lens which is in line with the beam known as the forward scatter channel (FSC). The FSC intensity gives the particles size and can give information used to distinguish between cellular debris and living cells. The side scatter channel (SSC) is perpendicular to the beam and provides information about the granular content within a particle. Both FSC and SSC are unique for each particle and a combination of the two may be used to differentiate between different cell types in a heterogeneous sample. <br />
<br />
[[Image:Fsc.ssc.JPG|center|600 px]]<br />
<br />
<p style="font-size:12pt">'''Cell Sorting'''</p><br />
Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. The rate of flow sorting at 10 000 cells/second provides a method for sorting a heterogeneous mixture of biological cells into separate storage containers. It is based upon the specific light scattering and fluorescent characteristics of each cell. It is an extremely useful scientific instrument, as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.<br />
<br />
<br />
<p style="font-size:12pt">'''How Cell Sorting Works'''</p><br />
After the cells have passed through the laser beam and the detectors, a vibrating mechanism causes the stream of cells to break into individual droplets. An electrical charge is placed at the point where the stream breaks into droplets immediately prior to the fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. The droplets then travel through a strong electrostatic field and are deflected based on their charge into waiting sample tubes. The number of cells and level of fluorescence in each tube is then known.<br />
<br />
<br />
<p style="font-size:12pt">'''Variables Considered for Our Project When Using the Flow Cytometry.''' </p><br />
During our experiment our choice of fluorochrome was restricted by the possibility of spectral overlap. When two or more fluorochromes are used during a single experiment there is a chance that their emission profiles will coincide, making measurement of the true fluorescence emitted by each particle very difficult. Therefore careful consideration of the excitation and emission wavelengths of the Green Fluorescent Protein and the Cyan Fluorescent Protein was carried out prior to the experiment to ensure there was no overlap.<br />
<br />
<br />
<p style="font-size:12pt">'''Data Received From Flow Cytometry and Analysis '''</p> <br />
The graph shown below is an example of a single-parameter histogram obtained from the FACS during our experiment. These graphs display a single measurement parameter; the relative fluorescence (as shown above) or light scatter intensity on the x-axis and the number of events (cell count) on the y-axis. This graph is very useful for evaluating the total number of cells in a sample that have the physical properties selected for or which express the marker of interest (as is the case with our project). The graph involves flow analysis on a mixed population of cells (some expressing GFP and some are not) this results in several peaks on the histogram. In order to identify the positive dataset, a positive and a negative control is used for positive identification of the peak corresponding to the cells which were and which were not expressing GFP.<br />
<br />
<br />
[[Image:Ka1.JPG|center|450 px]]<br />
<br />
<br />
Below is an example of a density plot taken during one of our experiments. In this plot, the particle counts are shown by dot density. Each cell recorded i.e. one of the dots shown above, is referred to as an event. The green colour represents larger number of events and the red one even more. The different colours are used to create a three-dimensional feel.<br />
<br />
<br />
[[Image:Ka2.JPG|center|450 px]]<br />
<br />
<br />
In preparation of the Flow cytometry analysis we;<br />
<br />
1. Washed and resuspended samples in PBS at a density of 10<sup style="font-size:10px">5</sup>-10^<sup style="font-size:10px">7</sup> cells/ml.<br><br />
2. Less than 1 ml was required for analysis and cells were stored on ice until analysed then vortexed before analysed.<br />
<br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/FACS_analysis_of_fluorescent_proteinsFACS analysis of fluorescent proteins2010-10-27T19:30:03Z<p>Porter: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
<br />
<h1>'''Flow cytometry Analysis of fluorescence Proteins'''</h1><br />
<br />
<p style="font-size:10pt">The flow cytometer used by our team was the Becton Dickinson 'LSRII '</p><br />
<br />
Please note that as a technique, flow cytometry was used in many of our experiments although this is frequently referred to in our wiki text as FACS (Fluorescent activated cell sorting) analysis . However, we stress that in fact no cell sorting was performed in our experiments. <br />
<br />
[[Image:Adbn_FACS.jpg|center|300 px]]<br />
<br />
<br />
<p style="font-size:12pt">'''Flow cytometry and Its Advantages'''</p><br />
Flow cytometry (FCM) is a technique used for counting and examining individual microscopic particles such as cells on the basis of their fluorescence. One of its unique features is that it measures fluorescence per cell or particle, contrasting with spectrophotometry which measures absorption and transmission of wavelengths as a bulk volume of the sample.<br />
<br />
<br />
<p style="font-size:12pt">'''How Flow Cytometry Works'''</p> <br />
The sample is injected into the center of the sheath stream of flow cytometer in a liquid state; therefore the particles are distributed randomly. The fluidics system is then responsible for separating out the particles into an ordered stream of single particles.<br />
<br />
<br />
<br />
[[Image:FACS_FC.jpg|center|450 px]]<br />
<br />
<br />
<br />
After hydrodynamic focusing, the cells or particles of interest pass through the laser beam therefore intercepting and scattering the light which excites the fluorochromes to a higher energy state. The energy is then released as a photon of light with spectral properties unique to specific fluorochromes. Light scattered in the forward direction (as shown in the below diagram) is collected by a lens which is in line with the beam known as the forward scatter channel (FSC). The FSC intensity gives the particles size and can give information used to distinguish between cellular debris and living cells. The side scatter channel (SSC) is perpendicular to the beam and provides information about the granular content within a particle. Both FSC and SSC are unique for each particle and a combination of the two may be used to differentiate between different cell types in a heterogeneous sample. <br />
<br />
[[Image:Fsc.ssc.JPG|center|600 px]]<br />
<br />
<p style="font-size:12pt">'''Cell Sorting'''</p><br />
Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. The rate of flow sorting at 10 000 cells/second provides a method for sorting a heterogeneous mixture of biological cells into separate storage containers. It is based upon the specific light scattering and fluorescent characteristics of each cell. It is an extremely useful scientific instrument, as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.<br />
<br />
<br />
<p style="font-size:12pt">'''How Cell Sorting Works'''</p><br />
After the cells have passed through the laser beam and the detectors, a vibrating mechanism causes the stream of cells to break into individual droplets. An electrical charge is placed at the point where the stream breaks into droplets immediately prior to the fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. The droplets then travel through a strong electrostatic field and are deflected based on their charge into waiting sample tubes. The number of cells and level of fluorescence in each tube is then known.<br />
<br />
<br />
<p style="font-size:12pt">'''Variables Considered for Our Project When Using the Flow Cytometry.''' </p><br />
During our experiment our choice of fluorochrome was restricted by the possibility of spectral overlap. When two or more fluorochromes are used during a single experiment there is a chance that their emission profiles will coincide, making measurement of the true fluorescence emitted by each particle very difficult. Therefore careful consideration of the excitation and emission wavelengths of the Green Fluorescent Protein and the Cyan Fluorescent Protein was carried out prior to the experiment to ensure there was no overlap.<br />
<br />
<br />
<p style="font-size:12pt">'''Data Received From Flow Cytometry and Analysis '''</p> <br />
The graph shown below is an example of a single-parameter histogram obtained from the FACS during our experiment. These graphs display a single measurement parameter; the relative fluorescence (as shown above) or light scatter intensity on the x-axis and the number of events (cell count) on the y-axis. This graph is very useful for evaluating the total number of cells in a sample that have the physical properties selected for or which express the marker of interest (as is the case with our project). The graph involves flow analysis on a mixed population of cells (some expressing GFP and some are not) this results in several peaks on the histogram. In order to identify the positive dataset, a positive and a negative control is used for positive identification of the peak corresponding to the cells which were and which were not expressing GFP.<br />
<br />
<br />
[[Image:Ka1.JPG|center|450 px]]<br />
<br />
<br />
Below is an example of a density plot taken during one of our experiments. In this plot, the particle counts are shown by dot density. Each cell recorded i.e. one of the dots shown above, is referred to as an event. The green colour represents larger number of events and the red one even more. The different colours are used to create a three-dimensional feel.<br />
<br />
<br />
[[Image:Ka2.JPG|center|450 px]]<br />
<br />
<br />
In preparation of the Flow cytometry analysis we;<br />
<br />
1. Washed and resuspended samples in PBS at a density of 10^5-10^7 cells/ml.<br><br />
2. Less than 1 ml was required for analysis and cells were stored on ice until analysed then vortexed before analysed.<br />
<br />
<br><br><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_ScotlandTeam:Aberdeen Scotland2010-10-26T12:11:11Z<p>Porter: </p>
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<h1>Why ayeSwitch?</h1><br />
<p><br />
Over the course of the summer, the University of Aberdeen iGEM team engineered a novel genetic toggle switch in yeast which is regulated at the translational level and allows mutually exclusive expression of either green or cyan fluorescent protein. Using cell cytometry (FACS) and fluorimetry, we successfully demonstrated gene expression and translational regulation of a fusion of mRNA binding proteins and fluorescent proteins. Deterministic and stochastic models including experimental results and published parameter values predicted that the probability of successful bistability for the switch is 0.96%, but that this can theoretically be improved to a maximum of 51.27% by limiting the variation range of the most sensitive parameters. The models also predicted that to generate switch-like behaviour, co-operative binding of the mRNA binding protein to its mRNA stem loop was essential. These results suggest that a translationally regulated genetic toggle switch is a viable and novel engineering concept applicable to medicinal, environmental and technological problems.<br />
</p><br />
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<h1>Our Sponsors:</h1> <br />
<p><br />
Aberdeen iGEM 2010 gratefully acknowledges the financial support of the following organisations:<br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Project_OverviewTeam:Aberdeen Scotland/Project Overview2010-10-26T12:09:33Z<p>Porter: </p>
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<h1>Project Overview</h1><br />
<h3>Introduction</h3><br />
<p> <br />
For this year’s iGEM competition the Aberdeen team has worked on developing a translationally controlled toggle switch embedded in yeast.<a href="#ref1"><sup style="font-size:10px">[1]</sup></a> Genetic toggle switches are a vital component for synthetic biology circuits , enabling functional control of biological functions. The majority of toggle switches used for iGEM are embedded in Escherichia coli and can only be controlled at the transcriptional level <a href="#ref2"><sup style="font-size:10px">[2]</sup></a><sup style="font-size:10px">,</sup><a href="#ref3"><sup style="font-size:10px">[3]</sup></a>. Our main goal was to create and model a novel gene circuit, wherein yeast cells can be switched between mutually exclusive fluorescent proteins under exposure to environmental factors. This switching behaviour would be regulated at the translational level, an innovation over previous systems that only demonstrated transcriptional regulation <a href="#ref4"><sup style="font-size:10px">[4]</sup></a><sup style="font-size:10px">,</sup><a href="#ref5"><sup style="font-size:10px">[5]</sup></a>.The novel genetic toggle switch operated by controlling gene expression at the translational level consisted of two gene expression constructs expressing an RNA-binding protein fused to either Green (GFP) or Cyan (CFP) fluorescent protein in the presence of appropriate inducer. When co-expressed in yeast, these translational fusions would be mutually inhibitory at the translational level, thereby forming a biological, ‘Toggle Switch’ system. <br />
</p><br />
<br><br />
<hr><br />
<h3>The AyeSwitch</h3><br />
<p>The toggle switch is shown by Fig 1 and was named the ‘AyeSwitch’. It is regulated by controlling the two constructs, GAL1p-[Npeptide-GFP] and CUP1p-[MS2-CFP], via inducible yeast promoters GAL1 or CUP1 in the presence or absence of galactose and Cu2+ ions respectively. <br />
</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/ff/Toggle_switch.jpg"><br />
</center><br />
<br><br />
<p><br />
For example, in the presence of galactose only, GAL1 is induced and there is expression of N-peptide-GFP protein. The subsequent addition of Cu2+ then induces the transcription of mRNA coding for MS2 coat binding protein and CFP. In addition to this, the mRNA also codes for a Bbox stem loop sequence that can be bound by N-peptide. </p><br />
<p><br><br />
Ideally, there is initial inhibition of MS2-CFP translation by Npeptide-GFP binding to the Bbox stem loop. Evolution of time corresponds to the ratio of MS2-CFP mRNA to N-peptide-GFP protein increasing allowing some MS2-CFP to be produced until CFP ‘switches ON’ as it gains dominance over GFP.</p><br />
<p><br />
Additionally, N-peptide-GFP protein translation can also be inhibited by MS2-CFP via MS2 protein binding to the MS2 stem loops on the N-peptide-GFP mRNA. This may help the switching ON of CFP and also means GFP would face a similar situation if the inducer was changed from Cu2+ to galactose.</p><br />
<p><br><br />
However, additional variables may come into play affecting the outcomes described above. It is likely that the concentration of each inducer present, the translational rate and binding efficiency of stem loop binding proteins to mRNA stem loop and degradation rate of proteins can also affect the outcome. Reversing the order of inducer present may also affect the outcome. </p><br />
<br><br />
<hr><br />
<h3>Modelling Characterisation of the Ayeswitch</h3><br />
<p><br />
Our team proposed a novel model to describe the functioning of the Aye-switch, based on ordinary differential equations (ODEs). The proposed system of ODEs was carefully and systematically studied both analytically and computationally. A bifurcation analysis was performed and the bistability of the system was investigated with respect to large variations in the parameters of the system. The deterministic simulations were compared with stochastic ones, using the Gillespie algorithm. The parameter space of the model was thoroughly investigated, using two different approaches: Monte-Carlo and directed evolution. These two approaches are very useful for a wide range of projects in synthetic biology. The theoretical predictions led to the proposition of optimised parameters for the Aye-switch that allow a very robust translational switch.</p><br />
<br><br />
<hr><br />
<h3>Experimental Characterisation of the AyeSwitch</h3><br />
<p><br />
The experimental work addressed these issues by initially characterising the promoters in terms of their dose response and time response using constructs GAL1-[GFP] and CUP1-[GFP]. These experiments were then extended to characterise GAL1p-[Npeptide-GFP] and CUP1p-[MS2-CFP] which discovered that CUP1p-[MS2-CFP] did not function as expected.</p><br><br />
<p><br />
The experimental work diverged from this point to troubleshoot CUP1p-[MS2-CFP], investigating the translation inhibition of GAL1p-[Npeptide-GFP] by MS2 coat protein using construct MET17p - [MS2], Bio-brick construction and testing of Bio-brick E2050 mOrange.</p><br />
<br><br />
<hr><br />
<h3>Troubleshooting CUP1p-[MS2-CFP]</h3><br />
<p><br />
Troubleshooting of CUP1p-[MS2-CFP] was carried out through a series of gene cassette replacement experiments testing the promoter and CFP sequences for functionality. The conclusions to these experiments suggest that the Bbox Stem loop, usually located in the 3’untranslated region but is in the 5’ untranslated region of our construct may be preventing the expression of downstream proteins. It may also be that the fusion of MS2 to CFP results in inappropriate protein folding, inhibiting expression.</p> <br />
<br><br />
<hr><br />
<h3>Verification of Translation Inhibition as a Regulatory Mechanism</h3><br />
<p><br />
It was shown that the translational inhibition of GAL1p-[Npeptide-GFP] by MS2 coat protein was possible, confirming that translational regulation is viable. Further work if time permitted would investigate if this inhibition could work in the context of a toggle switch.</p><br />
<br><br />
<hr><br />
<h3>Bio-brick construction and testing </h3><br />
<p><br />
In parallel, Bio-bricks were constructed and submitted to the Registry of parts whilst testing of the Bio-brick E2050 mOrange using fluorimetry and FACS analysis lead to the conclusion that the mOrange sequence did not function within our GAL1p-[Npeptide-GFP] construct that was shown to be able to express GFP appropriately. </p><br />
<br><br><br />
<hr><br />
<br />
<br />
<h1>Attribution and Contributions</h1><br />
<h3>Biological circuit construction and testing </h3><br />
<p><br />
The students within the experimental section of the team were provided (by their host lab) with two yeast strains that had Gal1p-GFP and Cup1p-GFP integrated into the genome (see 'DNA constructs). They then used these constructs to analyse the properties of the CUP1 and GAL1 promoters. With some instructor oversight, the student team themselves then completely designed constructs Gal1p-(Npep-GFP) and Cup1p-(MS2-CFP), which were then synthesised by a synthetic DNA supply company. The students then tested these constructs, and further engineered them during the trouble-shooting phase of the project.<br><br />
All the experimental work described on the wiki, involving characterisation, testing and re-engineering of the bio-bricks, was carried out by the student members of the team. All the construction and sequencing of the four submitted bio-bricks was also carried out by members of the student team.<br />
<br><br><br />
<br />
<h3>Mathematical modelling of the AyeSwitch </h3><br />
<p><br />
The students within the theoretical section of the team carried out all the described modelling. Team activities were overseen by the Instructors, but all model coding and model analysis was performed by the students within the team.<br />
<br />
<br><br><br />
<hr><br />
<h3> References</h3><br><br />
<p><br />
<a name="ref1"></a><br />
<a href="http://www.nature.com/msb/journal/v2/n1/full/msb4100073.html"target="_blank"><b><sup style="font-size:10px">[1]</sup></b></a> Ernesto Andrianantoandro et al. Synthetic biology: new engineering rules for an emerging discipline Molecular Systems Biology 2:2006.0028</p><br><br />
<p><br />
<a name="ref2"></a><br />
<a href="http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html"target="_blank"><b><sup style="font-size:10px">[2]</sup></b></a> Timothy S. Gardner et al. Construction of a genetic toggle switch in Escherichia coli Nature 403, 339-342 (20 January 2000)</p><br><br />
<p><br />
<a name="ref3"></a><br />
<a href="http://www.cell.com/retrieve/pii/S0092867403003465"target="_blank"><b><sup style="font-size:10px">[3]</sup></b></a> Mariette R. Atkinson et al. Development of Genetic Circuitry Exhibiting Toggle Switch or Oscillatory Behavior in Escherichia coli Cell, Volume 113, Issue 5, 597-607, 30 May 2003 </p><br><br />
<p><br />
<a name="ref4"></a><br />
<a href="http://www.nature.com/emboj/journal/v17/n14/abs/7591108a.html"target="_blank"><b><sup style="font-size:10px">[4]</sup></b></a> Adam Platt and Richard J Reece The yeast galactose genetic switch is mediated by the formation of a Gal4p–Gal80p–Gal3p complex The EMBO Journal (1998) 17, 4086 - 4091 </p><br><br />
<p><br />
<a name="ref5"></a><br />
<a href="http://www.pnas.org/content/88/19/8597.abstract"target="_blank"><b><sup style="font-size:10px">[5]</sup></b></a> D W Griggs and M Johnston Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression PNAS October 1, 1991 vol. 88 no. 19 8597-8601</i></p><br />
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<h1> Protocols</h1><br />
<p><br />
For our iGEM2010 project, we have used a variety of techniques such as -<br />
<br><br><br />
<br />
Restriction digest of DNA plasmids <br />
<br><br />
Gel electropheresis of DNA products to check for correct length and quantity of DNA present<br />
<br> <br />
Selective culture of E-coli and Yeast in liquid and Agar Medium (LB and SD)<br />
<br><br />
PCR Amplification<br />
<br><br />
Transformation of shuttle vectors into yeast<br />
<br><br />
DNA extraction for E-coli by Spin Mini-Prep<br />
<br><br />
Microscopy, Fluorimetry and FACS<br />
</p><br />
<br><br><br />
<p><br />
Many of these techniques are standard protocols carried out regularly in the lab and many iGEM teams will be familiar with them.<br />
<br><br />
However, we have also used some additional techniques which may require further details. These are included as follows - <br />
<br><br><br />
</html><br />
<br />
<UL><br />
<LI><b>[https://2010.igem.org/Construction_of_plasmids_in_vivo_using-yeast_homologous_recombination Construction of Plasmids In Vivo using Yeast Homologous Recombination]</b><br />
<br><br><br />
<LI><b>[https://2010.igem.org/BioBrick_Construction BioBrick Construction ]</b><br />
<br><br><br />
<LI><b>[https://2010.igem.org/Induction_protocols_for_GAL1_CUP1_MET17_promoters Induction protocols for the GAL1, CUP1 and MET17 promoters]</b><br />
<br><br><br />
<LI><b>[https://2010.igem.org/FACS_analysis_of_fluorescent_proteins FACS analysis of Fluorescent Proteins]</b><br />
</UL><br />
<br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/ProtocolsTeam:Aberdeen Scotland/Protocols2010-10-26T12:06:00Z<p>Porter: </p>
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<h1> Protocols</h1><br />
<p><br />
For our iGEM2010 project, we have used a variety of techniques such as -<br />
<br><br><br />
<br />
Restriction digest of DNA plasmids <br />
<br><br />
Gel electropheresis of DNA products to check for correct length and quantity of DNA present<br />
<br> <br />
Selective culture of E-coli and Yeast in liquid and Agar Medium (LB and SD)<br />
<br><br />
PCR Amplification<br />
<br><br />
Transformation of shuttle vectors into yeast<br />
<br><br />
DNA extraction for E-coli by Spin Mini-Prep<br />
<br><br />
Microscopy, Fluorimetry and FACS<br />
</p><br />
<br><br><br />
<p><br />
Many of these techniques are standard protocols carried out regularly in the lab and many iGEM teams will be familiar with them.<br />
<br><br />
However, we have also used some additional techniques which may require further details. These are included as follows - <br />
<br><br><br />
<br />
<UL><br />
<LI><b>[https://2010.igem.org/Construction_of_plasmids_in_vivo_using-yeast_homologous_recombination Construction of Plasmids In Vivo using Yeast Homologous Recombination]</b><br />
<br><br><br />
<LI><b>[https://2010.igem.org/BioBrick_Construction BioBrick Construction ]</b><br />
<br><br><br />
<LI><b>[https://2010.igem.org/Induction_protocols_for_GAL1_CUP1_MET17_promoters Induction protocols for the GAL1, CUP1 and MET17 promoters]</b><br />
<br><br><br />
<LI><b>[https://2010.igem.org/FACS_analysis_of_fluorescent_proteins FACS analysis of Fluorescent Proteins]</b><br />
</UL><br />
<br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/ProtocolsTeam:Aberdeen Scotland/Protocols2010-10-26T12:05:27Z<p>Porter: </p>
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<html><br />
<h1> Protocols</h1><br />
<p><br />
For our iGEM2010 project, we have used a variety of techniques such as -<br />
<br><br><br />
<br />
Restriction digest of DNA plasmids <br />
<br><br />
Gel electropheresis of DNA products to check for correct length and quantity of DNA present<br />
<br> <br />
Selective culture of E-coli and Yeast in liquid and Agar Medium (LB and SD)<br />
<br><br />
PCR Amplification<br />
<br><br />
Transformation of shuttle vectors into yeast<br />
<br><br />
DNA extraction for E-coli by Spin Mini-Prep<br />
<br><br />
Microscopy, Fluorimetry and FACS<br />
</p><br />
<br><br><br />
<p><br />
Many of these techniques are standard protocols carried out regularly in the lab and many iGEM teams will be familiar with them.<br />
<br><br />
However, we have also used some additional techniques which may require further details. These are included as follows - <br />
<br><br><br />
</html><br />
<UL><br />
<LI><b>[https://2010.igem.org/Construction_of_plasmids_in_vivo_using-yeast_homologous_recombination Construction of Plasmids In Vivo using Yeast Homologous Recombination]</b><br />
<br><br><br />
<LI><b>[https://2010.igem.org/BioBrick_Construction BioBrick Construction ]</b><br />
<br><br><br />
<LI><b>[https://2010.igem.org/Induction_protocols_for_GAL1_CUP1_MET17_promoters Induction protocols for the GAL1, CUP1 and MET17 promoters]</b><br />
<br><br><br />
<LI><b>[https://2010.igem.org/FACS_analysis_of_fluorescent_proteins FACS analysis of Fluorescent Proteins]</b><br />
</UL><br />
<br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/ConstructsTeam:Aberdeen Scotland/Constructs2010-10-26T12:05:07Z<p>Porter: </p>
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<h1>The DNA Constructs</h1><br />
<br />
<p><b>The DNA constructs used in this project are organised into three main groups:</b></p><br />
<ol><br />
<LI>Promoter characterisation constructs <br><br />
<LI>Switch components <br><br />
<LI>Constructs for switch testing<br />
</ol><br />
<p><br />
<b><i>For each construct, we provide a brief description, and its intended use;</i></b><br />
</p><br />
<br><br><br />
<h2>1. Promoter characterisation constructs</h2><br />
<br />
<h4>GAL1p-[GFP]</h4><br />
<hr><br />
<p><b>Description:</b> This is a genomically-integrated construct in which the GFP protein was placed under control of the yeast GAL1 promoter (Fig. 1)<br />
</p><br><br />
<p><b>Main use:</b> to characterise the induction characteristics of the <i>GAL1</i> promoter, which is induced by galactose. <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i>See promoter activity assay results</i></a><br />
</p><br />
<br />
<br><h4>CUP1p-[GFP]</h4><hr><br />
<p><br />
<b>Description:</b> This is a genomically integrated construct in which the GFP protein was placed under control of the yeast CUP1 promoter.</p><br><br />
<p><b>Main use:</b> to characterise the induction characteristics of the <i>CUP1</i> promoter, which is induced by copper ions. <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i>See promoter activity assay results</i></a></p><br />
<center><img src="https://static.igem.org/mediawiki/2010/e/ef/CUP1_promoter_and_GAL1_promoter.jpg"/></center><br />
<br><br><br />
<br><br />
<br />
<br />
<h2>2.Switch components</h2><br />
<br />
<br><h4>CUP1p - [MS2-CFP]</h4><hr><br />
<br />
<p><b>Description:</b> This construct was designed to be regulated by yeast CUP1 promoter. Downstream to this is a Bbox mRNA sequence followed by a fusion protein consisting of MS2 coat binding protein and CFP.</p><br><br />
<br />
<p><b>Main use:</b> to be one half of the <b>AyeSwitch</b>. The Bbox mRNA sequence can be bound by N-peptide, (from GAL1p-Npep-GFP) which inhibits translation of MS2 coat binding protein and CFP. Translation of the MS2 coat binding protein allows inhibition of translation of Npep-GFP whilst CFP provides a means of quantification. </p><br />
<br><br />
<p>N.B. It was discovered that this construct did not exhibit CFP fluorescence as expected when induced with Cu2+.<a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i>See 'Results'</i></a></p><br />
<br />
<br><br><br><br />
<center><img src="https://static.igem.org/mediawiki/2010/f/f4/CUP1_promoter_and_Bbox_stem_loop.jpg"/></center><br />
<br><br><br><br />
<br />
<h4>GAL1p-[Npep-GFP]</h4><br />
<br />
<p><b>Description:</b> This construct was designed to be regulated by yeast GAL1 promoter. Downstream of this is a MS2 mRNA sequence followed by a fusion protein consisting of two N-peptide binding proteins and GFP.</p><br />
<br><br />
<br />
<p><b>Main use;</b> to be one half of the <b>AyeSwitch</b>. The MS2 mRNA sequence can be bound by MS2 coat binding protein (from CUP1p-[MS2-CFP]) which inhibitions translation of N-peptide binding protein and GFP. Translation of the N-peptide binding protein allows inhibition of translation of MS2-CFP whilst GFP provides a means of quantification. <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i>See 'Results'</i></a></p><br />
<br><br><br><br />
<center><img src="https://static.igem.org/mediawiki/2010/9/9c/GAL1_promoter_and_MS2_Stem_Loops.jpg"/></center><br />
<br><br><br><br />
<br />
<br />
<h4>Combining Gal1p-[Npep-GFP] and Cup1p-[MS2-CFP]: The <b>AyeSwitch</b></h4><br />
<br />
<p>From the figure immediately below, it can be seen that there the Gal1p-[Npep-GFP] and Cup1p-[MS2-CFP] constructs are mutually inhibiting at the translational level. This is because the Npeptide, as part of an Npep-GFP fusion protein, is able to specifically bind the B-box mRNA stem loop in the Cup1p-[MS2-CFP] mRNA, and conversely, the MS2 protein, as part of an MS2-CFP fusion protein, is able to specifically bind the MS2 mRNA stem loops in the Gal1p-[Npep-GFP] mRNA. Each construct can be controlled transcriptionally using, respectively, galactose or copper ions in the growth medium.</p><br />
<br />
<br><br><br><br />
<center><img src="https://static.igem.org/mediawiki/2010/f/ff/Toggle_switch.jpg"/></center><br />
<br><br><br><br />
<br />
<br />
<h2>3. Constructs for switch testing</h2><br />
<br><h4>MET17p - [MS2]</h4><br />
<br />
<p><b>Description:</b> This was a construct already avalilable in the host laboratory, in which the MS2 RNA-binding protein was placed under the control of an inducible <i>MET17</i> promoter. This promoter is induced in the absence of methionine in the growth medium, and repressed by its presence.</p><br />
<br><br />
<p><b>Main use;</b> The MS2 RNA binding protein binds MS2 RNA stem loops, such as those present in the GAL1p-[Npep-GFP] construct (see 'Switch components' above). Thus co-transforming GAL1p-[Npep-GFP] with MET17p - [MS2] in yeast would allow us to verify that MS2 protein binding to MS2 RNA stem loops would inhibit expression of N-pep-GFP at the translational level.</p><br />
<br />
<br><br><br><br />
<center><img src="https://static.igem.org/mediawiki/2010/d/d0/MET17_promoter.jpg"/></center><br />
<br><br><br><br />
<br />
<h4>TEF1p -[CFP]</h4><br />
<br />
<p><b>Description:</b> This construct consisted of a constitutively active TEF1 promoter controlling the expression of a CFP sequence downstream.</p><br />
<br><br />
<p><b>Main use:</b> for troubleshooting experiments. This construct was made by the team to allow us to verify that our fluorescent microscopes and cell cytometer (FACS) was able to successfully detect CFP expressed in yeast. It was used to confirm whether or not the CFP sequence from CUP1p - [MS2-CFP] were able to exhibit CFP fluorescence. <a href="https://2010.igem.org/Team:Aberdeen_Scotland/Results"><i>See 'Results/Troubleshooting'</i></a></p><br />
<br />
<br><br><br><br />
<center><img src="https://static.igem.org/mediawiki/2010/c/c7/TEF1_promoter.jpg"/></center><br />
<br><br><br><br />
<br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/FACS_Analysis_of_mOrange_recombinant_pRS415FACS Analysis of mOrange recombinant pRS4152010-10-26T12:02:16Z<p>Porter: </p>
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<h1>Testing Biobrick E2050 Part 2 - FACS Analysis of mOrange recombinant GAL1p-[Npep-GFP]<br />
</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment was to test if mOrange inserted into GAL1p-[Npep-GFP], (GAL1p-[Npep-mOran]) in place of green fluorescent protein, (GFP) will fluoresce when galactose is added.</p><br />
<h3>Hypothesis</h3><br />
<p>Since GFP has been detected when GAL1p-[Npep-GFP] is induced with galactose, in-frame insertion of mOrange DNA sequence in place of GFP in GAL1p-[Npep-GFP] should produce orange fluorescence when galactose is added. This will allow confirmation of whether the Bio-brick E2050 will work.<br><br />
<br />
</p><br />
<h3>Protocol</h3><br />
<p>Starter cultures of BY4741ΔTrp, BY4741 GAL1p-[Npep-GFP] and BY4741 GAL1p-[Npep-mOran] were incubated overnight in SD medium and the OD600 was measured. These were then used to set-up overnight experimental cultures to have an OD600=0.6 for the following day. The following were set-up as shown by (Table 1).</p> <br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/2c/MOra_test_2_table_1.jpg"/><br />
</center><br />
<br />
<p> The OD600 was measured the following day and samples were normalised by spinning down in a centrifuge and washing (x2) with PBS buffer to an OD600=0.6. These were then used for the FACs analysis. The filters used were FITC and PE. These were chosen as they were the best available filters for measuring GFP and mOrange respectively as can be seen from the following data obtained from the BD Fluoresence Spectrum Viewer (Fig.1).</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/69/BD_Viewer.jpg"/><br />
</center><br />
<br />
<br><br />
<br><html><br />
<h3>Results</h3><br />
<p>From the FACS analysis, it was shown that sample 4 had 5.75% of the cell population analysed fluorescing whilst sample 6 had 16.6% of the cell population analysed fluorescing. As a result, the analysis focussed on sample 6 and its corresponding negative, sample 7.</p><br><br />
<br />
<p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p><br><br />
<br />
<p>The results are shown in Fig.2(i) and it can be seen that the untransfected and negative control both have a single peak which corresponds to autofluorescence from yeast with no GFP expression. For the positive control (GAL1p-[Npep-GFP] BY4741 + Galactose), there is a slight first peak followed by a taller second peak which suggests that a certain percentage of the analysed population (23.3%) were not fluorescing. It is likely that this could be because they are damaged yeast cells, did not carry the desired plasmid GAL1p-[Npep-GFP] or that the Gal promoter was not induced for unknown reasons. However, the larger second peak (76.7%) showed that there is a large proportion of the population that are able to express GFP as desired.</p><br> <br />
<br />
<p>An analysis for GAL1p-[Npep-mOran] sample 6 and 7 using FITC to show that there was no GFP fluorescence was also carried out as shown in Fig.2(ii). However, a small peak was observed for sample 6, which was unexpected. This may have been because the FITC filters overlap the emission spectra of mOrange as shown previously, (BD Spectrum Viewer). Further analysis using the PE-A filter specific for mOrange however show that there is a less well defined and smaller peak for sample 6 than would be expected if there was mOrange fluorescence. This is shown in Fig.3(ii), which suggests that the fluorescence detected is GFP, which was not expected.</p><br><br />
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<p>In a previous experiment, PCR screening was used to establish that the transformed colonies were positive for GAL1p-[Npep-mOran], which makes the chances of a BY4741 transformed with uncut and non-homologously recombined GAL1p-[Npep-GFP] with mOrange insert unlikely.</p><br><br />
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<p>Despite this, the observation of a small peak for both FITC and PE-A with the features as discussed above suggests that there are some yeast cells within the GAL1p-[Npep-mOran] BY4741 samples expressing GFP rather than mOrange. Which implies that the homologous recombination was not 100% efficient.</p> <br />
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<p>Conversely, 83.4% of the analysed cell population did not show any fluorescence, which suggests that either mOrange was not successfully homologously recombined or that it could not be expressed by BY4741. If uncut GAL1p-[Npep-GFP] had been transformed in place of GAL1p-[Npep-mOran] it is likely that a greater GFP fluorescence would have been detected, which was not the case. The cut GAL1p-[Npep-GFP] does not have any complementary ends that could re-ligate. Therefore it is unlikely that these are transformed and selected.</p><br><br />
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<p>Ideally, a positive control for detecting mOrange would allow verification of the specificity of PE-A detection of mOrange and a DNA sequence of transformant of GAL1p-[Npep-mOran] would confirm whether or not the yeast carried the appropriate recombined plasmids.</p><br> <br />
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<h3>Conclusions</h3><br />
<p>From the discussion of the results, the absence of any mOrange fluorescence from the FACS data suggests that the Bio-brick E2050 did not work. However, unexpected GFP fluorescence in GAL1p-[Npep-mOran] BY4741 was detected, which suggests that the homologous recombination of mOrange for GFP in GAL1p-[Npep-GFP] was not 100% efficient and further experimentation is required to confirm this result. <br />
</p><br><br />
<h3>References</h3><br />
<p><br />
<sup style="font-size:10px">[1]</sup><a href="http://www.bdbiosciences.com/external_files/media/spectrumviewer/index.jsp">Click here to visit the BD Website</a></p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Homologous_Recombination_of_E2050_into_pRS415_Construct_in_Place_of_GFP_ProteinHomologous Recombination of E2050 into pRS415 Construct in Place of GFP Protein2010-10-26T12:01:24Z<p>Porter: </p>
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<h1>Testing Bio-brick E2050 Part 1 - Homologous Recombination of E2050 into GAL1p-[Npep-GFP] Construct in Place of GFP Protein</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment was to homologously recombine the DNA sequence of Bio-brick E2050 mOrange (E2050), in place of green fluorescent protein (GFP), in GAL1p-[Npep-GFP] by transforming with yeast BY4741ΔTrp. This allowed testing of E2050 in further experiments.</p><br />
<h3>Hypothesis</h3><br />
<p>Since GFP has been detected when GAL1p-[Npep-GFP] is induced with galactose, if the E2050 is inserted in place of GFP in GAL1p-[Npep-GFP], then orange fluorescence should be detected under similar conditions for GFP expression. Homologous recombination carried out by yeast is one technique that can be used to conveniently replace the GFP sequence with E2050 in GAL1p-[Npep-GFP]. <br><br />
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<h3>Protocol</h3><br />
<p>The Bio-brick E2050 was rescued from the DNA Distribution Kit provided by iGEM by transforming into sub-cloning DH5α E-coli strain. The plasmids were then extracted using a Qiagen Mini-Prep kit following manufacturer’s protocol. Testing for correct plasmid rescue was done by cutting extracted DNA with restriction enzymes XbaI and SpeI which are unique cut sites required to remove the DNA sequence that codes for mOrange from the bio-brick plasmid pSB2K3. Verification of correct sequence was by checking the length of insert and vector by gel electrophoresis of restriction digest. The expected fragment lengths are 744bp and 4425bp respectively.</p> <br><br />
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<p>To homologously recombine the E2050 mOrange insert into GAL1p-[Npep-GFP] in place of GFP, the mOrange inserts were amplified by PCR using primers designed with 45bp overhangs that were homologous to the region of plasmid immediately prior and after the GFP sequence in GAL1p-[Npep-GFP]. The sequence to be amplified was checked to ensure that mOrange would be translated in-frame when integrated into GAL1p-[Npep-GFP].</p> <br><br />
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<p>In parallel, GAL1p-[Npep-GFP] constructs were restriction cut at unique sites using NheI and SmaI to remove the GFP coding sequence. Both digests were checked by gel electrophoresis to ensure the correct length of PCR product and cut vector was obtained (739bp and 965bp respectively).</p> <br><br />
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<p>For the homologous recombination, this was done by transforming yeast BY4741 auxotrophic for methionine, leucine, histidine, uracil and tryptophan with mOrange PCR products and cut GAL1p-[Npep-GFP] vector from previously. These would be homologously recombined by the yeast. The resultant transformants were selected by growing on SD agar with 1% each of methionine, histidine, uracil and tryptophan added since GAL1p-[Npep-GFP] has a selection marker for leucine encoded which is left intact throughout the experiment.</p> <br><br />
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<p>To confirm that homologously recombined plasmids were selectively cultured, PCR colony screening was carried out on selected colonies from transformation plates.</p> <br><br />
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<h3>Results</h3><br />
<p>From (Table 1.) the results of the selective culture of transformants show that the positive and negative controls worked as expected with BY4741 growing when all nutrients are present and not growing in the absence of essential amino acids. The expected transformants also grew on selection plates 1 & 2 at the three volumes plated (50µl, 100 µl and 200 µl) with increasing density of colonies.</P> <br><br />
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<p>Since a transformation with ‘cut GAL1p-[Npep-GFP] only’ was not carried out, no information about the likelihood of uncut GAL1p-[Npep-GFP] from the restriction digest being transformed was obtained. To overcome this, individual colonies were selected and plated out for culture. PCR colony screening was then used to check that these had the mOrange insert transformed. Twenty colonies were selected, cultured and two colonies were PCR screened using primers for mOrange. (i.e. The primers used previously to generate mOrange with 45bp overhangs) The results are shown by (Table 2.) and confirm that homologous recombination of mOrange PCR products had been successfully integrated into cut GAL1p-[Npep-GFP]. <br />
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<h3>Conclusions</h3><br />
<p>From the discussion of the results it has been shown that E2050 mOrange has been successfully rescued and amplified. These were then used as substrate for PCR amplification to generate product with homologous ends that were homologously recombined with cut GAL1p-[Npep-GFP], (GFP sequence removed) in a transformation process using BY4741ΔTrp. PCR colony screening using mOrange primers confirmed that colonies carrying the recombinant GAL1p-[Npep-GFP] had been successfully transformed into BY4741. <br />
</p><br><br />
<h3>References</h3><br />
<p>Hinnen A, Hicks JB and Fink GR. Transformation of yeast.<br />
Proc Natl Acad Sci, 75 1978 (1929-33)</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/GFP_decayTeam:Aberdeen Scotland/GFP decay2010-10-26T12:00:31Z<p>Porter: </p>
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<h1> Characterising the glucose repression of GAL1 promoter in the GAL1p-[Npeptide-GFP] construct </h1><br />
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<h3>Aim</h3><br />
<p>To test the effect of glucose on repression of the GAL1 promoter, and thus on shut-off of GFP expression from construct GAL1p-[Npeptide-GFP] construct over time.</p><br />
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<h3>Hypothesis</h3> <br />
<p>The presence of glucose should quickly repress the GAL1 promoter and therefore result in the overall reduction of the GFP intensity present within the cells; measurement of the rate of decay should identify the relative stability of the GFP protein</p><br />
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<h3>Protocol</h3><br />
<p>1. Yeast transformed with the GAL1p-[Npeptide-GFP] construct were innoculated overnight in 5 mls of synthetic defined medium with amino acids; his (0.2 %), met (0.2%), ura (0.2%), trp (0.2 %) and Raffinose (2 %) as the carbon source.<br><br><br />
2. Following overnight growth the cells were subcultured in fresh, pre-warmed SD medium (50 mls) containing galactose (a range of concentrations: see Results below) to obtain a predicted OD600 of 0.3 by 10 am the following morning.<br><br><br />
3. The following morning, at an OD600 of 0.3, a sample (1 ml) was taken before and after the addition of glucose (2 %). Samples were then taken every 20 minutes thereafter for a period of 167 minutes. All samples were then pelleted (13000 rpm, 5 mins, 4 degreesC), washed once with PBS buffer and stored on ice. Once collected all samples were then dispenced in PBS and diuted by a factor of 1/20 for FACS analysis.<br />
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<h3>Results</h3><p><br />
Cells grown on galactose and expressing GFP were switched to growth on medium containing glucose. Following the resultant switch-off of the GAL1 promoter, GFP decay was monitored.<br><br><br />
Panel A (below) shows FACS analysis, with the peak to the left indicating GFP expressing cells and a peak to the right showing non GFP expressing cells. The FACS analysis clearly shows that the highest GFP expression (bottom light blue line) is observed after incubation overnight with galactose, with glucose present. It can be observed that after the addition of glucose (all lines above the blue) that there is a continuous increase in the number of cells not expressing GFP over time.<br><br><br />
Panel B shows this data in summarised, averaged form. It reveals that the average GFP intensity of the cells decreased steadily with time after the glucose addition, hence showing that the glucose has rapidly repressed the GAL1 promoter,and inhibited the expression of GFP. The half-life of this decay was approximately 140 minutes, which corresponded to approximately the doubling time of the cell culture, indicating that cell division was the primary reason for GFP disappearance, rather than active GFP turnover.</p><br />
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<h3>Conclusion</h3><p><br />
The presence of glucose rapidly inhibits the GAL1 promoter from expressing GFP and the average GFP intensity within a cell reduces by over 50 % within 140 minutes, consistent with cell division being the primary source of GFP depletion. This confirmed the fact that GFP is widely considered to be an extremely stable protein.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415MS2 Coat-Protein Effect on Expression of GFP in pRS4152010-10-26T11:58:14Z<p>Porter: </p>
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<h1> Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein </h1><br />
<h3>Aim</h3><br />
<p>The characterisation of the effect of MS2 on the expression of GFP by GAL1p-[Npeptide-GFP] will allow more accurate modelling of the system and will allow us to determine with more precision the probability of success of the cross-inhibition of the switch. Expressing MS2 using MET17p - [MS2] will allow us to monitor the effect of MS2 without the complication of the λ-N-peptide produced by GAL1p-[Npeptide-GFP] in turn inhibiting the expression of MS2.</p><br />
<h3>Hypothesis</h3> <br />
<p>The expression of MS2 by MET17p - [MS2]will result in a decrease in the level of expression of GFP by GAL1p-[Npeptide-GFP]. The inhibition will show a linear correlation with the level of expression of MS2.</p><br />
<h3>Protocol</h3><br />
<p>During this experiment double transformants of BY4742 containing GAL1p-[Npeptide-GFP] and MET17p - [MS2] were used. Single transformants of BY4742, containing only GAL1p-[Npeptide-GFP], were used to provide the negative and positive controls for the expression of GFP.</p><br><br />
<p>The double transformants were first cultured overnight in specific conditions in order to establish the desired pre-conditions. The cells were then washed and re-cultured in a different specific set of conditions which would allow the characterisation of the effect of MS2.</p><br><br />
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<p>* 500μM Met was used as this concentration has been used in other experiments to successfully completely switch of the Met17 promoter<a href="#ref1"><sup style="font-size:10px">[1]</sup></a>. <br><br />
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The different pre-established conditions allow us to determine whether the history of the sample affects the final result.<br><br />
Final samples were then washed and normalised before being analysed using microscopy, Fluospar Optima readings and FACS analysis.</p><br><br />
<h3>Results</h3><br />
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<h5><u>Microscopy</u><h5><br><br />
<p>The microscopy analysis revealed that, in none of the samples, the GFP expression had been completely inhibited. All samples (bar the negative control) showed green fluorescence. The microscope did not allow us to determine if there was any variation however in the levels of GFP in each specific sample. </p><br />
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<h5><u>Fluospar Optima Readings</u></h5><br><br />
The fluorimeter readings correlated the microscopy results by recording fluorescence in all samples except the – control.<br><br />
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<p>The recorded fluorescence values for the respective samples showed that there was indeed some variation in the levels of GFP (Fig 1). In both the ‘MS2 Dom’ and the ‘Race’ sample the GFP level was lower than in the + control indicating that the expression of GFP had indeed been inhibited (a 20% decrease for the ‘Race’ sample and an 11% decrease for the ‘MS2’ sample). The ‘GFP Dom’ sample however showed an approximate 8% increase in GFP fluorescence when compared to the + control. Although this is a bit unexpected is could be due to the fact that the GFP expression was initiated in the 1˚ set of conditions whereas it took place in the 2˚ for the + control. However it appears that no inhibition took place indicating that once GFP is being expressed the amount present of MS2 as expressed by MET17p - [MS2] is not able to significantly inhibit the level of GFP fluorescence.</p><br />
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<h5><u>FACS analysis</u></h5><br />
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<p>The first FACS analysis experiment involved running samples from the same initial cultures (see Table 1). The results showed that the presence of the MS2 coat protein was having an effect on the expression of GFP. All three test samples revealed lower levels of GFP when compared to the positive control indicating that MS2 was inhibiting the expression of GFP (see Fig 3). As expected the sample where GFP had been allowed to dominate prior to expression of MS2 (GFPdom sample) showed the highest level of GFP in the test samples and equally the sample where MS2 dominated prior to the expression of GFP (MS2do sample) showed the lowest level of GFP expression.</p><br><br />
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<p>The second FACS analysis experiment was aimed to determine whether the inhibition of GFP expression was in any way dependent on the levels of MS2. The following cultures were set up containing transformants containing both GAL1p-[Npeptide-GFP] and MET17p - [MS2] with varying amounts of Methionine. The reasoning is that the varying levels of methionine will translate into varying amounts of MS2 being produced as the Met17 is repressed.</p><br><br />
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<p>The results showed that the inhibition of GFP expression by GAL1p-[Npeptide-GFP] by MS2 previously seen (see Fig.3) is indeed dependent on the concentration of MS2.<br><br />
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We can see a linear relationship between GFP levels and MS2 concentrations (see Fig.4). The observed level of GFP is at its lowest with no methionine being present. No methionine present translates as the Met17 promoter being unrepressed meaning that the MS2 expression rate is at its maximum. The levels of GFP gradually increase along with an increasing concentration of methionine (this translates as the Met17 promoter gradually being repressed until MS2 is no longer being expressed).</p><br />
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<h3>References</h3><br />
<a name="ref1"></a><br />
<p>[1] Dominik Mumberg, Rolf MulIer and Martin Funk*<br><br />
Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression<br><br />
Nucleic Acids Research, 1994, Vol. 22, No. 25 5767-5768</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Galactose_dose_response_of_Gal1_Promoter_in_pRS415Team:Aberdeen Scotland/Galactose dose response of Gal1 Promoter in pRS4152010-10-26T11:57:27Z<p>Porter: </p>
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<h1>Measurement of dose responsiveness of the GAL1 promoter to galactose using construct GAL1p-(Npep-GFP)</h1><br />
<h3>Aim</h3><br />
<p><br />
Previous dose response experiments using the fluorometer revealed that full GAL1 promoter induction was achieved at concentrations above 0.5% (data not shown). We wanted to examine the dose responsive behaviour of the GAL1 promoter across a full range of concentrations. Therefore the dose response experiments were repeated using lower concentrations of this inducing agent. We have therefore tested media containing between 0.02% and 2% of galactose. <br />
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<h3>Protocol</h3><br />
<p><br />
1. Yeast transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and raffinose (2 %) as the carbon source. <br><br><br />
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2. The following evening this cell culture was sub-cultured into a flask containing pre-warmed SD medium (50 mls) with 2% raffinose, and one of a range of concentrations of galactose between 0.02% and 2% of galactose, to achieve an optical density at 600nm of 0.6 by 9.00 am the following morning. <br><br />
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3. Samples were washed into PBS, and diluted 1/20 in preparation for FACS analysis.<br />
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<h3>Results</h3><br />
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(a) Flow cytometry was used to quantify GFP fluorescence, with an excitation wavelength of 488 nm, and an emission filter of 510 nm, for cells grown on medium containing galactose concentrations between 0.05% and 2% w/v </p><br />
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The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases in response to the percentage of galactose in the growth medium. The GAL1 promoter in our construct showed a high degree of sensitivity to the inducing agent, with concentrations as low as 0.01% having significant inducing potential. <br />
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(b) In further experiments (panels A and B, immediately below), we sought to test the ability of even lower concentrations of galactose to induce the Gal1 promoter. Concentrations between 0.02% and 0.07% were tested, revealing that even at 0.02%, galactose was able to induce the GAL1 promoter. Panel A shows the raw FACS data, with panel B indicating the quantitation of this data to indicate the inducing effect of different galactose concentrations. The study also revealed that as long as pre-culture in liquid medium was carried out with raffinose (2%) as a carbon source, the prior maintenance of the stock yeast on agar medium containing glucose or raffinose did not affect the ability of the promoter to be subsequently induced by galactose (see panel B below, compare blue symbols [glucose] with red symbols [raffinose]).<br />
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<h3>Conclusion</h3><br />
<p><br />
The experiment clearly showed that the percentage of cells expressing GFP was exquisitely sensitive to the presence of galactose, with the dose response saturating above 0.1% galactose. Testing of low concentrations also showed that the promoter was extremely sensitive, with concentrations as low as 0.02% w/v causing detectable induction of the promoter. This therefore clearly shows that the GAL1 promoter is highly sensitive, but that as a synthetic biology part, it may not exhibit ideal linear responses to inducing agent for some applications. The observed GFP expression response suggests that the GAL1 promoter behaves as an analogue switch across only a very narrow range of inducer concentrations. <br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_Gal1_Promoter_in_pRS415Team:Aberdeen Scotland/Timed Induction of Gal1 Promoter in pRS4152010-10-26T11:57:09Z<p>Porter: </p>
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<h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment was to test the response of the GAL1 promoter in the presence of galactose over time, by measuring the expression of GFP, the downstream gene. Construct Gal1p-(Npep-GFP) was used in the experiments described here <br />
</p><br />
<br />
<h3>Protocol</h3><br />
<p><br />
1. Yeast transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and Raffinose (2 %) as the carbon source. <br><br><br />
<br />
2. The following evening 861 µl of this cell culture were sub-cultured into a flask containing pre-warmed SD medium (50 mls) to achieve an optical density at 600nm of 0.3 by 10am the following morning. <br><br />
<br />
<br><br />
3. At OD 600 of 0.30, a 1 ml sample was taken to represent the t=0 min sample, and then galactose addded to a final concentration of 0.1 % w/v to begin the promoter induction process. Samples were then taken every 20 minutes thereafter for a period of 170 minutes. All samples were pelleted (13000 rpm, 5min, 4 degrees C), washed once with PBS buffer and stored on ice. Once collected all samples were then dispensed in PBS and diluted by a factor of 1/20 for flow cytometry analysis.<br />
<br><br><br />
<br />
<h3>Results</h3><br />
<p><br />
FACS data showing the changes to the GFP expression (peak to right) and non GFP expressing cells (peak to left) over time as a result of galactose being added.<br />
<br />
There are two significant peaks. The peak to the left of the graph represents the number of cells which did not express GFP and the peak to the right the number of the cells which did express GFP. <br />
The highest peak to the left is produced by the cells before adding galactose and the peak to the right is not present thus there is no GFP expression by the cells at time zero of the experiment, hence no natural GFP expression by the cells.<br />
As time increases there is an increased number of cells which are expressing GFP in the presence of galactose inducer, shown by the gradual increase of the peak to the right over time. The visible peak starts appearing 60 mins after adding galactose (the light blue line). </p><br />
</html><br />
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<center><br />
[[Image: Gal-facs.jpg|500 px]]<br />
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<br><br><br />
[[Image: Gal-facs2.jpg|500 px]]<br />
</center><br />
<html><br />
<br><br><br />
<p><br />
The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases over time after galactose has been added. The graph does not reach a plateau opver the time of the experiment. This may suggest that the cells have not expressed to their maximum capacity. Therefore to conclude, further experiments may be repeated with an increased galactose concentration or an increased period of time over which the experiment was carried out.</p><br />
<br><br><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
The experiment clearly showed that the percentage of cells expressing GFP increased to 68% after 167 minutes from the time that 0.1 % galactose was added to the culture medium. This therefore clearly showing that galactose has successfully induced the expression of the GFP from the GAL1-(Npep-GFP) construct. Expression induction was almost linear over this time, and after 167 minutes, the expression induction had still not reached a plateau. <br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_Gal1_Promoter_in_pRS415Team:Aberdeen Scotland/Timed Induction of Gal1 Promoter in pRS4152010-10-26T11:56:53Z<p>Porter: </p>
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<h1>Measurement of induction of the GAL1 promoter over time in construct GAL1p-(Npep-GFP)</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment was to test the response of the GAL1 promoter in the presence of galactose over time, by measuring the expression of GFP, the downstream gene. Construct Gal1p-(Npep-GFP) was used in the experiments described here <br />
</p><br />
<br />
<h3>Protocol</h3><br />
<p><br />
1. Yeast transformed with a plasmid carrying the GAL1p-(Npep-GFP) construct was inoculated overnight into 5 ml of synthetic defined (SD) medium with amino acids: his (0.2 %), met (0.2 %), ura (0.2 %), trp (0.2 %) and Raffinose (2 %) as the carbon source. <br><br><br />
<br />
2. The following evening 861 µl of this cell culture were sub-cultured into a flask containing pre-warmed SD medium (50 mls) to achieve an optical density at 600nm of 0.3 by 10am the following morning. <br><br />
<br />
<br><br />
3. At OD 600 of 0.30, a 1 ml sample was taken to represent the t=0 min sample, and then galactose addded to a final concentration of 0.1 % w/v to begin the promoter induction process. Samples were then taken every 20 minutes thereafter for a period of 170 minutes. All samples were pelleted (13000 rpm, 5min, 4 degrees C), washed once with PBS buffer and stored on ice. Once collected all samples were then dispensed in PBS and diluted by a factor of 1/20 for flow cytometry analysis.<br />
<br><br><br />
<br />
<h3>Results</h3><br />
<p><br />
FACS data showing the changes to the GFP expression (peak to right) and non GFP expressing cells (peak to left) over time as a result of galactose being added.<br />
<br />
There are two significant peaks. The peak to the left of the graph represents the number of cells which did not express GFP and the peak to the right the number of the cells which did express GFP. <br />
The highest peak to the left is produced by the cells before adding galactose and the peak to the right is not present thus there is no GFP expression by the cells at time zero of the experiment, hence no natural GFP expression by the cells.<br />
As time increases there is an increased number of cells which are expressing GFP in the presence of galactose inducer, shown by the gradual increase of the peak to the right over time. The visible peak starts appearing 60 mins after adding galactose (the light blue line). </p><br />
</html><br />
<br><br><br />
<br />
<center><br />
[[Image: Gal-facs.jpg|500 px]]<br />
<br />
<br><br><br />
[[Image: Gal-facs2.jpg|500 px]]<br />
</center><br />
<html><br />
<br><br><br />
<p><br />
The graph above summarises the FACS data, and shows that the intensity of GFP expressing cells increases over time after galactose has been added. The graph does not reach a plateau opver the time of the experiment. This may suggest that the cells have not expressed to their maximum capacity. Therefore to conclude, further experiments may be repeated with an increased galactose concentration or an increased period of time over which the experiment was carried out.</p><br />
<br><br><br />
<br />
<h3>Conclusion</h3><br />
<p><br />
The experiment clearly showed that the percentage of cells expressing GFP increased to 68% after 167 minutes from the time that 0.1 % galactose was added to the culture medium. This therefore clearly showing that galactose has successfully induced the expression of the GFP from the GAL1-(Npep-GFP) construct. Expression induction was almost linear over this time, and after 167 minutes, the expression induction had still not reached a plateau. <br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Copper_Dose_Response_of_the_CUP1_Promoter_Using_N4Team:Aberdeen Scotland/Copper Dose Response of the CUP1 Promoter Using N42010-10-26T11:56:29Z<p>Porter: </p>
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<h1>Quantitative Determination of the Response of the CUP1p - [GFP] to Varying Concentration of CuSO4</h1><br />
<br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to characterise the CUP1 promoter present in CUP1p - [GFP] by determining whether<br />
it displays a dose response quality. The determined characteristics of this promoter can then be applied to the promoter present in CUP1p - [MS2-CFP] in order to allow more precise modelling of the switch.</p><br />
<br />
<h3>Hypothesis</h3><br />
<p>The CUP1 promoter exhibits a linear relationship between the concentration of CuSO4 and the level of GFP expression when CuSO4 concentrations range from 0µM to 100µM. At concentrations of 100µM and higher the expression level of GFP will have reached a steady state and will remain unchanged despite increasing CuSO4 concentrations.</p><br />
<br />
<h3>Protocol</h3><br />
<p>A genomically integrated GFP gene under control of a CUP1 promoter was used to characterise the control properties of this promoter. This construct is referred to as CUP1p-[GFP] and was transformed into the yeast strain BY4741 for analysis.<br />
<br><br />
Three separate starter cultures were prepared of CUP1p-[GFP] in 5mL SD medium. The following tubes were then prepared in triplicate.</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/3/3a/Table1_for_Quantitative_determination_of_the_response_of_the_CUP1..._.png"/><br />
</center><br />
<p>Each one of these tubes was inoculated with N4 from each stater culture in order to provide triplicates of each concentratuion value. The cells were later harvested once the OD600 had reached 0.6. Triplicates from each culture were then loaded onto a 96 microtitre plate. The fluorescence of each sample was measured using a fluorometer running the "RussGFP" protocol.</p><br />
<h3>Results</h3><br />
<p>For full data spreadsheet<br><br />
IGEM 240610 JH+SL PCup Induction.xslx</p><br><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/20/Table2_for_Quantitative_determination_of_..._.png"/><br />
</center><br />
</p><br />
<h3>Conclusion</h3><br />
<p>The data suggests evidence of a dose dependent response. We can see from Figure 1 that the relationship between the GFP levels and the copper concentrations is linear when the concentrations of CuSO4 range from 0µM to 75µM. At concentrations higher than 75µM the response seems to reach a plateau and the GFP levels no longer increase along with increasing copper concentrations. Figure 2 shows us that these characteristics appear for each individual culture.</p><br />
<p>The data supports the hypothesis that the response of the copper promoter is dose dependent for a defined range of copper concentrations. The data however indicates that this range is smaller than our initial hypothesis suggested and that a plateau is reached with concentrations of 75μM as opposed to the initial 100μM.</p><br />
<h3>References</h3><br />
<p>[1]. Gorman JA, Clark PE, Lee MC, Debouck C and Rosenberg M<br><br />
Regulation of the yeast metallothionein gene<br><br />
Gene, 48 (1986 13-22)<br><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_the_CUP1_Promoter_Using_N4Team:Aberdeen Scotland/Timed Induction of the CUP1 Promoter Using N42010-10-26T11:56:08Z<p>Porter: </p>
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<h1>Timed Induction of GFP Expression by the CUP1p-[GFP]</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to determine how quickly the CUP1 promoter is fully induced by set concentrations of copper by measuring the levels of GFP being expressed by the yeast cells.This is done using the CUP1p-[GFP] construct.</p><br />
<h3>Hypothesis</h3><br />
<p>The CUP1 promoter will rapidly become fully induced following exposure to set concentrations of copper. Once it is fully induced the level of GFP expression will stabilise and will no longer increase despite more time passing.</p><br />
<h3>Protocol</h3><br />
<p>A genomically integrated GFP gene under control of a CUP1 promoter in the construct CUP1p-[GFP], was used to characterise the control properties of this promoter. This construct was transformed into the yeast strain BY4741 for analysis.<br><br><br />
Starter cultures of CUP1p-[GFP] in 5mL of SD medium + Raffinose were set up and incubated overnight. A 50mL flask containing SD Raff was then inoculated using the starter cultures and incubated overnight until the OD600 reached 0.3.<br><br><br />
At t=0 a sample from the flask was put on ice to provide a background reading of yeast’s natural fluorescence without any inducer. Copper was then added to the flask at a concentration of 100μM. Samples were then taken every 20 minutes and put on ice. All samples were then normalised to an OD600 of 0.75 and were washed and re-suspended in PBS before being analysed in the fluorometer (the programme RussGFP” was used for the analysis).</p><br />
<h3>Results</h3><br />
For full data spreadsheet<br><br />
iGEM 2.6.10 JH+SL N4 Timed Induction.xslx<br><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/PCUP1_response_graph.jpeg"/><br />
</center><br />
</p><br />
<h3>Conclusion</h3><br />
<p>Figure 1 indicates that full induction was reached after approximately 80 minutes and that the level of GFP expression seems to reach a plateau after this and no longer increases. This supports our initial hypothesis.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/Timed_Induction_of_the_CUP1_Promoter_Using_N4Team:Aberdeen Scotland/Timed Induction of the CUP1 Promoter Using N42010-10-26T11:55:45Z<p>Porter: </p>
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<h1>Timed Induction of GFP Expression by the CUP1p-[GFP]</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to determine how quickly the CUP1 promoter is fully induced by set concentrations of copper by measuring the levels of GFP being expressed by the yeast cells.This is done using the CUP1p-[GFP] construct.</p><br />
<h3>Hypothesis</h3><br />
<p>The CUP1 promoter will rapidly become fully induced following exposure to set concentrations of copper. Once it is fully induced the level of GFP expression will stabilise and will no longer increase despite more time passing.</p><br />
<h3>Protocol</h3><br />
<p>A genomically integrated GFP gene under control of a CUP1 promoter in the construct CUP1p-[GFP], was used to characterise the control properties of this promoter. This construct was transformed into the yeast strain BY4741 for analysis.<br><br><br />
Starter cultures of CUP1p-[GFP] in 5mL of SD medium + Raffinose were set up and incubated overnight. A 50mL flask containing SD Raff was then inoculated using the starter cultures and incubated overnight until the OD600 reached 0.3.<br><br><br />
At t=0 a sample from the flask was put on ice to provide a background reading of yeast’s natural fluorescence without any inducer. Copper was then added to the flask at a concentration of 100μM. Samples were then taken every 20 minutes and put on ice. All samples were then normalised to an OD600 of 0.75 and were washed and re-suspended in PBS before being analysed in the fluorometer (the programme RussGFP” was used for the analysis).</p><br />
<h3>Results</h3><br />
For full data spreadsheet<br><br />
iGEM 2.6.10 JH+SL N4 Timed Induction.xslx<br><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/a/ae/PCUP1_response_graph.jpeg"/><br />
</center><br />
</p><br />
<h3>Conclusion</h3><br />
<p>Figure 1 indicates that full induction was reached after approximately 80 minutes and that the level of GFP expression seems to reach a plateau after this and no longer increases. This supports our initial hypothesis.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/4._Replacing_the_CUP1_promoter_in_pRS414_with_the_CUP1-2_promoter_from_the_N4_construct4. Replacing the CUP1 promoter in pRS414 with the CUP1-2 promoter from the N4 construct2010-10-26T11:55:22Z<p>Porter: </p>
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<h1>Checking the Copper Promoter in CUP1p - [MS2-CFP] by Replacing it with the CUP1-2 Promoter from the CUP1p-[GFP]<br />
Construct</h1><br />
<h3>Aim</h3><br />
<p>When comparing the sequence of the promoter present in CUP1p - [MS2-CFP] to the sequence of the promoter in CUP1p-[GFP] we noticed that the CUP1p-[GFP] sequence contained 50 base pairs in its associated 5’UTR that were not present in the CUP1p - [MS2-CFP] 5’UTR sequence. We have shown that the CUP1p-[GFP] promoter works (Characterisation of Cup1 Promoter experiments). By replacing the promoter in CUP1p - [MS2-CFP] with the promoter and associated 5’UTR from CUP1p-[GFP] we can determine whether or not CUP1p - [MS2-CFP] had a defective or incomplete promoter which resulted in no expression of CFP.</p><br />
<h3>Hypothesis</h3><br />
<p>The Cup promoter present in CUP1p-[GFP] contains fifty base pairs in its associated 5’UTR that are not present in the CUP1p - [MS2-CFP] construct which are responsible for the pRS414 construct not expressing CFP properly.</p><br />
<h3>Protocol</h3><br />
<p>The CUP1p - [MS2-CFP] construct was digested using the restriction enzymes Bgl2 and Pst1 in order to remove the existing Cup1 promoter. The promoter present in CUP1p-[GFP] (Cup1-2) and the associated 5’UTR were then PCR amplified using primers designed to add complementary overhangs to the gapped CUP1p - [MS2-CFP] construct to allow homologous recombination.</p> <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/8/81/PRS414_construct.jpg"/><br />
</center><br />
<br><p>The gapped CUP1p - [MS2-CFP] vector and the PCR amplified CUP1-2 promoter were then co-transformed into yeast (BY4741ΔTrp strain) and incubated over several days. The resulting transformants were cultured in SD medium containing CuSO4 at concentrations high enough to reach full induction of the promoter. Final samples were washed and re-suspended in PBS and then analysed using a microscope fitted with CFP filters.<br><br />
<h3>Results</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/Absence_of_CFP_fluoresence.jpg"/><br />
</center><br />
<p>Microscope analysis revealed no fluorescence coming from the cell samples under the CFP excitation and filter settings. Controls containing pGAL/GFP showed clear fluorescence however only background fluorescence from the yeast cells was observed coming from the cells containing Cup1/CFP. 20 different samples were tested using different colonies form the transformation plate each time however all results came up negative. As we know that the promoter repaired into the CUP1p - [MS2-CFP] construct worked (see Characterisation of Cup1 Promoter experiments) we can conclude that the initial lack of CFP expression observed was not due to a faulty promoter but must stem from either the Bbox stem loop or the fusion of MS2 to CFP. <br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/3._N-GFP_Swap_Experiment3. N-GFP Swap Experiment2010-10-26T11:54:59Z<p>Porter: </p>
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<h1>Replacement of MS2-[CFP] in CUP1p - [MS2-CFP] with Npep-GFP from GAL1p-[Npep-GFP] to Determine Functionality of Promoter/5'leader/Binding Stem Loop Sequence</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to see if the CUP1p - [MS2-CFP] construct possesses a working promoter/5’leader/binding stem loop sequence by replacing the MS2-CFP part of the construct with the Npep-GFP sequence from GAL1p-[Npep-GFP]. We have shown that Npep-GFP is expressed by GAL1p-[Npep-GFP] therefore if we can express it in CUP1p - [MS2-CFP] we can determine the functionality of the promoter/5'leader/binding stem loop sequence.</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/7/77/Joint_diagram_of_pRS414%2BpRS415.jpg"/><br />
</center><br />
<h3>Hypothesis</h3><br />
<p>The reason why CUP1p - [MS2-CFP] is not expressing CFP lies within the promoter/5’leader/stem loop sequence.</p><br><br />
<h3>Protocol</h3><br />
<p>The CUP1p - [MS2-CFP] construct was digested using the restriction enzymes Cla1 and Nde1 in order to remove the MS2-CFP section of CUP1p - [MS2-CFP].<br><br><br />
In parallel, the Npeptide-GFP section of GAL1p-[Npep-GFP] was PCR amplified using primers designed to add 45 base pair long overhangs that would allow homologous recombination of the PCR product into the gapped CUP1p - [MS2-CFP]. The gapped CUP1p - [MS2-CFP] and the PCR product were co-transformed into yeast (BY4741ΔTrp strain) in order to allow the homologous recombination to take place. The resulting transformants were then screened using Colony PCR in order to make sure that the swap had taken place.<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/b8/N-GFP_swap_construct.jpg"/><br />
</center><br />
<p>The colonies where the swap had been successful were then cultured overnight in SD medium containing Raffinose and CuSO4 (100µM). Samples were then normalised and resuspended in PBS and loaded onto a 96 well microtitre plate before being analysed using the FACS machine.</p><br />
<br />
<br><br />
<h3>Results</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/c/cc/Fluor_graph_for_N-GFP_swap.jpg"/><br />
</center><br />
<p>From Fig 4. we can determine the level of background fluorescence of yeast cells using the results from the negative control “GAL1p-[Npep-GFP]”. We can also see the expected levels of GFP in a working construct from the positive control of “pRS415 + 2% Gal”.<br><br><br />
The two samples from colony 10, which are in presence of Cu2+ inducer, show readings that correspond with the levels of background fluorescence of yeast cells. This indicates that although the swap was successful the CUP1p - [MS2-CFP] construct is still not expressing properly.</p><br><br />
<br />
<h3>Conclusion</h3><br />
<p>We can conclude from these results that the problem resulting in CUP1p - [MS2-CFP] not being functional is related to either the Copper promoter or to the 5’UTR and Bbox stem loop. This experiment does not rule out however that the MS2-CFP part of the construct is also defective.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/2._Confirming_the_CFP_sequence_is_functional2. Confirming the CFP sequence is functional2010-10-26T11:54:23Z<p>Porter: </p>
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<br />
<h1>Confirming Functionality of CFP Sequence</h1><br />
<h3>Aim</h3><br />
<p>As part of the experiments to repair the CUP1p - [MS2-CFP] construct we need to determine if the CFP sequence used is functional or not. By confirming that the CFP protein is functional we can narrow down where the fault lies in the construct. If the CFP sequence was not functional this could mean that the construct was expressing properly but we simply could not detect this.</p><br />
<h3>Hypothesis</h3><br />
<p>The CFP sequence is accurate and the reason why no expression of CFP is being observed is because the protein is not being synthesised properly or not all.</p><br />
<h3>Protocol</h3><br />
<p>The TEF1p -[CFP] plasmid has been used to drive the expression of fluorescent proteins. The TEF1 promoter has been shown to constitutively express GFP at high levels.<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/1/1e/Diagram_of_YCP_lac_22_Fl.jpg"/><br />
</center><br />
<p>A restriction digest was performed using the enzymes Nde1 and Xba1 in order to remove GFP from the construct. The CFP sequence in CUP1p - [MS2-CFP] was then PCR amplified using primers to generate complementary overhangs to allow homologous recombination into the gaped TEF1p -[CFP] vector.<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/0/05/Construct_generated_to_test_CFP.jpg"/><br />
</center><br />
<p>The gapped vector and the PCR product were co-transformed into yeast (BY4741ΔTrp strain) and incubated for several days. The resulting transformants were then cultured overnight in SD medium. Sample were washed and re-suspended in PBS and then observed under a microscope fitted with CFP filters.</p><br />
<br><br />
<h3>Results</h3><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/f0/Evidence_of_CFP_fluo.jpg"/><br />
</center><br />
<p>We can see from Fig.3 that the CFP protein is being properly expressed. This means that the sequence used for CFP in the CUP1p - [MS2-CFP] construct is accurate and should be working. During this experiment the sequence of MS2-CFP was also checked to ensure that during the design both proteins had remained in frame.<br />
<br><br><br />
We can conclude from this experiment that the reason we are not seeing any CFP expression in pRS414 is because the CFP protein is not being produced properly.</p><br />
<br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP1. Confirmation using microscope and fluorometer analysis that the pRS414 construct was not expressing CFP2010-10-26T11:53:46Z<p>Porter: </p>
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{{:Team:Aberdeen_Scotland/Title}}<br />
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<br />
<h1>Confirmation Using Microscope and Fluorometer Analysis that CUP1p-[MS2-CFP] was Not Expressing CFP</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to determine whether the CUP1p-[MS2-CFP] construct can express CFP in the presence of CuSO4. The detection of CFP using the fluorometer and the microscope would confirm whether the construct as a whole was functional or not.</p><br />
<h3>Hypothesis</h3><br />
<p>The observed lack of CFP expression is due to experimental errors and using appropriate medium and proper inducing agent, the CUP1p-[MS2-CFP] construct will express CFP.</p><br />
<h3>Protocol</h3><br />
<p>The Cyan filters on the fluorometer were first checked to ensure that they could detect CFP. Pacific Blue beads (a fluorescent dye used by the FACS machine) has an excitation and emission profile similar to CFP and was therefore used as a control. A range of concentration of Pacific Blue (diluted in PBS and also in SD medium) was loaded onto a 96 well microtitre plate and analysed using the fluorometer. The resulting reading (Fig.1) indicated that the filters in place were able to detect CFP.<br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/d/d8/Table_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/><br />
</center><br />
<br><br />
<p>In order to check whether CUP1p-[MS2-CFP] was expressing CFP, cultures of BY4741 containing CUP1p-[MS2-CFP] were prepared using the old/previously used medium and inducer (concentration of 50 and 10µM copper were used) and also using freshly made medium and inducer stocks. The cultures were incubated at 30°C and then samples were analysed using the fluorometer and a microscope equipped with CFP filters.</p><br />
<h3>Results</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/5/57/Table2_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/><br />
</center><br />
<br><br />
<p>The fluorometer readings indicated a small increase in fluorescence in samples containing Cu2+. However, the readings were still very close to the readings obtained for the background fluorescence of the yeast cells.<br />
<br><br />
The microscope observations indicated that no CFP fluorescence was present in any of the samples prepared.</p><br />
<h3>Conclusion</h3><br />
<p>We conclude from these results that the lack of CFP expression is not due to experimental error and that the fault must lie with one or several of the components making up the CUP1p-[MS2-CFP] construct.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Experimental_LayoutExperimental Layout2010-10-26T11:53:30Z<p>Porter: </p>
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{{:Team:Aberdeen_Scotland/Title}}<br />
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<h1>Troubleshooting the CUP1p-[MS2-CFP] construct</h1><br />
<h3>Aim</h3><br />
<p>Following the analysis of the two promoters using CUP1p-GFP and GAL1p-GFP the next experiments planned would start to characterise the interactions of both constructs (CUP1p-[MS2-CFP] and GAL1p-[Npep-GFP]) and more specifically the mutual repression that would be taking place. Experiments using FACS analysis were set up in order to measure various levels of CFP and GFP depending on different conditions (i.e. different ranges of inducers, different ratios of pRS415 to pRS414 etc.). However the FACS machine was unable to detect any GFP or CFP in our calibration samples.</p><br />
<h3>Hypothesis</h3><br />
<p>Our initial hypothesis was that the lack of GFP and CFP expression was due to experimental error during the set up of the cultures (this proved to be correct as far as pRS415 was concerned where the lack of expression was traced back to a faulty stock of inducing agent).<br><br><br />
The second hypothesis put forward to explain the lack of CFP expression was that one of the parts that made up pRS414 was defective.<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/2c/Diagram_of_pRS414.jpg"/><br />
</center><br />
</p><br />
<h3>Protocol</h3><br />
<p>A series of experiments were set up in order to determine the functionality of various parts of CUP1p-[MS2-CFP] with a view to repair the construct.<br><br><br />
<br />
<a href="https://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP">1. Confirmation using microscope and fluorometer analysis that the CUP1p-[MS2-CFP] construct was not expressing CFP</a><br><br />
<br />
<a href="https://2010.igem.org/2._Confirming_the_CFP_sequence_is_functional"> 2. Confirming Functionality of CFP Sequence </a><br><br />
<br />
<a href="https://2010.igem.org/3._N-GFP_Swap_Experiment"> 3. Replacement of MS2-[CFP] in CUP1p - [MS2-CFP] with Npep-GFP from GAL1p-[Npep-GFP] to Determine Functionality of Promoter/5'leader/Binding Stem Loop Sequence </a><br><br />
<br />
<a href="https://2010.igem.org/4._Replacing_the_CUP1_promoter_in_pRS414_with_the_CUP1-2_promoter_from_the_N4_construct">4. Replacing the CUP1 promoter in CUP1p-[MS2-CFP] with the CUP1-2 promoter from the CUP1p-GFP construct</a><br><br />
<br />
<br><br />
<h3>Conclusion</h3><br />
<p>The first experiment did not detect any CFP fluorescent whilst the second experiment indicated that the CFP sequence used was accurate and in another vector resulted in fluorescence. The third experiment revealed that a fault lies with either the CUP1 promoter or/and the Bbox stem loop however the fourth experiment showed that the promoter did not seem to be contributing to the lack of expression.<br><br><br />
Our data suggests that the reason why CUP1p-[MS2-CFP] is not producing any CFP is due to the presence of the Bbox Stem loop in the 5’UTR. Although Bbox stem loops have already be used in conjunction with the λ-N peptide as markers for mRNA, it is possible that it has never been placed in 5’UTR but is rather located to a 3’UTR. As a result we cannot be sure that the loop can be melted and could therefore be preventing the expression of the following proteins.<br><br><br />
Our experiments however, do not rule out the possibility that the fusion of MS2 to CFP is defective and is resulting in an unstable protein that is being turned-over rapidly. It is also possible that the fusion is preventing the CFP portion from fluorescencing due to improper folding.<br />
</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/FACS_Analysis_of_mOrange_recombinant_pRS415FACS Analysis of mOrange recombinant pRS4152010-10-26T11:51:51Z<p>Porter: </p>
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{{:Team:Aberdeen_Scotland/Title}}<br />
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<h1>Testing Biobrick E2050 Part 2 - FACS Analysis of mOrange recombinant GAL1p-[Npep-GFP]<br />
</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment was to test if mOrange inserted into GAL1p-[Npep-GFP], (GAL1p-[Npep-mOran]) in place of green fluorescent protein, (GFP) will fluoresce when galactose is added.</p><br />
<h3>Hypothesis</h3><br />
<p>Since GFP has been detected when GAL1p-[Npep-GFP] is induced with galactose, in-frame insertion of mOrange DNA sequence in place of GFP in GAL1p-[Npep-GFP] should produce orange fluorescence when galactose is added. This will allow confirmation of whether the Bio-brick E2050 will work.<br><br />
<br />
</p><br />
<h3>Protocol</h3><br />
<p>Starter cultures of BY4741ΔTrp, BY4741 GAL1p-[Npep-GFP] and BY4741 GAL1p-[Npep-mOran] were incubated overnight in SD medium and the OD600 was measured. These were then used to set-up overnight experimental cultures to have an OD600=0.6 for the following day. The following were set-up as shown by (Table 1).</p> <br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/2c/MOra_test_2_table_1.jpg"/><br />
</center><br />
<br />
<p> The OD600 was measured the following day and samples were normalised by spinning down in a centrifuge and washing (x2) with PBS buffer to an OD600=0.6. These were then used for the FACs analysis. The filters used were FITC and PE. These were chosen as they were the best available filters for measuring GFP and mOrange respectively as can be seen from the following data obtained from the BD Fluoresence Spectrum Viewer (Fig.1).</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/69/BD_Viewer.jpg"/><br />
</center><br />
<br />
<br><br />
<br><html><br />
<h3>Results</h3><br />
<p>From the FACS analysis, it was shown that sample 4 had 5.75% of the cell population analysed fluorescing whilst sample 6 had 16.6% of the cell population analysed fluorescing. As a result, the analysis focussed on sample 6 and its corresponding negative, sample 7.</p><br><br />
<br />
<p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p><br><br />
<br />
<p>The results are shown in Fig.2(i) and it can be seen that the untransfected and negative control both have a single peak which corresponds to autofluorescence from yeast with no GFP expression. For the positive control (GAL1p-[Npep-GFP] BY4741 + Galactose), there is a slight first peak followed by a taller second peak which suggests that a certain percentage of the analysed population (23.3%) were not fluorescing. It is likely that this could be because they are damaged yeast cells, did not carry the desired plasmid GAL1p-[Npep-GFP] or that the Gal promoter was not induced for unknown reasons. However, the larger second peak (76.7%) showed that there is a large proportion of the population that are able to express GFP as desired.</p><br> <br />
<br />
<p>An analysis for GAL1p-[Npep-mOran] sample 6 and 7 using FITC to show that there was no GFP fluorescence was also carried out as shown in Fig.2(ii). However, a small peak was observed for sample 6, which was unexpected. This may have been because the FITC filters overlap the emission spectra of mOrange as shown previously, (BD Spectrum Viewer). Further analysis using the PE-A filter specific for mOrange however show that there is a less well defined and smaller peak for sample 6 than would be expected if there was mOrange fluorescence. This is shown in Fig.3(ii), which suggests that the fluorescence detected is GFP, which was not expected.</p><br><br />
<br />
<p>In a previous experiment, PCR screening was used to establish that the transformed colonies were positive for GAL1p-[Npep-mOran], which makes the chances of a BY4741 transformed with uncut and non-homologously recombined GAL1p-[Npep-GFP] with mOrange insert unlikely.</p><br><br />
<br />
<p>Despite this, the observation of a small peak for both FITC and PE-A with the features as discussed above suggests that there are some yeast cells within the GAL1p-[Npep-mOran] BY4741 samples expressing GFP rather than mOrange. Which implies that the homologous recombination was not 100% efficient.</p> <br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/61/MOr_FACS.jpg"/><br />
</center><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/MOr_FACS_2.jpg"/><br />
</center><br />
<br />
<p>Conversely, 83.4% of the analysed cell population did not show any fluorescence, which suggests that either mOrange was not successfully homologously recombined or that it could not be expressed by BY4741. If uncut GAL1p-[Npep-GFP] had been transformed in place of GAL1p-[Npep-mOran] it is likely that a greater GFP fluorescence would have been detected, which was not the case. The cut GAL1p-[Npep-GFP] does not have any complementary ends that could re-ligate. Therefore it is unlikely that these are transformed and selected.</p><br><br />
<br />
<p>Ideally, a positive control for detecting mOrange would allow verification of the specificity of PE-A detection of mOrange and a DNA sequence of transformant of GAL1p-[Npep-mOran] would confirm whether or not the yeast carried the appropriate recombined plasmids.</p><br> <br />
<br />
</html><br />
<br><html><br />
<h3>Conclusions</h3><br />
<p>From the discussion of the results, the absence of any mOrange fluorescence from the FACS data suggests that the Bio-brick E2050 did not work. However, unexpected GFP fluorescence in GAL1p-[Npep-mOran] BY4741 was detected, which suggests that the homologous recombination of mOrange for GFP in GAL1p-[Npep-GFP] was not 100% efficient and further experimentation is required to confirm this result. <br />
</p><br><br />
<h3>References</h3><br />
<p><br />
<sup style="font-size:10px">[1]</sup><a href="http://www.bdbiosciences.com/external_files/media/spectrumviewer/index.jsp">Click here to visit the BD Website</a></p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/FACS_Analysis_of_mOrange_recombinant_pRS415FACS Analysis of mOrange recombinant pRS4152010-10-26T11:51:18Z<p>Porter: </p>
<hr />
<div>{{:Team:Aberdeen_Scotland/css}}<br />
{{:Team:Aberdeen_Scotland/Title}}<br />
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<h1>Testing Biobrick E2050 Part 2 - FACS Analysis of mOrange recombinant GAL1p-[Npep-GFP]<br />
</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment was to test if mOrange inserted into GAL1p-[Npep-GFP], (GAL1p-[Npep-mOran]) in place of green fluorescent protein, (GFP) will fluoresce when galactose is added.</p><br />
<h3>Hypothesis</h3><br />
<p>Since GFP has been detected when GAL1p-[Npep-GFP] is induced with galactose, in-frame insertion of mOrange DNA sequence in place of GFP in GAL1p-[Npep-GFP] should produce orange fluorescence when galactose is added. This will allow confirmation of whether the Bio-brick E2050 will work.<br><br />
<br />
</p><br />
<h3>Protocol</h3><br />
<p>Starter cultures of BY4741ΔTrp, BY4741 GAL1p-[Npep-GFP] and BY4741 GAL1p-[Npep-mOran] were incubated overnight in SD medium and the OD600 was measured. These were then used to set-up overnight experimental cultures to have an OD600=0.6 for the following day. The following were set-up as shown by (Table 1).</p> <br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/2c/MOra_test_2_table_1.jpg"/><br />
</center><br />
<br />
<p> The OD600 was measured the following day and samples were normalised by spinning down in a centrifuge and washing (x2) with PBS buffer to an OD600=0.6. These were then used for the FACs analysis. The filters used were FITC and PE. These were chosen as they were the best available filters for measuring GFP and mOrange respectively as can be seen from the following data obtained from the BD Fluoresence Spectrum Viewer (Fig.1).</p><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/69/BD_Viewer.jpg"/><br />
</center><br />
<br />
<br><br />
<br><html><br />
<h3>Results</h3><br />
<p>From the FACS analysis, it was shown that sample 4 had 5.75% of the cell population analysed fluorescing whilst sample 6 had 16.6% of the cell population analysed fluorescing. As a result, the analysis focussed on sample 6 and its corresponding negative, sample 7.</p><br><br />
<br />
<p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p><br><br />
<br />
<p>The results are shown in Fig.2(i) and it can be seen that the untransfected and negative control both have a single peak which corresponds to autofluorescence from yeast with no GFP expression. For the positive control (GAL1p-[Npep-GFP] BY4741 + Galactose), there is a slight first peak followed by a taller second peak which suggests that a certain percentage of the analysed population (23.3%) were not fluorescing. It is likely that this could be because they are damaged yeast cells, did not carry the desired plasmid GAL1p-[Npep-GFP] or that the Gal promoter was not induced for unknown reasons. However, the larger second peak (76.7%) showed that there is a large proportion of the population that are able to express GFP as desired.</p><br> <br />
<br />
<p>An analysis for GAL1p-[Npep-mOran] sample 6 and 7 using FITC to show that there was no GFP fluorescence was also carried out as shown in Fig.2(ii). However, a small peak was observed for sample 6, which was unexpected. This may have been because the FITC filters overlap the emission spectra of mOrange as shown previously, (BD Spectrum Viewer). Further analysis using the PE-A filter specific for mOrange however show that there is a less well defined and smaller peak for sample 6 than would be expected if there was mOrange fluorescence. This is shown in Fig.3(ii), which suggests that the fluorescence detected is GFP, which was not expected.</p><br><br />
<br />
<p>In a previous experiment, PCR screening was used to establish that the transformed colonies were positive for GAL1p-[Npep-mOran], which makes the chances of a BY4741 transformed with uncut and non-homologously recombined GAL1p-[Npep-GFP] with mOrange insert unlikely.</p><br><br />
<br />
<p>Despite this, the observation of a small peak for both FITC and PE-A with the features as discussed above suggests that there are some yeast cells within the GAL1p-[Npep-mOran] BY4741 samples expressing GFP rather than mOrange. Which implies that the homologous recombination was not 100% efficient.</p> <br />
<br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/61/MOr_FACS.jpg"/><br />
</center><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/MOr_FACS_2.jpg"/><br />
</center><br />
<br />
<p>Conversely, 83.4% of the analysed cell population did not show any fluorescence, which suggests that either mOrange was not successfully homologously recombined or that it could not be expressed by BY4741. If uncut GAL1p-[Npep-GFP] had been transformed in place of GAL1p-[Npep-mOran] it is likely that a greater GFP fluorescence would have been detected, which was not the case. The cut GAL1p-[Npep-GFP] does not have any complementary ends that could re-ligate. Therefore it is unlikely that these are transformed and selected.</p><br><br />
<br />
<p>Ideally, a positive control for detecting mOrange would allow verification of the specificity of PE-A detection of mOrange and a DNA sequence of transformant of GAL1p-[Npep-mOran] would confirm whether or not the yeast carried the appropriate recombined plasmids.</p><br> <br />
<br />
</html><br />
<br><html><br />
<h3>Conclusions</h3><br />
<p>From the discussion of the results, the absence of any mOrange fluorescence from the FACS data suggests that the Bio-brick E2050 did not work. However, unexpected GFP fluorescence in GAL1p-[Npep-mOran] BY4741 was detected, which suggests that the homologous recombination of mOrange for GFP in GAL1p-[Npep-GFP] was not 100% efficient and further experimentation is required to confirm this result. <br />
</p><br><br />
<h3>References<br />
<p><br />
<sup style="font-size:10px">[1]</sup><a href="http://www.bdbiosciences.com/external_files/media/spectrumviewer/index.jsp">Click here to visit the BD Website</a></p><br />
<br><br><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/FACS_Analysis_of_mOrange_recombinant_pRS415FACS Analysis of mOrange recombinant pRS4152010-10-26T11:49:01Z<p>Porter: </p>
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<h1>Testing Biobrick E2050 Part 2 - FACS Analysis of mOrange recombinant GAL1p-[Npep-GFP]<br />
</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment was to test if mOrange inserted into GAL1p-[Npep-GFP], (GAL1p-[Npep-mOran]) in place of green fluorescent protein, (GFP) will fluoresce when galactose is added.</p><br />
<h3>Hypothesis</h3><br />
<p>Since GFP has been detected when GAL1p-[Npep-GFP] is induced with galactose, in-frame insertion of mOrange DNA sequence in place of GFP in GAL1p-[Npep-GFP] should produce orange fluorescence when galactose is added. This will allow confirmation of whether the Bio-brick E2050 will work.<br><br />
<br />
</p><br />
<h3>Protocol</h3><br />
<p>Starter cultures of BY4741ΔTrp, BY4741 GAL1p-[Npep-GFP] and BY4741 GAL1p-[Npep-mOran] were incubated overnight in SD medium and the OD600 was measured. These were then used to set-up overnight experimental cultures to have an OD600=0.6 for the following day. The following were set-up as shown by (Table 1).</p> <br />
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<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/2c/MOra_test_2_table_1.jpg"/><br />
</center><br />
<br />
<p> The OD600 was measured the following day and samples were normalised by spinning down in a centrifuge and washing (x2) with PBS buffer to an OD600=0.6. These were then used for the FACs analysis. The filters used were FITC and PE. These were chosen as they were the best available filters for measuring GFP and mOrange respectively as can be seen from the following data obtained from the BD Fluoresence Spectrum Viewer (Fig.1).</p><br />
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<center><br />
<img src="https://static.igem.org/mediawiki/2010/6/69/BD_Viewer.jpg"/><br />
</center><br />
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<h3>Results</h3><br />
<p>From the FACS analysis, it was shown that sample 4 had 5.75% of the cell population analysed fluorescing whilst sample 6 had 16.6% of the cell population analysed fluorescing. As a result, the analysis focussed on sample 6 and its corresponding negative, sample 7.</p><br><br />
<br />
<p>To check the background fluorescence of BY4741ΔTrp, untransfected cells were analysed. To show that GFP could be detected, positive and negative controls of GAL1p-[Npep-GFP] BY4741 which should fluoresce green if galactose is added were also analysed. This analysis was carried out using FITC filters which are specific for GFP.</p><br><br />
<br />
<p>The results are shown in Fig.2(i) and it can be seen that the untransfected and negative control both have a single peak which corresponds to autofluorescence from yeast with no GFP expression. For the positive control (GAL1p-[Npep-GFP] BY4741 + Galactose), there is a slight first peak followed by a taller second peak which suggests that a certain percentage of the analysed population (23.3%) were not fluorescing. It is likely that this could be because they are damaged yeast cells, did not carry the desired plasmid GAL1p-[Npep-GFP] or that the Gal promoter was not induced for unknown reasons. However, the larger second peak (76.7%) showed that there is a large proportion of the population that are able to express GFP as desired.</p><br> <br />
<br />
<p>An analysis for GAL1p-[Npep-mOran] sample 6 and 7 using FITC to show that there was no GFP fluorescence was also carried out as shown in Fig.2(ii). However, a small peak was observed for sample 6, which was unexpected. This may have been because the FITC filters overlap the emission spectra of mOrange as shown previously, (BD Spectrum Viewer). Further analysis using the PE-A filter specific for mOrange however show that there is a less well defined and smaller peak for sample 6 than would be expected if there was mOrange fluorescence. This is shown in Fig.3(ii), which suggests that the fluorescence detected is GFP, which was not expected.</p><br><br />
<br />
<p>In a previous experiment, PCR screening was used to establish that the transformed colonies were positive for GAL1p-[Npep-mOran], which makes the chances of a BY4741 transformed with uncut and non-homologously recombined GAL1p-[Npep-GFP] with mOrange insert unlikely.</p><br><br />
<br />
<p>Despite this, the observation of a small peak for both FITC and PE-A with the features as discussed above suggests that there are some yeast cells within the GAL1p-[Npep-mOran] BY4741 samples expressing GFP rather than mOrange. Which implies that the homologous recombination was not 100% efficient.</p> <br />
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<img src="https://static.igem.org/mediawiki/2010/6/61/MOr_FACS.jpg"/><br />
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<center><br />
<img src="https://static.igem.org/mediawiki/2010/c/c3/MOr_FACS_2.jpg"/><br />
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<p>Conversely, 83.4% of the analysed cell population did not show any fluorescence, which suggests that either mOrange was not successfully homologously recombined or that it could not be expressed by BY4741. If uncut GAL1p-[Npep-GFP] had been transformed in place of GAL1p-[Npep-mOran] it is likely that a greater GFP fluorescence would have been detected, which was not the case. The cut GAL1p-[Npep-GFP] does not have any complementary ends that could re-ligate. Therefore it is unlikely that these are transformed and selected.</p><br><br />
<br />
<p>Ideally, a positive control for detecting mOrange would allow verification of the specificity of PE-A detection of mOrange and a DNA sequence of transformant of GAL1p-[Npep-mOran] would confirm whether or not the yeast carried the appropriate recombined plasmids.</p><br> <br />
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<h3>Conclusions</h3><br />
<p>From the discussion of the results, the absence of any mOrange fluorescence from the FACS data suggests that the Bio-brick E2050 did not work. However, unexpected GFP fluorescence in GAL1p-[Npep-mOran] BY4741 was detected, which suggests that the homologous recombination of mOrange for GFP in GAL1p-[Npep-GFP] was not 100% efficient and further experimentation is required to confirm this result. <br />
</p><br />
<p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Homologous_Recombination_of_E2050_into_pRS415_Construct_in_Place_of_GFP_ProteinHomologous Recombination of E2050 into pRS415 Construct in Place of GFP Protein2010-10-26T11:41:03Z<p>Porter: </p>
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<h1>Testing Bio-brick E2050 Part 1 - Homologous Recombination of E2050 into GAL1p-[Npep-GFP] Construct in Place of GFP Protein</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment was to homologously recombine the DNA sequence of Bio-brick E2050 mOrange (E2050), in place of green fluorescent protein (GFP), in GAL1p-[Npep-GFP] by transforming with yeast BY4741ΔTrp. This allowed testing of E2050 in further experiments.</p><br />
<h3>Hypothesis</h3><br />
<p>Since GFP has been detected when GAL1p-[Npep-GFP] is induced with galactose, if the E2050 is inserted in place of GFP in GAL1p-[Npep-GFP], then orange fluorescence should be detected under similar conditions for GFP expression. Homologous recombination carried out by yeast is one technique that can be used to conveniently replace the GFP sequence with E2050 in GAL1p-[Npep-GFP]. <br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/b1/MOr_GFP_swap.jpg"/><br />
</center><br />
</p><br />
<h3>Protocol</h3><br />
<p>The Bio-brick E2050 was rescued from the DNA Distribution Kit provided by iGEM by transforming into sub-cloning DH5α E-coli strain. The plasmids were then extracted using a Qiagen Mini-Prep kit following manufacturer’s protocol. Testing for correct plasmid rescue was done by cutting extracted DNA with restriction enzymes XbaI and SpeI which are unique cut sites required to remove the DNA sequence that codes for mOrange from the bio-brick plasmid pSB2K3. Verification of correct sequence was by checking the length of insert and vector by gel electrophoresis of restriction digest. The expected fragment lengths are 744bp and 4425bp respectively.</p> <br><br />
<br />
<p>To homologously recombine the E2050 mOrange insert into GAL1p-[Npep-GFP] in place of GFP, the mOrange inserts were amplified by PCR using primers designed with 45bp overhangs that were homologous to the region of plasmid immediately prior and after the GFP sequence in GAL1p-[Npep-GFP]. The sequence to be amplified was checked to ensure that mOrange would be translated in-frame when integrated into GAL1p-[Npep-GFP].</p> <br><br />
<br />
<p>In parallel, GAL1p-[Npep-GFP] constructs were restriction cut at unique sites using NheI and SmaI to remove the GFP coding sequence. Both digests were checked by gel electrophoresis to ensure the correct length of PCR product and cut vector was obtained (739bp and 965bp respectively).</p> <br><br />
<br />
<p>For the homologous recombination, this was done by transforming yeast BY4741 auxotrophic for methionine, leucine, histidine, uracil and tryptophan with mOrange PCR products and cut GAL1p-[Npep-GFP] vector from previously. These would be homologously recombined by the yeast. The resultant transformants were selected by growing on SD agar with 1% each of methionine, histidine, uracil and tryptophan added since GAL1p-[Npep-GFP] has a selection marker for leucine encoded which is left intact throughout the experiment.</p> <br><br />
<br />
<p>To confirm that homologously recombined plasmids were selectively cultured, PCR colony screening was carried out on selected colonies from transformation plates.</p> <br><br />
<br><br />
<br><html><br />
<h3>Results</h3><br />
<p>From (Table 1.) the results of the selective culture of transformants show that the positive and negative controls worked as expected with BY4741 growing when all nutrients are present and not growing in the absence of essential amino acids. The expected transformants also grew on selection plates 1 & 2 at the three volumes plated (50µl, 100 µl and 200 µl) with increasing density of colonies.</P> <br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/7/7c/MOra_test_table_1.jpg"/><br />
</center><br />
<br />
<br />
<p>Since a transformation with ‘cut GAL1p-[Npep-GFP] only’ was not carried out, no information about the likelihood of uncut GAL1p-[Npep-GFP] from the restriction digest being transformed was obtained. To overcome this, individual colonies were selected and plated out for culture. PCR colony screening was then used to check that these had the mOrange insert transformed. Twenty colonies were selected, cultured and two colonies were PCR screened using primers for mOrange. (i.e. The primers used previously to generate mOrange with 45bp overhangs) The results are shown by (Table 2.) and confirm that homologous recombination of mOrange PCR products had been successfully integrated into cut GAL1p-[Npep-GFP]. <br />
</p><br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/1/12/MOra_test_table_2.jpg"/><br />
</center><br />
<br />
</html><br />
<br><html><br />
<h3>Conclusions</h3><br />
<p>From the discussion of the results it has been shown that E2050 mOrange has been successfully rescued and amplified. These were then used as substrate for PCR amplification to generate product with homologous ends that were homologously recombined with cut GAL1p-[Npep-GFP], (GFP sequence removed) in a transformation process using BY4741ΔTrp. PCR colony screening using mOrange primers confirmed that colonies carrying the recombinant GAL1p-[Npep-GFP] had been successfully transformed into BY4741. <br />
</p><br><br />
<h3>References</h3><br />
<p>Hinnen A, Hicks JB and Fink GR. Transformation of yeast.<br />
Proc Natl Acad Sci, 75 1978 (1929-33)</p><br />
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<a href="https://2010.igem.org/FACS_Analysis_of_mOrange_recombinant_pRS415">Continue to Biobrick E2050 Test Part 2&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2010/3/36/Right_arrow.png"></a><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Homologous_Recombination_of_E2050_into_pRS415_Construct_in_Place_of_GFP_ProteinHomologous Recombination of E2050 into pRS415 Construct in Place of GFP Protein2010-10-26T11:40:11Z<p>Porter: </p>
<hr />
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{{:Team:Aberdeen_Scotland/Title}}<br />
<html><br />
<h1>Testing Bio-brick E2050 Part 1 - Homologous Recombination of E2050 into GAL1p-[Npep-GFP] Construct in Place of GFP Protein</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment was to homologously recombine the DNA sequence of Bio-brick E2050 mOrange (E2050), in place of green fluorescent protein (GFP), in GAL1p-[Npep-GFP] by transforming with yeast BY4741ΔTrp. This allowed testing of E2050 in further experiments.</p><br />
<h3>Hypothesis</h3><br />
<p>Since GFP has been detected when GAL1p-[Npep-GFP] is induced with galactose, if the E2050 is inserted in place of GFP in GAL1p-[Npep-GFP], then orange fluorescence should be detected under similar conditions for GFP expression. Homologous recombination carried out by yeast is one technique that can be used to conveniently replace the GFP sequence with E2050 in GAL1p-[Npep-GFP]. <br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/b1/MOr_GFP_swap.jpg"/><br />
</center><br />
</p><br />
<h3>Protocol</h3><br />
<p>The Bio-brick E2050 was rescued from the DNA Distribution Kit provided by iGEM by transforming into sub-cloning DH5α E-coli strain. The plasmids were then extracted using a Qiagen Mini-Prep kit following manufacturer’s protocol. Testing for correct plasmid rescue was done by cutting extracted DNA with restriction enzymes XbaI and SpeI which are unique cut sites required to remove the DNA sequence that codes for mOrange from the bio-brick plasmid pSB2K3. Verification of correct sequence was by checking the length of insert and vector by gel electrophoresis of restriction digest. The expected fragment lengths are 744bp and 4425bp respectively.</p> <br><br />
<br />
<p>To homologously recombine the E2050 mOrange insert into GAL1p-[Npep-GFP] in place of GFP, the mOrange inserts were amplified by PCR using primers designed with 45bp overhangs that were homologous to the region of plasmid immediately prior and after the GFP sequence in GAL1p-[Npep-GFP]. The sequence to be amplified was checked to ensure that mOrange would be translated in-frame when integrated into GAL1p-[Npep-GFP].</p> <br><br />
<br />
<p>In parallel, GAL1p-[Npep-GFP] constructs were restriction cut at unique sites using NheI and SmaI to remove the GFP coding sequence. Both digests were checked by gel electrophoresis to ensure the correct length of PCR product and cut vector was obtained (739bp and 965bp respectively).</p> <br><br />
<br />
<p>For the homologous recombination, this was done by transforming yeast BY4741 auxotrophic for methionine, leucine, histidine, uracil and tryptophan with mOrange PCR products and cut GAL1p-[Npep-GFP] vector from previously. These would be homologously recombined by the yeast. The resultant transformants were selected by growing on SD agar with 1% each of methionine, histidine, uracil and tryptophan added since GAL1p-[Npep-GFP] has a selection marker for leucine encoded which is left intact throughout the experiment.</p> <br><br />
<br />
<p>To confirm that homologously recombined plasmids were selectively cultured, PCR colony screening was carried out on selected colonies from transformation plates.</p> <br><br />
<br><br />
<br><html><br />
<h3>Results</h3><br />
<p>From (Table 1.) the results of the selective culture of transformants show that the positive and negative controls worked as expected with BY4741 growing when all nutrients are present and not growing in the absence of essential amino acids. The expected transformants also grew on selection plates 1 & 2 at the three volumes plated (50µl, 100 µl and 200 µl) with increasing density of colonies.</P> <br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/7/7c/MOra_test_table_1.jpg"/><br />
</center><br />
<br />
<br />
<p>Since a transformation with ‘cut GAL1p-[Npep-GFP] only’ was not carried out, no information about the likelihood of uncut GAL1p-[Npep-GFP] from the restriction digest being transformed was obtained. To overcome this, individual colonies were selected and plated out for culture. PCR colony screening was then used to check that these had the mOrange insert transformed. Twenty colonies were selected, cultured and two colonies were PCR screened using primers for mOrange. (i.e. The primers used previously to generate mOrange with 45bp overhangs) The results are shown by (Table 2.) and confirm that homologous recombination of mOrange PCR products had been successfully integrated into cut GAL1p-[Npep-GFP]. <br />
</p><br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/1/12/MOra_test_table_2.jpg"/><br />
</center><br />
<br />
</html><br />
<br><html><br />
<h3>Conclusions</h3><br />
<p>From the discussion of the results it has been shown that E2050 mOrange has been successfully rescued and amplified. These were then used as substrate for PCR amplification to generate product with homologous ends that were homologously recombined with cut GAL1p-[Npep-GFP], (GFP sequence removed) in a transformation process using BY4741ΔTrp. PCR colony screening using mOrange primers confirmed that colonies carrying the recombinant GAL1p-[Npep-GFP] had been successfully transformed into BY4741. <br />
</p><br><br />
<h3>References</h3><br />
<p>Hinnen A, Hicks JB and Fink GR. Transformation of yeast.<br />
Proc Natl Acad Sci, 75 1978 (1929-33)</p><br />
<br />
</html><br />
<br />
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<a href="https://2010.igem.org/FACS_Analysis_of_mOrange_recombinant_pRS415">Continue to Biobrick E2050 Test Part 2&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2010/3/36/Right_arrow.png"></a><br />
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</html></div>Porterhttp://2010.igem.org/4._Replacing_the_CUP1_promoter_in_pRS414_with_the_CUP1-2_promoter_from_the_N4_construct4. Replacing the CUP1 promoter in pRS414 with the CUP1-2 promoter from the N4 construct2010-10-26T11:36:31Z<p>Porter: </p>
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<br />
<h1>Checking the Copper Promoter in CUP1p - [MS2-CFP] by Replacing it with the CUP1-2 Promoter from the CUP1p-[GFP]<br />
Construct</h1><br />
<h3>Aim</h3><br />
<p>When comparing the sequence of the promoter present in CUP1p - [MS2-CFP] to the sequence of the promoter in CUP1p-[GFP] we noticed that the CUP1p-[GFP] sequence contained 50 base pairs in its associated 5’UTR that were not present in the CUP1p - [MS2-CFP] 5’UTR sequence. We have shown that the CUP1p-[GFP] promoter works (Characterisation of Cup1 Promoter experiments). By replacing the promoter in CUP1p - [MS2-CFP] with the promoter and associated 5’UTR from CUP1p-[GFP] we can determine whether or not CUP1p - [MS2-CFP] had a defective or incomplete promoter which resulted in no expression of CFP.</p><br />
<h3>Hypothesis</h3><br />
<p>The Cup promoter present in CUP1p-[GFP] contains fifty base pairs in its associated 5’UTR that are not present in the CUP1p - [MS2-CFP] construct which are responsible for the pRS414 construct not expressing CFP properly.</p><br />
<h3>Protocol</h3><br />
<p>The CUP1p - [MS2-CFP] construct was digested using the restriction enzymes Bgl2 and Pst1 in order to remove the existing Cup1 promoter. The promoter present in CUP1p-[GFP] (Cup1-2) and the associated 5’UTR were then PCR amplified using primers designed to add complementary overhangs to the gapped CUP1p - [MS2-CFP] construct to allow homologous recombination.</p> <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/8/81/PRS414_construct.jpg"/><br />
</center><br />
<br><p>The gapped CUP1p - [MS2-CFP] vector and the PCR amplified CUP1-2 promoter were then co-transformed into yeast (BY4741ΔTrp strain) and incubated over several days. The resulting transformants were cultured in SD medium containing CuSO4 at concentrations high enough to reach full induction of the promoter. Final samples were washed and re-suspended in PBS and then analysed using a microscope fitted with CFP filters.<br><br />
<h3>Results</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/0/0a/Absence_of_CFP_fluoresence.jpg"/><br />
</center><br />
<p>Microscope analysis revealed no fluorescence coming from the cell samples under the CFP excitation and filter settings. Controls containing pGAL/GFP showed clear fluorescence however only background fluorescence from the yeast cells was observed coming from the cells containing Cup1/CFP. 20 different samples were tested using different colonies form the transformation plate each time however all results came up negative. As we know that the promoter repaired into the CUP1p - [MS2-CFP] construct worked (see Characterisation of Cup1 Promoter experiments) we can conclude that the initial lack of CFP expression observed was not due to a faulty promoter but must stem from either the Bbox stem loop or the fusion of MS2 to CFP. <br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/2._Confirming_the_CFP_sequence_is_functional2. Confirming the CFP sequence is functional2010-10-26T11:36:11Z<p>Porter: </p>
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<h1>Confirming Functionality of CFP Sequence</h1><br />
<h3>Aim</h3><br />
<p>As part of the experiments to repair the CUP1p - [MS2-CFP] construct we need to determine if the CFP sequence used is functional or not. By confirming that the CFP protein is functional we can narrow down where the fault lies in the construct. If the CFP sequence was not functional this could mean that the construct was expressing properly but we simply could not detect this.</p><br />
<h3>Hypothesis</h3><br />
<p>The CFP sequence is accurate and the reason why no expression of CFP is being observed is because the protein is not being synthesised properly or not all.</p><br />
<h3>Protocol</h3><br />
<p>The TEF1p -[CFP] plasmid has been used to drive the expression of fluorescent proteins. The TEF1 promoter has been shown to constitutively express GFP at high levels.<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/1/1e/Diagram_of_YCP_lac_22_Fl.jpg"/><br />
</center><br />
<p>A restriction digest was performed using the enzymes Nde1 and Xba1 in order to remove GFP from the construct. The CFP sequence in CUP1p - [MS2-CFP] was then PCR amplified using primers to generate complementary overhangs to allow homologous recombination into the gaped TEF1p -[CFP] vector.<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/0/05/Construct_generated_to_test_CFP.jpg"/><br />
</center><br />
<p>The gapped vector and the PCR product were co-transformed into yeast (BY4741ΔTrp strain) and incubated for several days. The resulting transformants were then cultured overnight in SD medium. Sample were washed and re-suspended in PBS and then observed under a microscope fitted with CFP filters.</p><br />
<br><br />
<h3>Results</h3><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/f0/Evidence_of_CFP_fluo.jpg"/><br />
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<p>We can see from Fig.3 that the CFP protein is being properly expressed. This means that the sequence used for CFP in the CUP1p - [MS2-CFP] construct is accurate and should be working. During this experiment the sequence of MS2-CFP was also checked to ensure that during the design both proteins had remained in frame.<br />
<br><br><br />
We can conclude from this experiment that the reason we are not seeing any CFP expression in pRS414 is because the CFP protein is not being produced properly.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/3._N-GFP_Swap_Experiment3. N-GFP Swap Experiment2010-10-26T11:35:44Z<p>Porter: </p>
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<h1>Replacement of MS2-[CFP] in CUP1p - [MS2-CFP] with Npep-GFP from GAL1p-[Npep-GFP] to Determine Functionality of Promoter/5'leader/Binding Stem Loop Sequence</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to see if the CUP1p - [MS2-CFP] construct possesses a working promoter/5’leader/binding stem loop sequence by replacing the MS2-CFP part of the construct with the Npep-GFP sequence from GAL1p-[Npep-GFP]. We have shown that Npep-GFP is expressed by GAL1p-[Npep-GFP] therefore if we can express it in CUP1p - [MS2-CFP] we can determine the functionality of the promoter/5'leader/binding stem loop sequence.</p><br />
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<img src="https://static.igem.org/mediawiki/2010/7/77/Joint_diagram_of_pRS414%2BpRS415.jpg"/><br />
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<h3>Hypothesis</h3><br />
<p>The reason why CUP1p - [MS2-CFP] is not expressing CFP lies within the promoter/5’leader/stem loop sequence.</p><br><br />
<h3>Protocol</h3><br />
<p>The CUP1p - [MS2-CFP] construct was digested using the restriction enzymes Cla1 and Nde1 in order to remove the MS2-CFP section of CUP1p - [MS2-CFP].<br><br><br />
In parallel, the Npeptide-GFP section of GAL1p-[Npep-GFP] was PCR amplified using primers designed to add 45 base pair long overhangs that would allow homologous recombination of the PCR product into the gapped CUP1p - [MS2-CFP]. The gapped CUP1p - [MS2-CFP] and the PCR product were co-transformed into yeast (BY4741ΔTrp strain) in order to allow the homologous recombination to take place. The resulting transformants were then screened using Colony PCR in order to make sure that the swap had taken place.<br />
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<img src="https://static.igem.org/mediawiki/2010/b/b8/N-GFP_swap_construct.jpg"/><br />
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<p>The colonies where the swap had been successful were then cultured overnight in SD medium containing Raffinose and CuSO4 (100µM). Samples were then normalised and resuspended in PBS and loaded onto a 96 well microtitre plate before being analysed using the FACS machine.</p><br />
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<br><br />
<h3>Results</h3><br />
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<img src="https://static.igem.org/mediawiki/2010/c/cc/Fluor_graph_for_N-GFP_swap.jpg"/><br />
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<p>From Fig 4. we can determine the level of background fluorescence of yeast cells using the results from the negative control “GAL1p-[Npep-GFP]”. We can also see the expected levels of GFP in a working construct from the positive control of “pRS415 + 2% Gal”.<br><br><br />
The two samples from colony 10, which are in presence of Cu2+ inducer, show readings that correspond with the levels of background fluorescence of yeast cells. This indicates that although the swap was successful the CUP1p - [MS2-CFP] construct is still not expressing properly.</p><br><br />
<br />
<h3>Conclusion</h3><br />
<p>We can conclude from these results that the problem resulting in CUP1p - [MS2-CFP] not being functional is related to either the Copper promoter or to the 5’UTR and Bbox stem loop. This experiment does not rule out however that the MS2-CFP part of the construct is also defective.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/2._Confirming_the_CFP_sequence_is_functional2. Confirming the CFP sequence is functional2010-10-26T11:35:14Z<p>Porter: </p>
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<h1>Confirming Functionality of CFP Sequence</h1><br />
<h3>Aim</h3><br />
<p>As part of the experiments to repair the CUP1p - [MS2-CFP] construct we need to determine if the CFP sequence used is functional or not. By confirming that the CFP protein is functional we can narrow down where the fault lies in the construct. If the CFP sequence was not functional this could mean that the construct was expressing properly but we simply could not detect this.</p><br />
<h3>Hypothesis</h3><br />
<p>The CFP sequence is accurate and the reason why no expression of CFP is being observed is because the protein is not being synthesised properly or not all.</p><br />
<h3>Protocol</h3><br />
<p>The TEF1p -[CFP] plasmid has been used to drive the expression of fluorescent proteins. The TEF1 promoter has been shown to constitutively express GFP at high levels.<br />
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<img src="https://static.igem.org/mediawiki/2010/1/1e/Diagram_of_YCP_lac_22_Fl.jpg"/><br />
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<p>A restriction digest was performed using the enzymes Nde1 and Xba1 in order to remove GFP from the construct. The CFP sequence in CUP1p - [MS2-CFP] was then PCR amplified using primers to generate complementary overhangs to allow homologous recombination into the gaped TEF1p -[CFP] vector.<br><br />
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<img src="https://static.igem.org/mediawiki/2010/0/05/Construct_generated_to_test_CFP.jpg"/><br />
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<p>The gapped vector and the PCR product were co-transformed into yeast (BY4741ΔTrp strain) and incubated for several days. The resulting transformants were then cultured overnight in SD medium. Sample were washed and re-suspended in PBS and then observed under a microscope fitted with CFP filters.</p><br />
<br><br />
<h3>Results</h3><br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/f0/Evidence_of_CFP_fluo.jpg"/><br />
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<p>We can see from Fig.3 that the CFP protein is being properly expressed. This means that the sequence used for CFP in the CUP1p - [MS2-CFP] construct is accurate and should be working. During this experiment the sequence of MS2-CFP was also checked to ensure that during the design both proteins had remained in frame.<br />
<br><br><br />
We can conclude from this experiment that the reason we are not seeing any CFP expression in pRS414 is because the CFP protein is not being produced properly.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Experimental_LayoutExperimental Layout2010-10-26T11:34:15Z<p>Porter: </p>
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<h1>Troubleshooting the CUP1p-[MS2-CFP] construct</h1><br />
<h3>Aim</h3><br />
<p>Following the analysis of the two promoters using CUP1p-GFP and GAL1p-GFP the next experiments planned would start to characterise the interactions of both constructs (CUP1p-[MS2-CFP] and GAL1p-[Npep-GFP]) and more specifically the mutual repression that would be taking place. Experiments using FACS analysis were set up in order to measure various levels of CFP and GFP depending on different conditions (i.e. different ranges of inducers, different ratios of pRS415 to pRS414 etc.). However the FACS machine was unable to detect any GFP or CFP in our calibration samples.</p><br />
<h3>Hypothesis</h3><br />
<p>Our initial hypothesis was that the lack of GFP and CFP expression was due to experimental error during the set up of the cultures (this proved to be correct as far as pRS415 was concerned where the lack of expression was traced back to a faulty stock of inducing agent).<br><br><br />
The second hypothesis put forward to explain the lack of CFP expression was that one of the parts that made up pRS414 was defective.<br><br />
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<img src="https://static.igem.org/mediawiki/2010/2/2c/Diagram_of_pRS414.jpg"/><br />
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<h3>Protocol</h3><br />
<p>A series of experiments were set up in order to determine the functionality of various parts of CUP1p-[MS2-CFP] with a view to repair the construct.<br><br><br />
<br />
<a href="https://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP">1. Confirmation using microscope and fluorometer analysis that the CUP1p-[MS2-CFP] construct was not expressing CFP</a><br><br />
<br />
<a href="https://2010.igem.org/2._Confirming_the_CFP_sequence_is_functional"> 2. Confirming Functionality of CFP Sequence </a><br><br />
<br />
<a href="https://2010.igem.org/3._N-GFP_Swap_Experiment"> 3. Replacement of MS2-[CFP] in CUP1p - [MS2-CFP] with Npep-GFP from GAL1p-[Npep-GFP] to Determine Functionality of Promoter/5'leader/Binding Stem Loop Sequence </a><br><br />
<br />
<a href="https://2010.igem.org/4._Replacing_the_CUP1_promoter_in_pRS414_with_the_CUP1-2_promoter_from_the_N4_construct">4. Replacing the CUP1 promoter in CUP1p-[MS2-CFP] with the CUP1-2 promoter from the CUP1p-GFP construct</a><br><br />
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<br><br />
<h3>Conclusion</h3><br />
<p>The first experiment did not detect any CFP fluorescent whilst the second experiment indicated that the CFP sequence used was accurate and in another vector resulted in fluorescence. The third experiment revealed that a fault lies with either the CUP1 promoter or/and the Bbox stem loop however the fourth experiment showed that the promoter did not seem to be contributing to the lack of expression.<br><br><br />
Our data suggests that the reason why CUP1p-[MS2-CFP] is not producing any CFP is due to the presence of the Bbox Stem loop in the 5’UTR. Although Bbox stem loops have already be used in conjunction with the λ-N peptide as markers for mRNA, it is possible that it has never been placed in 5’UTR but is rather located to a 3’UTR. As a result we cannot be sure that the loop can be melted and could therefore be preventing the expression of the following proteins.<br><br><br />
Our experiments however, do not rule out the possibility that the fusion of MS2 to CFP is defective and is resulting in an unstable protein that is being turned-over rapidly. It is also possible that the fusion is preventing the CFP portion from fluorescencing due to improper folding.<br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/Team:Aberdeen_Scotland/GFP_decayTeam:Aberdeen Scotland/GFP decay2010-10-26T11:33:20Z<p>Porter: </p>
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<h1> Characterising the glucose repression of GAL1 promoter in the GAL1p-[Npeptide-GFP] construct </h1><br />
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<h3>Aim</h3><br />
<p>To test the effect of glucose on repression of the GAL1 promoter, and thus on shut-off of GFP expression from construct GAL1p-[Npeptide-GFP] construct over time.</p><br />
<br />
<h3>Hypothesis</h3> <br />
<p>The presence of glucose should quickly repress the GAL1 promoter and therefore result in the overall reduction of the GFP intensity present within the cells; measurement of the rate of decay should identify the relative stability of the GFP protein</p><br />
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<h3>Protocol</h3><br />
<p>1. Yeast transformed with the GAL1p-[Npeptide-GFP] construct were innoculated overnight in 5 mls of synthetic defined medium with amino acids; his (0.2 %), met (0.2%), ura (0.2%), trp (0.2 %) and Raffinose (2 %) as the carbon source.<br><br><br />
2. Following overnight growth the cells were subcultured in fresh, pre-warmed SD medium (50 mls) containing galactose (a range of concentrations: see Results below) to obtain a predicted OD600 of 0.3 by 10 am the following morning.<br><br><br />
3. The following morning, at an OD600 of 0.3, a sample (1 ml) was taken before and after the addition of glucose (2 %). Samples were then taken every 20 minutes thereafter for a period of 167 minutes. All samples were then pelleted (13000 rpm, 5 mins, 4 degreesC), washed once with PBS buffer and stored on ice. Once collected all samples were then dispenced in PBS and diuted by a factor of 1/20 for FACS analysis.<br />
</p><br><br />
<br />
<h3>Results</h3><p><br />
Cells grown on galactose and expressing GFP were switched to growth on medium containing glucose. Following the resultant switch-off of the GAL1 promoter, GFP decay was monitored.<br><br><br />
Panel A (below) shows FACS analysis, with the peak to the left indicating GFP expressing cells and a peak to the right showing non GFP expressing cells. The FACS analysis clearly shows that the highest GFP expression (bottom light blue line) is observed after incubation overnight with galactose, with glucose present. It can be observed that after the addition of glucose (all lines above the blue) that there is a continuous increase in the number of cells not expressing GFP over time.<br><br><br />
Panel B shows this data in summarised, averaged form. It reveals that the average GFP intensity of the cells decreased steadily with time after the glucose addition, hence showing that the glucose has rapidly repressed the GAL1 promoter,and inhibited the expression of GFP. The half-life of this decay was approximately 140 minutes, which corresponded to approximately the doubling time of the cell culture, indicating that cell division was the primary reason for GFP disappearance, rather than active GFP turnover.</p><br />
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<h3>Conclusion</h3><p><br />
The presence of glucose rapidly inhibits the GAL1 promoter from expressing GFP and the average GFP intensity within a cell reduces by over 50 % within 140 minutes, consistent with cell division being the primary source of GFP depletion. This confirmed the fact that GFP is widely considered to be an extremely stable protein.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415MS2 Coat-Protein Effect on Expression of GFP in pRS4152010-10-26T11:30:44Z<p>Porter: </p>
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<h1> Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein </h1><br />
<h3>Aim</h3><br />
<p>The characterisation of the effect of MS2 on the expression of GFP by GAL1p-[Npeptide-GFP] will allow more accurate modelling of the system and will allow us to determine with more precision the probability of success of the cross-inhibition of the switch. Expressing MS2 using MET17p - [MS2] will allow us to monitor the effect of MS2 without the complication of the λ-N-peptide produced by GAL1p-[Npeptide-GFP] in turn inhibiting the expression of MS2.</p><br />
<h3>Hypothesis</h3> <br />
<p>The expression of MS2 by MET17p - [MS2]will result in a decrease in the level of expression of GFP by GAL1p-[Npeptide-GFP]. The inhibition will show a linear correlation with the level of expression of MS2.</p><br />
<h3>Protocol</h3><br />
<p>During this experiment double transformants of BY4742 containing GAL1p-[Npeptide-GFP] and MET17p - [MS2] were used. Single transformants of BY4742, containing only GAL1p-[Npeptide-GFP], were used to provide the negative and positive controls for the expression of GFP.</p><br><br />
<p>The double transformants were first cultured overnight in specific conditions in order to establish the desired pre-conditions. The cells were then washed and re-cultured in a different specific set of conditions which would allow the characterisation of the effect of MS2.</p><br><br />
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<p>* 500μM Met was used as this concentration has been used in other experiments to successfully completely switch of the Met17 promoter<a href="#ref1"><sup style="font-size:10px">[1]</sup></a>. <br><br />
<br><br />
The different pre-established conditions allow us to determine whether the history of the sample affects the final result.<br><br />
Final samples were then washed and normalised before being analysed using microscopy, Fluospar Optima readings and FACS analysis.</p><br><br />
<h3>Results</h3><br />
<br />
<h5><u>Microscopy</u><h5><br><br />
<p>The microscopy analysis revealed that, in none of the samples, the GFP expression had been completely inhibited. All samples (bar the negative control) showed green fluorescence. The microscope did not allow us to determine if there was any variation however in the levels of GFP in each specific sample. </p><br />
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<h5><u>Fluospar Optima Readings</u></h5><br><br />
The fluorimeter readings correlated the microscopy results by recording fluorescence in all samples except the – control.<br><br />
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<p>The recorded fluorescence values for the respective samples showed that there was indeed some variation in the levels of GFP (Fig 1). In both the ‘MS2 Dom’ and the ‘Race’ sample the GFP level was lower than in the + control indicating that the expression of GFP had indeed been inhibited (a 20% decrease for the ‘Race’ sample and an 11% decrease for the ‘MS2’ sample). The ‘GFP Dom’ sample however showed an approximate 8% increase in GFP fluorescence when compared to the + control. Although this is a bit unexpected is could be due to the fact that the GFP expression was initiated in the 1˚ set of conditions whereas it took place in the 2˚ for the + control. However it appears that no inhibition took place indicating that once GFP is being expressed the amount present of MS2 as expressed by MET17p - [MS2] is not able to significantly inhibit the level of GFP fluorescence.</p><br />
<br><br />
<h5><u>FACS analysis</u></h5><br />
<br><br />
<p>The first FACS analysis experiment involved running samples from the same initial cultures (see Table 1). The results showed that the presence of the MS2 coat protein was having an effect on the expression of GFP. All three test samples revealed lower levels of GFP when compared to the positive control indicating that MS2 was inhibiting the expression of GFP (see Fig 3). As expected the sample where GFP had been allowed to dominate prior to expression of MS2 (GFPdom sample) showed the highest level of GFP in the test samples and equally the sample where MS2 dominated prior to the expression of GFP (MS2do sample) showed the lowest level of GFP expression.</p><br><br />
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<p>The second FACS analysis experiment was aimed to determine whether the inhibition of GFP expression was in any way dependent on the levels of MS2. The following cultures were set up containing transformants containing both GAL1p-[Npeptide-GFP] and MET17p - [MS2] with varying amounts of Methionine. The reasoning is that the varying levels of methionine will translate into varying amounts of MS2 being produced as the Met17 is repressed.</p><br><br />
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<p>The results showed that the inhibition of GFP expression by GAL1p-[Npeptide-GFP] by MS2 previously seen (see Fig.3) is indeed dependent on the concentration of MS2.<br><br />
<br><br />
We can see a linear relationship between GFP levels and MS2 concentrations (see Fig.4). The observed level of GFP is at its lowest with no methionine being present. No methionine present translates as the Met17 promoter being unrepressed meaning that the MS2 expression rate is at its maximum. The levels of GFP gradually increase along with an increasing concentration of methionine (this translates as the Met17 promoter gradually being repressed until MS2 is no longer being expressed).</p><br />
<br />
<h3>References</h3><br />
<a name="ref1"></a><br />
<p>[1] Dominik Mumberg, Rolf MulIer and Martin Funk*<br><br />
Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression<br><br />
Nucleic Acids Research, 1994, Vol. 22, No. 25 5767-5768</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/MS2_Coat-Protein_Effect_on_Expression_of_GFP_in_pRS415MS2 Coat-Protein Effect on Expression of GFP in pRS4152010-10-26T11:29:57Z<p>Porter: </p>
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<h1> Characterising the translational repression of GAL1p-[Npeptide-GFP] by trans expression of the MS2 protein </h1><br />
<h3>Aim</h3><br />
<p>The characterisation of the effect of MS2 on the expression of GFP by GAL1p-[Npeptide-GFP] will allow more accurate modelling of the system and will allow us to determine with more precision the probability of success of the cross-inhibition of the switch. Expressing MS2 using MET17p - [MS2] will allow us to monitor the effect of MS2 without the complication of the λ-N-peptide produced by GAL1p-[Npeptide-GFP] in turn inhibiting the expression of MS2.</p><br />
<h3>Hypothesis</h3> <br />
<p>The expression of MS2 by MET17p - [MS2]will result in a decrease in the level of expression of GFP by GAL1p-[Npeptide-GFP]. The inhibition will show a linear correlation with the level of expression of MS2.</p><br />
<h3>Protocol</h3><br />
<p>During this experiment double transformants of BY4742 containing GAL1p-[Npeptide-GFP] and MET17p - [MS2] were used. Single transformants of BY4742, containing only GAL1p-[Npeptide-GFP], were used to provide the negative and positive controls for the expression of GFP.</p><br><br />
<p>The double transformants were first cultured overnight in specific conditions in order to establish the desired pre-conditions. The cells were then washed and re-cultured in a different specific set of conditions which would allow the characterisation of the effect of MS2.</p><br><br />
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<p>* 500μM Met was used as this concentration has been used in other experiments to successfully completely switch of the Met17 promoter<a href="#ref1"><sup style="font-size:10px">[1]</sup></a>. <br><br />
<br><br />
The different pre-established conditions allow us to determine whether the history of the sample affects the final result.<br><br />
Final samples were then washed and normalised before being analysed using microscopy, Fluospar Optima readings and FACS analysis.</p><br><br />
<h3>Results</h3><br />
<br />
<h5><u>Microscopy</u><h5><br><br />
<p>The microscopy analysis revealed that, in none of the samples, the GFP expression had been completely inhibited. All samples (bar the negative control) showed green fluorescence. The microscope did not allow us to determine if there was any variation however in the levels of GFP in each specific sample. </p><br />
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<h5><u>Fluospar Optima Readings</u></h5><br><br />
The fluorimeter readings correlated the microscopy results by recording fluorescence in all samples except the – control.<br><br />
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<br><br />
<p>The recorded fluorescence values for the respective samples showed that there was indeed some variation in the levels of GFP (Fig 1). In both the ‘MS2 Dom’ and the ‘Race’ sample the GFP level was lower than in the + control indicating that the expression of GFP had indeed been inhibited (a 20% decrease for the ‘Race’ sample and an 11% decrease for the ‘MS2’ sample). The ‘GFP Dom’ sample however showed an approximate 8% increase in GFP fluorescence when compared to the + control. Although this is a bit unexpected is could be due to the fact that the GFP expression was initiated in the 1˚ set of conditions whereas it took place in the 2˚ for the + control. However it appears that no inhibition took place indicating that once GFP is being expressed the amount present of MS2 as expressed by MET17p - [MS2] is not able to significantly inhibit the level of GFP fluorescence.</p><br />
<br><br />
<h5><u>FACS analysis</u></h5><br />
<br><br />
<p>The first FACS analysis experiment involved running samples from the same initial cultures (see Table 1). The results showed that the presence of the MS2 coat protein was having an effect on the expression of GFP. All three test samples revealed lower levels of GFP when compared to the positive control indicating that MS2 was inhibiting the expression of GFP (see Fig 3). As expected the sample where GFP had been allowed to dominate prior to expression of MS2 (GFPdom sample) showed the highest level of GFP in the test samples and equally the sample where MS2 dominated prior to the expression of GFP (MS2do sample) showed the lowest level of GFP expression.</p><br><br />
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<br><br />
<p>The second FACS analysis experiment was aimed to determine whether the inhibition of GFP expression was in any way dependent on the levels of MS2. The following cultures were set up containing transformants containing both GAL1p-[Npeptide-GFP] and MET17p - [MS2] with varying amounts of Methionine. The reasoning is that the varying levels of methionine will translate into varying amounts of MS2 being produced as the Met17 is repressed.</p><br><br />
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<br><br><br />
<p>The results showed that the inhibition of GFP expression by GAL1p-[Npeptide-GFP] by MS2 previously seen (see Fig.3) is indeed dependent on the concentration of MS2.<br><br />
<br><br />
We can see a linear relationship between GFP levels and MS2 concentrations (see Fig.4). The observed level of GFP is at its lowest with no methionine being present. No methionine present translates as the Met17 promoter being unrepressed meaning that the MS2 expression rate is at its maximum. The levels of GFP gradually increase along with an increasing concentration of methionine (this translates as the Met17 promoter gradually being repressed until MS2 is no longer being expressed).</p><br />
<br />
<h3>References</h3><br />
<a name="ref1"></a><br />
<p>[1] Dominik Mumberg, Rolf MulIer and Martin Funk*<br><br />
Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression<br><br />
Nucleic Acids Research, 1994, Vol. 22, No. 25 5767-5768</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP1. Confirmation using microscope and fluorometer analysis that the pRS414 construct was not expressing CFP2010-10-26T11:19:54Z<p>Porter: </p>
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<br />
<h1>Confirmation Using Microscope and Fluorometer Analysis that CUP1p-[MS2-CFP] was Not Expressing CFP</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to determine whether the CUP1p-[MS2-CFP] construct can express CFP in the presence of CuSO4. The detection of CFP using the fluorometer and the microscope would confirm whether the construct as a whole was functional or not.</p><br />
<h3>Hypothesis</h3><br />
<p>The observed lack of CFP expression is due to experimental errors and using appropriate medium and proper inducing agent, the CUP1p-[MS2-CFP] construct will express CFP.</p><br />
<h3>Protocol</h3><br />
<p>The Cyan filters on the fluorometer were first checked to ensure that they could detect CFP. Pacific Blue beads (a fluorescent dye used by the FACS machine) has an excitation and emission profile similar to CFP and was therefore used as a control. A range of concentration of Pacific Blue (diluted in PBS and also in SD medium) was loaded onto a 96 well microtitre plate and analysed using the fluorometer. The resulting reading (Fig.1) indicated that the filters in place were able to detect CFP.<br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/d/d8/Table_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/><br />
</center><br />
<br><br />
<p>In order to check whether CUP1p-[MS2-CFP] was expressing CFP, cultures of BY4741 containing CUP1p-[MS2-CFP] were prepared using the old/previously used medium and inducer (concentration of 50 and 10µM copper were used) and also using freshly made medium and inducer stocks. The cultures were incubated at 30°C and then samples were analysed using the fluorometer and a microscope equipped with CFP filters.</p><br />
<h3>Results</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/5/57/Table2_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/><br />
</center><br />
<br><br />
<p>The fluorometer readings indicated a small increase in fluorescence in samples containing Cu2+. However, the readings were still very close to the readings obtained for the background fluorescence of the yeast cells.<br />
<br><br />
The microscope observations indicated that no CFP fluorescence was present in any of the samples prepared.</p><br />
<h3>Conclusion</h3><br />
<p>We conclude from these results that the lack of CFP expression is not due to experimental error and that the fault must lie with one or several of the components making up the CUP1p-[MS2-CFP] construct.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP1. Confirmation using microscope and fluorometer analysis that the pRS414 construct was not expressing CFP2010-10-26T11:19:41Z<p>Porter: </p>
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{{:Team:Aberdeen_Scotland/Title}}<br />
<html><br />
<br />
<h1>Confirmation Using Microscope and Fluorometer Analysis that CUP1p-[MS2-CFP] was Not Expressing CFP</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to determine whether the CUP1p-[MS2-CFP] construct can express CFP in the presence of CuSO4. The detection of CFP using the fluorometer and the microscope would confirm whether the construct as a whole was functional or not.</p><br />
<h3>Hypothesis</h3><br />
<p>The observed lack of CFP expression is due to experimental errors and using appropriate medium and proper inducing agent, the CUP1p-[MS2-CFP] construct will express CFP.</p><br />
<h3>Protocol</h3><br />
<p>The Cyan filters on the fluorometer were first checked to ensure that they could detect CFP. Pacific Blue beads (a fluorescent dye used by the FACS machine) has an excitation and emission profile similar to CFP and was therefore used as a control. A range of concentration of Pacific Blue (diluted in PBS and also in SD medium) was loaded onto a 96 well microtitre plate and analysed using the fluorometer. The resulting reading (Fig.1) indicated that the filters in place were able to detect CFP.<br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/d/d8/Table_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/><br />
</center><br />
<br><br />
<p>In order to check whether CUP1p-[MS2-CFP] was expressing CFP, cultures of BY4741 containing CUP1p-[MS2-CFP] were prepared using the old/previously used medium and inducer (concentration of 50 and 10µM copper were used) and also using freshly made medium and inducer stocks. The cultures were incubated at 30°C and then samples were analysed using the fluorometer and a microscope equipped with CFP filters.</p><br />
<h3>Results</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/5/57/Table2_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/><br />
</center><br />
<br><br />
<p>The fluorometer readings indicated a small increase in fluorescence in samples containing Cu2+. However, the readings were still very close to the readings obtained for the background fluorescence of the yeast cells.<br />
<br><br />
The microscope observations indicated that no CFP fluorescence was present in any of the samples prepared.</p><br />
<h3>Conclusion</h3><br />
<p>We conclude from these results that the lack of CFP expression is not due to experimental error and that the fault must lie with one or several of the components making up the CUP1p-[MS2-CFP] construct.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP1. Confirmation using microscope and fluorometer analysis that the pRS414 construct was not expressing CFP2010-10-26T11:18:57Z<p>Porter: </p>
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{{:Team:Aberdeen_Scotland/Title}}<br />
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<br />
<h1>Confirmation Using Microscope and Fluorometer Analysis that CUP1p-[MS2-CFP] was Not Expressing CFP</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to determine whether the CUP1p-[MS2-CFP] construct can express CFP in the presence of CuSO4. The detection of CFP using the fluorometer and the microscope would confirm whether the construct as a whole was functional or not.</p><br />
<h3>Hypothesis</h3><br />
<p>The observed lack of CFP expression is due to experimental errors and using appropriate medium and proper inducing agent, the CUP1p-[MS2-CFP] construct will express CFP.</p><br />
<h3>Protocol</h3><br />
<p>The Cyan filters on the fluorometer were first checked to ensure that they could detect CFP. Pacific Blue beads (a fluorescent dye used by the FACS machine) has an excitation and emission profile similar to CFP and was therefore used as a control. A range of concentration of Pacific Blue (diluted in PBS and also in SD medium) was loaded onto a 96 well microtitre plate and analysed using the fluorometer. The resulting reading (Fig.1) indicated that the filters in place were able to detect CFP.<br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/d/d8/Table_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/><br />
</center><br />
<br><br />
<p>In order to check whether CUP1p-[MS2-CFP] was expressing CFP, cultures of BY4741 containing CUP1p-[MS2-CFP] were prepared using the old/previously used medium and inducer (concentration of 50 and 10µM copper were used) and also using freshly made medium and inducer stocks. The cultures were incubated at 30°C and then samples were analysed using the fluorometer and a microscope equipped with CFP filters.</p><br />
<h3>Results</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/5/57/Table2_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/><br />
</center><br />
<br><br />
<p>The fluorometer readings indicated a small increase in fluorescence in samples containing Cu2+. However, the readings were still very close to the readings obtained for the background fluorescence of the yeast cells.<br />
<br><br />
The microscope observations indicated that no CFP fluorescence was present in any of the samples prepared.</p><br />
<h3>Conclusion</h3><br />
<p>We conclude from these results that the lack of CFP expression is not due to experimental error and that the fault must lie with one or several of the components making up the CUP1p-[MS2-CFP] construct.</p><br />
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{{:Team:Aberdeen_Scotland/Footer}}</div>Porterhttp://2010.igem.org/1._Confirmation_using_microscope_and_fluorometer_analysis_that_the_pRS414_construct_was_not_expressing_CFP1. Confirmation using microscope and fluorometer analysis that the pRS414 construct was not expressing CFP2010-10-26T11:17:52Z<p>Porter: </p>
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{{:Team:Aberdeen_Scotland/Title}}<br />
<html><br />
<br />
<h1>Confirmation Using Microscope and Fluorometer Analysis that CUP1p-[MS2-CFP] was Not Expressing CFP</h1><br />
<h3>Aim</h3><br />
<p>The aim of this experiment is to determine whether the CUP1p-[MS2-CFP] construct can express CFP in the presence of CuSO4. The detection of CFP using the fluorometer and the microscope would confirm whether the construct as a whole was functional or not.</p><br />
<h3>Hypothesis</h3><br />
<p>The observed lack of CFP expression is due to experimental errors and using appropriate medium and proper inducing agent, the CUP1p-[MS2-CFP] construct will express CFP.</p><br />
<h3>Protocol</h3><br />
<p>The Cyan filters on the fluorometer were first checked to ensure that they could detect CFP. Pacific Blue beads (a fluorescent dye used by the FACS machine) has an excitation and emission profile similar to CFP and was therefore used as a control. A range of concentration of Pacific Blue (diluted in PBS and also in SD medium) was loaded onto a 96 well microtitre plate and analysed using the fluorometer. The resulting reading (Fig.1) indicated that the filters in place were able to detect CFP.<br />
<br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/d/d8/Table_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/><br />
</center><br />
<br><br />
<p>In order to check whether CUP1p-[MS2-CFP] was expressing CFP, cultures of BY4741 containing CUP1p-[MS2-CFP] were prepared using the old/previously used medium and inducer (concentration of 50 and 10µM copper were used) and also using freshly made medium and inducer stocks. The cultures were incubated at 30°C and then samples were analysed using the fluorometer and a microscope equipped with CFP filters.</p><br />
<h3>Results</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/5/57/Table2_for_Confirmation_that_PRS414_was_not_expressing_CFP_.png"/><br />
</center><br />
<br><br />
<p>The fluorometer readings indicated a small increase in fluorescence in samples containing Cu2+. However, the readings were still very close to the readings obtained for the background fluorescence of the yeast cells.<br />
<br><br />
The microscope observations indicated that no CFP fluorescence was present in any of the samples prepared.</p><br />
<h3>Conclusion</h3><br />
<p>We conclude from these results that the lack of CFP expression is not due to experimental error and that the fault must lie with one or several of the components making up the CUP1p-[MS2-CFP] construct.</p><br />
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