http://2009.igem.org/wiki/index.php?title=Special:Contributions/Awalbridge&feed=atom&limit=50&target=Awalbridge&year=&month=2009.igem.org - User contributions [en]2024-03-29T14:09:30ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Cambridge/ModellingTeam:Cambridge/Modelling2009-10-22T03:20:34Z<p>Awalbridge: </p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
=Modelling=<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop}}<br />
[[#Introduction | Introduction ]]<br />
[[#Basic Phage Activators | Basic Phage Activators ]]<br />
[[#Extended Systems | Extended Systems ]]<br />
[[#Modelling Basics | Modelling Basics ]]<br />
[[#References | References]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Introduction ==<br />
<br />
Our initial work was based around creation of a system that allows switching on of output at different calibrated input signal levels. Creating a model allows the feasibility of the proposed systems to be tested. A basic model of the original amplifier system was put forward, building on both our data and the Cambridge 2007 data. <br />
<br />
===Modelling the phage activator system===<br />
<br />
This is the basic 'amplifier' system that consists of an input sensitive promoter system,a protein activator and sensitive promoter. It can therefore be divided into two boxes, the approach taken in putting forward an initial model.<br />
<br />
====The pBAD promoter====<br />
An arabinose input acts as an inducer, permitting transcription, by binding the AraC transcription factor. This is a dual transcription factor; when unbound to arabinose a dimer restricts access of polymersase to reduce basal levels of transcription, upon binding arabinose the conformation changes and the dimer permits binding of polymerase. [[#References | [1]]]<br />
<br />
To model this situation, araC is first assumed to take the role of a repressor that reversibly binds and unbinds a site on the DNA. If it binds arabinose, it is sequestered and cannot bind the DNA. Here, an input function is created, after Alon [[#References | [2]]]. This gives the rate of transcription from the promoter dependent on the concentration of arabinose. Since mRNA is then translated at a roughly constant rate, it is related with a multiplicative constant to the rate of protein production, in this case activator and RFP.<br />
<br />
[[Image:Cambridge Eq1.gif | center]]<br />
<br />
This gives the rate of transcription as a function of X* which represents the concentration of active repressor, unbound to arabinose. B is the maximum rate of transcription, here this rate is when induced by arabinose at highest concentration. K_d is the dissociation constant (see modelling derivations). Parameters must be found by a parameter scan for sensible values or by comparing to already gathered data.<br />
<br />
The concentration of 'active' repressor is given as a function of arabinose concentration by:<br />
<br />
[[Image:Cambridge Eq2.gif | center]]<br />
<br />
where X^T is the total amount of araC available, bound or unbound. A is arabinose concentration. n is the number of arabinose molecules binding to each molecule of the repressor, and K is a binding constant. n was taken to be two by assuming that each araC dimer needs two molecules to be bound before it can permit transcription.<br />
<br />
<br />
Combining these two gives the overall input function, which has leaky transcription included at A = 0, seen in the actual results.<br />
<br />
====The Activator and its Promoter====<br />
<br />
This is based on a similar idea. Activator is made by transcription from pBAD, the mRNA is then translated (the potential time delays will be taken into account). The activity of the phage promoter is dependent on activator concentration according to:<br />
<br />
[[Image:Cambridge Eq3.gif | center ]]<br />
<br />
Assuming that translation rates remain constant, the rate of GFP and RFP production would be expected to be multiples of the above promoter activities/ input functions (which represent rate of transcription)<br />
<br />
The aim of this area of work is to fit the activator plate reader to curves in an attempt to better charactertise them in terms of hill function parameters after Canton [[#References | [3]]].<br />
<br />
====Entire System====<br />
<br />
The entire system, although a collected of nested hill functions, has an overall sigmoidal response to input concentration. This means that the full system can be modelled as a hill function, with appropriate parameters obtained by experimental analysis.<br />
<br />
===Making a Latch===<br />
<br />
A switch that remains on once stimulated would be useful if it was only necessary, say, to see if a hazardous contaminant had ever been present in a sample (it could still be there in low levels etc.). A method proposed is positive feedback; an activator placed downstream of its own promoter (as well as the reporter/ pigment) will, in theory, keep pigment production going. The rate of activator production from its own promoter is given in equation 3 above, which is dependent on activator concentration itself.<br />
<br />
===Modelling the proposed switching levels system===<br />
This idea was summarised in the diagram and discussion included in week 4 dry work:[[Image:Cambridge_thresholds.png ]]<br />
<br />
Most of these model systems will rely on the very basic equations outlined above, with extensions to add levels of complexity, such as the transport of arabinose into a cell. <br />
<br />
==Basic Phage Activators==<br />
<br />
The first step is to take a look at the preliminary data to try to find appropriate parameters. An overall aim for the modelling of this system is to refine the model to each component and attempt to predict how different combinations of components will behave. The data collection separates the promoter and activator by use of both RFP and GFP reporters.<br />
<br />
The basic model for the relationship between input concentration and GFP output rate of production is a Hill Function. The aim is to characterise each activator-promoter system and fit it to this basic model:<br />
<br />
[[Image:Cambridge_Eq3.gif | center]]<br />
<br />
The three parameters we require are the maximum output rate, the dissociation or binding constant and the hill coefficient.<br />
<br />
Having studied the behaviour of the actual phage activator systems we discovered that each construct 'switched on' at different input levels; they have different effective binding constants. This gives us the behaviour required for a threshold system without much further development.<br />
<br />
===Investigating the Model===<br />
<br />
Before data was available we changed parameters to investigate the effects and to check that our programs were working as expected. Week 6, in particular Tuesday, summarises this work.<br />
<br />
==Extended Systems==<br />
<br />
Despite the ability of the activator systems to switch on at different levels, before this was apparent an exercise in modelling different methods of achieving such a system was carried out. There were three proposed mechanisms for such a system. All are based on the presence of both a repressor and an activator of some form. <br />
* 1. Repressor and Activator bind at different sites, but transcription only occurs if the activator is bound alone.<br />
*2. The repressor and activator bind at different sites but the binding of one prevents binding of the other (by blocking the site for example). <br />
<br />
These assume that the repressor and activator are both transcription factors. In effect, these are bistable switches. <br />
<br />
*3. The activator (input of some form) prevents the production of a repressor that is otherwise continually expressed in the cell.<br />
<br />
Looking at the effects of changing parameters was carried out during weeks 6 and 7.<br />
<br />
==Modelling Basics==<br />
<br />
In order to model the kinetics of gene transcription and translation, the approach taken is similar to that in enzyme kinetics. The transcription factors bind reversibly to the relevant site on the DNA and depending upon the particular case, transcription is promoted or repressed. <br />
<br />
The steps of working out the input function (which describes the rate of transcription as a function of the inducer concentration) of a gene are finding the concentration of active repressor (that unbound to the inducer, in this case, arabinose) and then finding the effect of that repressor on transcription.<br />
<br />
We have taken araC to act as a repressor in these models, although as mentioned, it has dual activity, remaining on the DNA after arabinose binding to promote transcription. <br />
<br />
====Binding of repressor to site on DNA====<br />
<br />
First, working out the probability of the DNA site being occupied or free in the presence of a concentration of transcription factor. The equation describing the reversible DNA-Factor complex [DnX] formation is given below. Note that more than one molecule of repressor, the number here is given by n.<br />
<br />
[[Image:Cambridge_modbasics1.gif | center | 140px]]<br />
<br />
k_1 is the forward rate constant, k_2 is for the reverse reaction.<br />
<br />
The probability of the DNA site being free of the repressor is given by [D]/[D_t] where D_t is the total amount of free DNA binding site available. Assuming a pseudo steady state where [Dnx] is stable, so that d[DnX]/dt = 0, and including the constraint that<br />
<br />
[[Image:Cambridge_modbasics2.gif |center| 140px]]<br />
<br />
allows the probability of finding a DNA site free of the repressor to be found by simple substitution and rearrangement of the rate equation below.<br />
<br />
[[Image:Cambridge_modbasics3.gif |center| 260px]]<br />
<br />
This gives the repressor dependent input function of the gene:<br />
<br />
[[Image:Cambridge Eq1.gif | center]]<br />
<br />
The constant K_d is effectively the nth root of k_2/k_1, as can be found by following through the derivations.<br />
<br />
====Sequestering of the repressor AraC by binding to arabinose====<br />
<br />
Again, a similar approach is taken as in enzyme kinetics. Effectively, all of the above derivation is followed but this time the amount of active repressor, X*, is found. This assumes that the amount of AraC repressor bound to arabinose is in pseudo steady state. The total effect of the two repressing systems becomes that of an activator and in effect the detail could be hidden to have a simple activated transcription model. This approach would be the best to pursue in characterising the activator systems. <br />
<br />
====The Phage Activator Systems====<br />
<br />
Including the pBAD promoter as described above, an additional level of complication needs to be taken account of with the phage activator systems. Here the activator promotes transcription, so the input function for the gene under the activator controlled promoter is found from the probability that the DNA binding site is occupied. This is [DnX]/[D_t] where [DnX] here represents the amount of activator-DNA binding site complex (note this is not the amount of free DNA sites this time). Otherwise, the derivation is as above, with the assumption again that [DnX] is in pseudo steady state.<br />
<br />
==References==<br />
<br />
1. [http://www.ncbi.nlm.nih.gov/pubmed/9600836 Apo-AraC actively seeks to loop. Schleif J.Mol. Biol (1988) 278, 529-538]<br />
<br />
2. AN INTRODUCTION TO SYSTEMS BIOLOGY Design Principles Of Biological Circuits. Uri Alon<br />
<br />
3. [http://partsregistry.org/Part:BBa_F2620 BBa_F2620 Parts Registry entry with characterisation.]<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Project/Amplification/CharacterisationTeam:Cambridge/Project/Amplification/Characterisation2009-10-22T03:08:36Z<p>Awalbridge: </p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
= The Sensitivity Tuner =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop2}}<br />
[[#Introduction | Introduction ]]<br />
[[#Characterisation | Characterisation]]<br />
[[#Data| Data]]<br />
[[#Discussion | Discussion]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Introduction ==<br />
<br />
The Cambridge 2007 iGEM team build 15 "amplifiers," constructs with RFP and GFP reporters that amplified the PoPS output of the promoter pBad/AraC (<partinfo>BBa_I0500</partinfo>), as described below:<br />
<br />
[[Image:amplifier07.jpg]]<br />
<br />
We re-designed these constructs to be PoPS converters as follows...<br />
<br />
[[Image:thresholddevice3.jpg]] <br />
<br />
...and generated our own set of Sensitivity Tuners:<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_K274370</partinfo> || <partinfo>BBa_K274380</partinfo>||<br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_K274371</partinfo><br />
|<partinfo>BBa_K274381</partinfo><br />
|<partinfo>BBa_K274391</partinfo><br />
|-<br />
! PP promoter<br />
|<br />
|<partinfo>BBa_K274382</partinfo><br />
|<partinfo>BBa_K274392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_K274374</partinfo><br />
|<partinfo>BBa_K274384</partinfo><br />
|<partinfo>BBa_K274394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_K274375</partinfo><br />
|<br />
|<partinfo>BBa_K274395</partinfo><br />
|}<br />
<br />
In order to characterize these phage activator/promoter constructs, we used the corresponding Cambridge 2007 amplifier as an illustration of how our Sensitivity Tuners alter the behaviour of pBad/AraC. These parts are very useful for characterisation as they contain fluorescent reporters; the parts we designed, which lack an input promoter and fluorescent reporters, are more useful parts for other iGEM teams to incorporate into their own projects. <br />
<br />
== Characterisation ==<br />
<br />
For characterisation, we moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with <partinfo>BBa_J69591</partinfo>, the standard promoter. We looked at a few major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.<br />
<br />
[[Image:Cambridge_hillfunction.gif]]<br />
<br />
[[Image:characterization3.jpg]]<br />
<br />
For our characterisation assay, we transformed the ''E. coli'' strain BW27783 with the Cambridge 2007 amplifiers (biobricks summarised in the table below):<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_I746370</partinfo> || <partinfo>BBa_I746380</partinfo>||<partinfo>BBa_I746390</partinfo><br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_I746371</partinfo><br />
|<partinfo>BBa_I746381</partinfo><br />
|<partinfo>BBa_I746391</partinfo><br />
|-<br />
! PP promoter<br />
|<partinfo>BBa_I746372</partinfo><br />
|<partinfo>BBa_I746382</partinfo><br />
|<partinfo>BBa_I746392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_I746374</partinfo><br />
|<partinfo>BBa_I746384</partinfo><br />
|<partinfo>BBa_I746394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_I746375</partinfo><br />
|<partinfo>BBa_I746385</partinfo><br />
|<partinfo>BBa_I746395</partinfo><br />
|}<br />
<br />
The BW27783 strain is ideal for assays using arabinose as it expresses arabinose transporters in the membrane constitutively (rather than in response to the detection of arabinose) and is unable to metabolise arabinose. We used a standard assay for each construct. We characterised cultures transformed with the amplifier constructs in exponential phase at the following arabinose concentrations: 0, 0.1, 0.5, 1, 10, 50, 100, and 500 uM. The controls that we ran for each assay included LB, untransformed BW27783, and J59691, the standard promoter. With the data we gathered, we were able to update the Registry pages of the Cambridge 2007 amplifiers, and also illustrated the ability of our Sensitivity Tuners to modulate the transcriptional system downstream of pBad/AraC.<br />
<br />
==Data==<br />
<br />
=== Parameters of Fitted Curves ===<br />
<br />
{| border="1"<br />
|+ <br />
! Construct !! Increase in Rate, a (RPU) !! Basal Rate, c (RPU) !! Switch Point, k (uM) !! Hill Coefficient, n<br />
|-<br />
!<partinfo>BBa_I746370</partinfo><br />
| 0.205 || 0.022 || 0.85 || 2.72<br />
|-<br />
!<partinfo>BBa_I746371</partinfo><br />
| 0.323 || 0.039 || 0.92 || 2.89<br />
|-<br />
!<partinfo>BBa_I746374</partinfo><br />
| 0.137 || 0.021 || 0.88 || 2.51<br />
|-<br />
!<partinfo>BBa_I746375</partinfo><br />
| 0.196 || 0.032 || 0.95 || 2.71<br />
|-<br />
!<partinfo>BBa_I746380</partinfo><br />
| 0.129 || 0.018 || 2.20 || 4.00<br />
|-<br />
!<partinfo>BBa_I746381</partinfo><br />
| 0.452 || 0.118 || 1.05 || 2.45<br />
|-<br />
!<partinfo>BBa_I746382</partinfo><br />
| 0.496 || 0.029 || 3.98 || 2.37<br />
|-<br />
!<partinfo>BBa_I746384</partinfo><br />
| 0.194 || 0.018 || 1.46 || 3.96<br />
|-<br />
!<partinfo>BBa_I746391</partinfo><br />
| 1.312 || 0.058 || 1.11 || 2.78<br />
|-<br />
!<partinfo>BBa_I746392</partinfo><br />
| 1.271 || 0.040 || 0.35 || 2.26<br />
|-<br />
!<partinfo>BBa_I746394</partinfo><br />
| 1.105 || 0.046 || 1.81 || 4.00<br />
|-<br />
!<partinfo>BBa_I746395</partinfo><br />
| 1.366 || 0.040 || 1.91 || 4.00<br />
|}<br />
<br />
=== Sample Graph ===<br />
<br />
[[Image:Cambridge_characterisation82.png]]<br />
<br />
== Discussion ==<br />
<br />
Using the plate reader data, we were able to successfully characterise a wide range of Sensitivity Tuners. The switching point varies across the devices by a factor of ten, showing that the constructs would be suitable for their purpose.<br />
<br />
The size of the response is shown to depend to a large extent on the activator. The choice of promoter has has less of an impact, although a discernible one.<br />
<br />
The basal rate was consistent across the constructs, with the exception of BBa_I747381. A closer look at the data shows that the larger response only happened on one of the three cultures, so can be assumed anomolous.<br />
<br />
The hill coefficients are largely within an accepted range, suggesting that 2 or 3 molecules of the activator are necessary to engage the promoter. In some cases, however, the resolution of concentrations was too small to achieve a sensible value for the hill coefficient. Here, the upper limit of the fitting script was reached; the parameter takes the value 4.00.<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/File:Cambridge_hillfunction.gifFile:Cambridge hillfunction.gif2009-10-22T02:53:21Z<p>Awalbridge: </p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Project/Amplification/CharacterisationTeam:Cambridge/Project/Amplification/Characterisation2009-10-22T02:47:15Z<p>Awalbridge: </p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
= The Sensitivity Tuner =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop2}}<br />
[[#Introduction | Introduction ]]<br />
[[#Characterisation | Characterisation]]<br />
[[#Data| Data]]<br />
[[#Discussion | Discussion]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Introduction ==<br />
<br />
The Cambridge 2007 iGEM team build 15 "amplifiers," constructs with RFP and GFP reporters that amplified the PoPS output of the promoter pBad/AraC (<partinfo>BBa_I0500</partinfo>), as described below:<br />
<br />
[[Image:amplifier07.jpg]]<br />
<br />
We re-designed these constructs to be PoPS converters as follows...<br />
<br />
[[Image:thresholddevice3.jpg]] <br />
<br />
...and generated our own set of Sensitivity Tuners:<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_K274370</partinfo> || <partinfo>BBa_K274380</partinfo>||<br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_K274371</partinfo><br />
|<partinfo>BBa_K274381</partinfo><br />
|<partinfo>BBa_K274391</partinfo><br />
|-<br />
! PP promoter<br />
|<br />
|<partinfo>BBa_K274382</partinfo><br />
|<partinfo>BBa_K274392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_K274374</partinfo><br />
|<partinfo>BBa_K274384</partinfo><br />
|<partinfo>BBa_K274394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_K274375</partinfo><br />
|<br />
|<partinfo>BBa_K274395</partinfo><br />
|}<br />
<br />
In order to characterize these phage activator/promoter constructs, we used the corresponding Cambridge 2007 amplifier as an illustration of how our Sensitivity Tuners alter the behaviour of pBad/AraC. These parts are very useful for characterisation as they contain fluorescent reporters; the parts we designed, which lack an input promoter and fluorescent reporters, are more useful parts for other iGEM teams to incorporate into their own projects. <br />
<br />
== Characterisation ==<br />
<br />
For characterisation, we moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with <partinfo>BBa_J69591</partinfo>, the standard promoter. We looked at a few major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.<br />
<br />
[[Image:characterization3.jpg]]<br />
<br />
For our characterization assay, we transformed the ''E. coli'' strain BW27783 with the Cambridge 2007 amplifiers (biobricks summarised in the table below):<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_I746370</partinfo> || <partinfo>BBa_I746380</partinfo>||<partinfo>BBa_I746390</partinfo><br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_I746371</partinfo><br />
|<partinfo>BBa_I746381</partinfo><br />
|<partinfo>BBa_I746391</partinfo><br />
|-<br />
! PP promoter<br />
|<partinfo>BBa_I746372</partinfo><br />
|<partinfo>BBa_I746382</partinfo><br />
|<partinfo>BBa_I746392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_I746374</partinfo><br />
|<partinfo>BBa_I746384</partinfo><br />
|<partinfo>BBa_I746394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_I746375</partinfo><br />
|<partinfo>BBa_I746385</partinfo><br />
|<partinfo>BBa_I746395</partinfo><br />
|}<br />
<br />
The BW27783 strain is ideal for assays using arabinose as it expresses arabinose transporters in the membrane constitutively (rather than in response to the detection of arabinose) and is unable to metabolise arabinose. We used a standard assay for each construct. We characterised cultures transformed with the amplifier constructs in exponential phase at the following arabinose concentrations: 0, 0.1, 0.5, 1, 10, 50, 100, and 500 uM. The controls that we ran for each assay included LB, untransformed BW27783, and J59691, the standard promoter. With the data we gathered, we were able to update the Registry pages of the Cambridge 2007 amplifiers, and also illustrated the ability of our Sensitivity Tuners to modulate the transcriptional system downstream of pBad/AraC.<br />
<br />
==Data==<br />
<br />
=== Maximum Rates against Arabinose Concentrations ===<br />
<br />
{| border="1"<br />
|+ <br />
! Construct !! Increase in Rate, a (RPU) !! Basal Rate, c (RPU) !! Switch Point, k (uM) !! Hill Coefficient, n<br />
|-<br />
!<partinfo>BBa_I746370</partinfo><br />
| 0.205 || 0.022 || 0.85 || 2.72<br />
|-<br />
!<partinfo>BBa_I746371</partinfo><br />
| 0.323 || 0.039 || 0.92 || 2.89<br />
|-<br />
!<partinfo>BBa_I746374</partinfo><br />
| 0.137 || 0.021 || 0.88 || 2.51<br />
|-<br />
!<partinfo>BBa_I746375</partinfo><br />
| 0.196 || 0.032 || 0.95 || 2.71<br />
|-<br />
!<partinfo>BBa_I746380</partinfo><br />
| 0.129 || 0.018 || 2.20 || 4.00<br />
|-<br />
!<partinfo>BBa_I746381</partinfo><br />
| 0.452 || 0.118 || 1.05 || 2.45<br />
|-<br />
!<partinfo>BBa_I746382</partinfo><br />
| 0.496 || 0.029 || 3.98 || 2.37<br />
|-<br />
!<partinfo>BBa_I746384</partinfo><br />
| 0.194 || 0.018 || 1.46 || 3.96<br />
|-<br />
!<partinfo>BBa_I746391</partinfo><br />
| 1.312 || 0.058 || 1.11 || 2.78<br />
|-<br />
!<partinfo>BBa_I746392</partinfo><br />
| 1.271 || 0.040 || 0.35 || 2.26<br />
|-<br />
!<partinfo>BBa_I746394</partinfo><br />
| 1.105 || 0.046 || 1.81 || 4.00<br />
|-<br />
!<partinfo>BBa_I746395</partinfo><br />
| 1.366 || 0.040 || 1.91 || 4.00<br />
|}<br />
<br />
<br />
[[Image:Cambridge_characterisation82.png]]<br />
<br />
<br />
<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/File:Cambridge_characterisation82.pngFile:Cambridge characterisation82.png2009-10-22T02:45:29Z<p>Awalbridge: </p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Project/Amplification/CharacterisationTeam:Cambridge/Project/Amplification/Characterisation2009-10-22T02:42:32Z<p>Awalbridge: </p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
= The Sensitivity Tuner =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop2}}<br />
[[#Introduction | Introduction ]]<br />
[[#Characterisation | Characterisation]]<br />
[[#Data| Data]]<br />
[[#Discussion | Discussion]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Introduction ==<br />
<br />
The Cambridge 2007 iGEM team build 15 "amplifiers," constructs with RFP and GFP reporters that amplified the PoPS output of the promoter pBad/AraC (<partinfo>BBa_I0500</partinfo>), as described below:<br />
<br />
[[Image:amplifier07.jpg]]<br />
<br />
We re-designed these constructs to be PoPS converters as follows...<br />
<br />
[[Image:thresholddevice3.jpg]] <br />
<br />
...and generated our own set of Sensitivity Tuners:<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_K274370</partinfo> || <partinfo>BBa_K274380</partinfo>||<br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_K274371</partinfo><br />
|<partinfo>BBa_K274381</partinfo><br />
|<partinfo>BBa_K274391</partinfo><br />
|-<br />
! PP promoter<br />
|<br />
|<partinfo>BBa_K274382</partinfo><br />
|<partinfo>BBa_K274392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_K274374</partinfo><br />
|<partinfo>BBa_K274384</partinfo><br />
|<partinfo>BBa_K274394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_K274375</partinfo><br />
|<br />
|<partinfo>BBa_K274395</partinfo><br />
|}<br />
<br />
In order to characterize these phage activator/promoter constructs, we used the corresponding Cambridge 2007 amplifier as an illustration of how our Sensitivity Tuners alter the behaviour of pBad/AraC. These parts are very useful for characterisation as they contain fluorescent reporters; the parts we designed, which lack an input promoter and fluorescent reporters, are more useful parts for other iGEM teams to incorporate into their own projects. <br />
<br />
== Characterisation ==<br />
<br />
For characterisation, we moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with <partinfo>BBa_J69591</partinfo>, the standard promoter. We looked at a few major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.<br />
<br />
[[Image:characterization3.jpg]]<br />
<br />
For our characterization assay, we transformed the ''E. coli'' strain BW27783 with the Cambridge 2007 amplifiers (biobricks summarised in the table below):<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_I746370</partinfo> || <partinfo>BBa_I746380</partinfo>||<partinfo>BBa_I746390</partinfo><br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_I746371</partinfo><br />
|<partinfo>BBa_I746381</partinfo><br />
|<partinfo>BBa_I746391</partinfo><br />
|-<br />
! PP promoter<br />
|<partinfo>BBa_I746372</partinfo><br />
|<partinfo>BBa_I746382</partinfo><br />
|<partinfo>BBa_I746392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_I746374</partinfo><br />
|<partinfo>BBa_I746384</partinfo><br />
|<partinfo>BBa_I746394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_I746375</partinfo><br />
|<partinfo>BBa_I746385</partinfo><br />
|<partinfo>BBa_I746395</partinfo><br />
|}<br />
<br />
The BW27783 strain is ideal for assays using arabinose as it expresses arabinose transporters in the membrane constitutively (rather than in response to the detection of arabinose) and is unable to metabolise arabinose. We used a standard assay for each construct. We characterised cultures transformed with the amplifier constructs in exponential phase at the following arabinose concentrations: 0, 0.1, 0.5, 1, 10, 50, 100, and 500 uM. The controls that we ran for each assay included LB, untransformed BW27783, and J59691, the standard promoter. With the data we gathered, we were able to update the Registry pages of the Cambridge 2007 amplifiers, and also illustrated the ability of our Sensitivity Tuners to modulate the transcriptional system downstream of pBad/AraC.<br />
<br />
==Data==<br />
<br />
=== Maximum Rates against Arabinose Concentrations ===<br />
<br />
{| border="1"<br />
|+ <br />
! Construct !! Increase in Rate, a (RPU) !! Basal Rate, c (RPU) !! Switch Point, k (uM) !! Hill Coefficient, n<br />
|-<br />
!<partinfo>BBa_I746370</partinfo><br />
| 0.205 || 0.022 || 0.85 || 2.72<br />
|-<br />
!<partinfo>BBa_I746371</partinfo><br />
| 0.323 || 0.039 || 0.92 || 2.89<br />
|-<br />
!<partinfo>BBa_I746374</partinfo><br />
| 0.137 || 0.021 || 0.88 || 2.51<br />
|-<br />
!<partinfo>BBa_I746375</partinfo><br />
| 0.196 || 0.032 || 0.95 || 2.71<br />
|-<br />
!<partinfo>BBa_I746380</partinfo><br />
| 0.129 || 0.018 || 2.20 || 4.00<br />
|-<br />
!<partinfo>BBa_I746381</partinfo><br />
| 0.452 || 0.118 || 1.05 || 2.45<br />
|-<br />
!<partinfo>BBa_I746382</partinfo><br />
| 0.496 || 0.029 || 3.98 || 2.37<br />
|-<br />
!<partinfo>BBa_I746384</partinfo><br />
| 0.194 || 0.018 || 1.46 || 3.96<br />
|-<br />
!<partinfo>BBa_I746391</partinfo><br />
| 1.312 || 0.058 || 1.11 || 2.78<br />
|-<br />
!<partinfo>BBa_I746392</partinfo><br />
| 1.271 || 0.040 || 0.35 || 2.26<br />
|-<br />
!<partinfo>BBa_I746394</partinfo><br />
| 1.105 || 0.046 || 1.81 || 4.00<br />
|-<br />
!<partinfo>BBa_I746395</partinfo><br />
| 1.366 || 0.040 || 1.91 || 4.00<br />
|}<br />
<br />
==== 80 ====<br />
[[Image:Cambridge_maxrates1.jpg | 600px]]<br />
<br />
==== 81 ====<br />
[[Image:Cambridge_maxrates2.jpg | 600px]]<br />
<br />
==== 82 ====<br />
[[Image:Cambridge_maxrates3.jpg | 600px]]<br />
<br />
==== 84 ====<br />
[[Image:Cambridge_maxrates4.jpg | 600px]]<br />
<br />
==== 85 ====<br />
[[Image:Cambridge_maxrates5.jpg | 600px]]<br />
<br />
==== 90 ====<br />
[[Image:Cambridge_maxrates6.jpg | 600px]]<br />
<br />
==== 92 ====<br />
[[Image:Cambridge_maxrates7.jpg | 600px]]<br />
<br />
==== 94 ====<br />
[[Image:Cambridge_maxrates8.jpg | 600px]]<br />
<br />
==== 95 ====<br />
[[Image:Cambridge_maxrates9.jpg | 600px]]<br />
<br />
<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Project/Amplification/CharacterisationTeam:Cambridge/Project/Amplification/Characterisation2009-10-22T02:37:34Z<p>Awalbridge: </p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
= The Sensitivity Tuner =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop2}}<br />
[[#Introduction | Introduction ]]<br />
[[#Characterisation | Characterisation]]<br />
[[#Data| Data]]<br />
[[#Discussion | Discussion]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Introduction ==<br />
<br />
The Cambridge 2007 iGEM team build 15 "amplifiers," constructs with RFP and GFP reporters that amplified the PoPS output of the promoter pBad/AraC (<partinfo>BBa_I0500</partinfo>), as described below:<br />
<br />
[[Image:amplifier07.jpg]]<br />
<br />
We re-designed these constructs to be PoPS converters as follows...<br />
<br />
[[Image:thresholddevice3.jpg]] <br />
<br />
...and generated our own set of Sensitivity Tuners:<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_K274370</partinfo> || <partinfo>BBa_K274380</partinfo>||<br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_K274371</partinfo><br />
|<partinfo>BBa_K274381</partinfo><br />
|<partinfo>BBa_K274391</partinfo><br />
|-<br />
! PP promoter<br />
|<br />
|<partinfo>BBa_K274382</partinfo><br />
|<partinfo>BBa_K274392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_K274374</partinfo><br />
|<partinfo>BBa_K274384</partinfo><br />
|<partinfo>BBa_K274394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_K274375</partinfo><br />
|<br />
|<partinfo>BBa_K274395</partinfo><br />
|}<br />
<br />
In order to characterize these phage activator/promoter constructs, we used the corresponding Cambridge 2007 amplifier as an illustration of how our Sensitivity Tuners alter the behaviour of pBad/AraC. These parts are very useful for characterisation as they contain fluorescent reporters; the parts we designed, which lack an input promoter and fluorescent reporters, are more useful parts for other iGEM teams to incorporate into their own projects. <br />
<br />
== Characterisation ==<br />
<br />
For characterisation, we moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with <partinfo>BBa_J69591</partinfo>, the standard promoter. We looked at a few major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.<br />
<br />
[[Image:characterization3.jpg]]<br />
<br />
For our characterization assay, we transformed the ''E. coli'' strain BW27783 with the Cambridge 2007 amplifiers (biobricks summarised in the table below):<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_I746370</partinfo> || <partinfo>BBa_I746380</partinfo>||<partinfo>BBa_I746390</partinfo><br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_I746371</partinfo><br />
|<partinfo>BBa_I746381</partinfo><br />
|<partinfo>BBa_I746391</partinfo><br />
|-<br />
! PP promoter<br />
|<partinfo>BBa_I746372</partinfo><br />
|<partinfo>BBa_I746382</partinfo><br />
|<partinfo>BBa_I746392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_I746374</partinfo><br />
|<partinfo>BBa_I746384</partinfo><br />
|<partinfo>BBa_I746394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_I746375</partinfo><br />
|<partinfo>BBa_I746385</partinfo><br />
|<partinfo>BBa_I746395</partinfo><br />
|}<br />
<br />
The BW27783 strain is ideal for assays using arabinose as it expresses arabinose transporters in the membrane constitutively (rather than in response to the detection of arabinose) and is unable to metabolise arabinose. We used a standard assay for each construct. We characterised cultures transformed with the amplifier constructs in exponential phase at the following arabinose concentrations: 0, 0.1, 0.5, 1, 10, 50, 100, and 500 uM. The controls that we ran for each assay included LB, untransformed BW27783, and J59691, the standard promoter. With the data we gathered, we were able to update the Registry pages of the Cambridge 2007 amplifiers, and also illustrated the ability of our Sensitivity Tuners to modulate the transcriptional system downstream of pBad/AraC.<br />
<br />
==Data==<br />
<br />
=== Maximum Rates against Arabinose Concentrations ===<br />
<br />
{| border="1"<br />
|+ <br />
! Construct !! Increase in Rate, a (RPU) !! Basal Rate, c (RPU) !! Switch Point, k (uM) !! Hill Coefficient, n<br />
|-<br />
!<partinfo>BBa_I746340</partinfo><br />
| 13.203 || 1.256 || 76.49 || 2.18<br />
|-<br />
!<partinfo>BBa_I746370</partinfo><br />
| 0.205 || 0.022 || 0.85 || 2.72<br />
|-<br />
!<partinfo>BBa_I746371</partinfo><br />
| 0.323 || 0.039 || 0.92 || 2.89<br />
|-<br />
!<partinfo>BBa_I746374</partinfo><br />
| 0.137 || 0.021 || 0.88 || 2.51<br />
|-<br />
!<partinfo>BBa_I746375</partinfo><br />
| 0.196 || 0.032 || 0.95 || 2.71<br />
|-<br />
!<partinfo>BBa_I746380</partinfo><br />
| 0.129 || 0.018 || 2.20 || 4.00<br />
|-<br />
!<partinfo>BBa_I746381</partinfo><br />
| 0.452 || 0.118 || 1.05 || 2.45<br />
|-<br />
!<partinfo>BBa_I746382</partinfo><br />
| 0.496 || 0.029 || 3.98 || 2.37<br />
|-<br />
!<partinfo>BBa_I746384</partinfo><br />
| 0.194 || 0.018 || 1.46 || 3.96<br />
|-<br />
!<partinfo>BBa_I746391</partinfo><br />
| 1.312 || 0.058 || 1.11 || 2.78<br />
|-<br />
!<partinfo>BBa_I746392</partinfo><br />
| 1.271 || 0.040 || 0.35 || 2.26<br />
|-<br />
!<partinfo>BBa_I746394</partinfo><br />
| 1.105 || 0.046 || 1.81 || 4.00<br />
|-<br />
!<partinfo>BBa_I746395</partinfo><br />
| 1.366 || 0.040 || 1.91 || 4.00<br />
|}<br />
<br />
==== 80 ====<br />
[[Image:Cambridge_maxrates1.jpg | 600px]]<br />
<br />
==== 81 ====<br />
[[Image:Cambridge_maxrates2.jpg | 600px]]<br />
<br />
==== 82 ====<br />
[[Image:Cambridge_maxrates3.jpg | 600px]]<br />
<br />
==== 84 ====<br />
[[Image:Cambridge_maxrates4.jpg | 600px]]<br />
<br />
==== 85 ====<br />
[[Image:Cambridge_maxrates5.jpg | 600px]]<br />
<br />
==== 90 ====<br />
[[Image:Cambridge_maxrates6.jpg | 600px]]<br />
<br />
==== 92 ====<br />
[[Image:Cambridge_maxrates7.jpg | 600px]]<br />
<br />
==== 94 ====<br />
[[Image:Cambridge_maxrates8.jpg | 600px]]<br />
<br />
==== 95 ====<br />
[[Image:Cambridge_maxrates9.jpg | 600px]]<br />
<br />
<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Project/Amplification/CharacterisationTeam:Cambridge/Project/Amplification/Characterisation2009-10-22T02:34:06Z<p>Awalbridge: </p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
= The Sensitivity Tuner =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop2}}<br />
[[#Introduction | Introduction ]]<br />
[[#Characterisation | Characterisation]]<br />
[[#Data| Data]]<br />
[[#Discussion | Discussion]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Introduction ==<br />
<br />
The Cambridge 2007 iGEM team build 15 "amplifiers," constructs with RFP and GFP reporters that amplified the PoPS output of the promoter pBad/AraC (<partinfo>BBa_I0500</partinfo>), as described below:<br />
<br />
[[Image:amplifier07.jpg]]<br />
<br />
We re-designed these constructs to be PoPS converters as follows...<br />
<br />
[[Image:thresholddevice3.jpg]] <br />
<br />
...and generated our own set of Sensitivity Tuners:<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_K274370</partinfo> || <partinfo>BBa_K274380</partinfo>||<br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_K274371</partinfo><br />
|<partinfo>BBa_K274381</partinfo><br />
|<partinfo>BBa_K274391</partinfo><br />
|-<br />
! PP promoter<br />
|<br />
|<partinfo>BBa_K274382</partinfo><br />
|<partinfo>BBa_K274392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_K274374</partinfo><br />
|<partinfo>BBa_K274384</partinfo><br />
|<partinfo>BBa_K274394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_K274375</partinfo><br />
|<br />
|<partinfo>BBa_K274395</partinfo><br />
|}<br />
<br />
In order to characterize these phage activator/promoter constructs, we used the corresponding Cambridge 2007 amplifier as an illustration of how our Sensitivity Tuners alter the behaviour of pBad/AraC. These parts are very useful for characterisation as they contain fluorescent reporters; the parts we designed, which lack an input promoter and fluorescent reporters, are more useful parts for other iGEM teams to incorporate into their own projects. <br />
<br />
== Characterisation ==<br />
<br />
For characterisation, we moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with <partinfo>BBa_J69591</partinfo>, the standard promoter. We looked at a few major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.<br />
<br />
[[Image:characterization3.jpg]]<br />
<br />
For our characterization assay, we transformed the ''E. coli'' strain BW27783 with the Cambridge 2007 amplifiers (biobricks summarised in the table below):<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_I746370</partinfo> || <partinfo>BBa_I746380</partinfo>||<partinfo>BBa_I746390</partinfo><br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_I746371</partinfo><br />
|<partinfo>BBa_I746381</partinfo><br />
|<partinfo>BBa_I746391</partinfo><br />
|-<br />
! PP promoter<br />
|<partinfo>BBa_I746372</partinfo><br />
|<partinfo>BBa_I746382</partinfo><br />
|<partinfo>BBa_I746392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_I746374</partinfo><br />
|<partinfo>BBa_I746384</partinfo><br />
|<partinfo>BBa_I746394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_I746375</partinfo><br />
|<partinfo>BBa_I746385</partinfo><br />
|<partinfo>BBa_I746395</partinfo><br />
|}<br />
<br />
The BW27783 strain is ideal for assays using arabinose as it expresses arabinose transporters in the membrane constitutively (rather than in response to the detection of arabinose) and is unable to metabolise arabinose. We used a standard assay for each construct. We characterised cultures transformed with the amplifier constructs in exponential phase at the following arabinose concentrations: 0, 0.1, 0.5, 1, 10, 50, 100, and 500 uM. The controls that we ran for each assay included LB, untransformed BW27783, and J59691, the standard promoter. With the data we gathered, we were able to update the Registry pages of the Cambridge 2007 amplifiers, and also illustrated the ability of our Sensitivity Tuners to modulate the transcriptional system downstream of pBad/AraC.<br />
<br />
==Data==<br />
<br />
=== Maximum Rates against Arabinose Concentrations ===<br />
<br />
{| border="1"<br />
|+ <br />
! Construct !! Increase in Rate, a (RPU) !! Basal Rate, c (RPU) !! Switch Point, k (uM) !! Hill Coefficient, n<br />
|-<br />
!<partinfo>BBa_I746340</partinfo><br />
| 13.203 | 1.256 | 76.49 | 2.18<br />
|-<br />
!<partinfo>BBa_I746370</partinfo><br />
| 0.205 | 0.022 | 0.85 | 2.72<br />
|-<br />
!<partinfo>BBa_I746371</partinfo><br />
| 0.323 | 0.039 | 0.92 | 2.89<br />
|-<br />
!<partinfo>BBa_I746374</partinfo><br />
| 0.137 | 0.021 | 0.88 | 2.51<br />
|-<br />
!<partinfo>BBa_I746375</partinfo><br />
| 0.196 | 0.032 | 0.95 | 2.71<br />
|-<br />
!<partinfo>BBa_I746380</partinfo><br />
| 0.129 | 0.018 | 2.20 | 4.00<br />
|-<br />
!<partinfo>BBa_I746381</partinfo><br />
| 0.452 | 0.118 | 1.05 | 2.45<br />
|-<br />
!<partinfo>BBa_I746382</partinfo><br />
| 0.496 | 0.029 | 3.98 | 2.37<br />
|-<br />
!<partinfo>BBa_I746384</partinfo><br />
| 0.194 | 0.018 | 1.46 | 3.96<br />
|-<br />
!<partinfo>BBa_I746391</partinfo><br />
| 1.312 | 0.058 | 1.11 | 2.78<br />
|-<br />
!<partinfo>BBa_I746392</partinfo><br />
| 1.271 | 0.040 | 0.35 | 2.26<br />
|-<br />
!<partinfo>BBa_I746394</partinfo><br />
| 1.105 | 0.046 | 1.81 | 4.00<br />
|-<br />
!<partinfo>BBa_I746395</partinfo><br />
| 1.366 | 0.040 | 1.91 | 4.00<br />
|}<br />
<br />
==== 80 ====<br />
[[Image:Cambridge_maxrates1.jpg | 600px]]<br />
<br />
==== 81 ====<br />
[[Image:Cambridge_maxrates2.jpg | 600px]]<br />
<br />
==== 82 ====<br />
[[Image:Cambridge_maxrates3.jpg | 600px]]<br />
<br />
==== 84 ====<br />
[[Image:Cambridge_maxrates4.jpg | 600px]]<br />
<br />
==== 85 ====<br />
[[Image:Cambridge_maxrates5.jpg | 600px]]<br />
<br />
==== 90 ====<br />
[[Image:Cambridge_maxrates6.jpg | 600px]]<br />
<br />
==== 92 ====<br />
[[Image:Cambridge_maxrates7.jpg | 600px]]<br />
<br />
==== 94 ====<br />
[[Image:Cambridge_maxrates8.jpg | 600px]]<br />
<br />
==== 95 ====<br />
[[Image:Cambridge_maxrates9.jpg | 600px]]<br />
<br />
<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Project/Amplification/CharacterisationTeam:Cambridge/Project/Amplification/Characterisation2009-10-22T02:29:38Z<p>Awalbridge: </p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
= The Sensitivity Tuner =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop2}}<br />
[[#Introduction | Introduction ]]<br />
[[#Characterisation | Characterisation]]<br />
[[#Data| Data]]<br />
[[#Discussion | Discussion]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Introduction ==<br />
<br />
The Cambridge 2007 iGEM team build 15 "amplifiers," constructs with RFP and GFP reporters that amplified the PoPS output of the promoter pBad/AraC (<partinfo>BBa_I0500</partinfo>), as described below:<br />
<br />
[[Image:amplifier07.jpg]]<br />
<br />
We re-designed these constructs to be PoPS converters as follows...<br />
<br />
[[Image:thresholddevice3.jpg]] <br />
<br />
...and generated our own set of Sensitivity Tuners:<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_K274370</partinfo> || <partinfo>BBa_K274380</partinfo>||<br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_K274371</partinfo><br />
|<partinfo>BBa_K274381</partinfo><br />
|<partinfo>BBa_K274391</partinfo><br />
|-<br />
! PP promoter<br />
|<br />
|<partinfo>BBa_K274382</partinfo><br />
|<partinfo>BBa_K274392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_K274374</partinfo><br />
|<partinfo>BBa_K274384</partinfo><br />
|<partinfo>BBa_K274394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_K274375</partinfo><br />
|<br />
|<partinfo>BBa_K274395</partinfo><br />
|}<br />
<br />
In order to characterize these phage activator/promoter constructs, we used the corresponding Cambridge 2007 amplifier as an illustration of how our Sensitivity Tuners alter the behaviour of pBad/AraC. These parts are very useful for characterisation as they contain fluorescent reporters; the parts we designed, which lack an input promoter and fluorescent reporters, are more useful parts for other iGEM teams to incorporate into their own projects. <br />
<br />
== Characterisation ==<br />
<br />
For characterisation, we moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with <partinfo>BBa_J69591</partinfo>, the standard promoter. We looked at a few major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.<br />
<br />
[[Image:characterization3.jpg]]<br />
<br />
For our characterization assay, we transformed the ''E. coli'' strain BW27783 with the Cambridge 2007 amplifiers (biobricks summarised in the table below):<br />
<br />
{| border="1"<br />
|+ <br />
! !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator<br />
|-<br />
! PF promoter<br />
| <partinfo>BBa_I746370</partinfo> || <partinfo>BBa_I746380</partinfo>||<partinfo>BBa_I746390</partinfo><br />
|-<br />
! PO promoter<br />
|<partinfo>BBa_I746371</partinfo><br />
|<partinfo>BBa_I746381</partinfo><br />
|<partinfo>BBa_I746391</partinfo><br />
|-<br />
! PP promoter<br />
|<partinfo>BBa_I746372</partinfo><br />
|<partinfo>BBa_I746382</partinfo><br />
|<partinfo>BBa_I746392</partinfo><br />
|-<br />
! Psid promoter<br />
|<partinfo>BBa_I746374</partinfo><br />
|<partinfo>BBa_I746384</partinfo><br />
|<partinfo>BBa_I746394</partinfo><br />
|-<br />
! PLL promoter<br />
|<partinfo>BBa_I746375</partinfo><br />
|<partinfo>BBa_I746385</partinfo><br />
|<partinfo>BBa_I746395</partinfo><br />
|}<br />
<br />
The BW27783 strain is ideal for assays using arabinose as it expresses arabinose transporters in the membrane constitutively (rather than in response to the detection of arabinose) and is unable to metabolise arabinose. We used a standard assay for each construct. We characterised cultures transformed with the amplifier constructs in exponential phase at the following arabinose concentrations: 0, 0.1, 0.5, 1, 10, 50, 100, and 500 uM. The controls that we ran for each assay included LB, untransformed BW27783, and J59691, the standard promoter. With the data we gathered, we were able to update the Registry pages of the Cambridge 2007 amplifiers, and also illustrated the ability of our Sensitivity Tuners to modulate the transcriptional system downstream of pBad/AraC.<br />
<br />
==Data==<br />
<br />
=== Maximum Rates against Arabinose Concentrations ===<br />
<br />
{| border="1"<br />
|+ <br />
! Construct !! Increase in Rate, a (RPU) !! Basal Rate, c (RPU) !! Switch Point, k (uM) !! Hill Coefficient, n<br />
|-<br />
!<partinfo>BBa_I746340</partinfo><br />
|13.203 |1.256 |76.49 |2.18<br />
|-<br />
!<partinfo>BBa_I746370</partinfo><br />
|0.205 |0.022 |0.85 |2.72<br />
|-<br />
!<partinfo>BBa_I746371</partinfo><br />
|0.323 |0.039 |0.92 |2.89<br />
|-<br />
!<partinfo>BBa_I746374</partinfo><br />
|0.137 |0.021 |0.88 |2.51<br />
|-<br />
!<partinfo>BBa_I746375</partinfo><br />
|0.196 |0.032 |0.95 |2.71<br />
|-<br />
!<partinfo>BBa_I746380</partinfo><br />
|0.129 |0.018 |2.20 |4.00<br />
|-<br />
!<partinfo>BBa_I746381</partinfo><br />
|0.452 |0.118 |1.05 |2.45<br />
|-<br />
!<partinfo>BBa_I746382</partinfo><br />
|0.496 |0.029 |3.98 |2.37<br />
|-<br />
!<partinfo>BBa_I746384</partinfo><br />
|0.194 |0.018 |1.46 |3.96<br />
|-<br />
!<partinfo>BBa_I746391</partinfo><br />
|1.312 |0.058 |1.11 |2.78<br />
|-<br />
!<partinfo>BBa_I746392</partinfo><br />
|1.271 |0.040 |0.35 |2.26<br />
|-<br />
!<partinfo>BBa_I746394</partinfo><br />
|1.105 |0.046 |1.81 |4.00<br />
|-<br />
!<partinfo>BBa_I746395</partinfo><br />
|1.366 |0.040 |1.91 |4.00<br />
|}<br />
<br />
==== 80 ====<br />
[[Image:Cambridge_maxrates1.jpg | 600px]]<br />
<br />
==== 81 ====<br />
[[Image:Cambridge_maxrates2.jpg | 600px]]<br />
<br />
==== 82 ====<br />
[[Image:Cambridge_maxrates3.jpg | 600px]]<br />
<br />
==== 84 ====<br />
[[Image:Cambridge_maxrates4.jpg | 600px]]<br />
<br />
==== 85 ====<br />
[[Image:Cambridge_maxrates5.jpg | 600px]]<br />
<br />
==== 90 ====<br />
[[Image:Cambridge_maxrates6.jpg | 600px]]<br />
<br />
==== 92 ====<br />
[[Image:Cambridge_maxrates7.jpg | 600px]]<br />
<br />
==== 94 ====<br />
[[Image:Cambridge_maxrates8.jpg | 600px]]<br />
<br />
==== 95 ====<br />
[[Image:Cambridge_maxrates9.jpg | 600px]]<br />
<br />
<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/ProtocolsTeam:Cambridge/Protocols2009-09-17T15:29:33Z<p>Awalbridge: /* Protocols */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Protocols =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop}}<br />
[[#Producing competent cells | Competent Cells]]<br />
[[#Competent cells Transformation | Transformation ]]<br />
[[#Carotene extraction with acetone | Carotene Extraction ]]<br />
[[#Agarose gel electrophoresis | Electrophoresis ]]<br />
[[#Finnzymes Phusion (TM) PCR | Finnzymes PCR]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
{{Template:Cambridgetemplatetop}}<br />
[[#PCR procedures | PCR Procedures]]<br />
[[#Glycerol Stocks | Glycerol Stocks]]<br />
[[# | ]]<br />
[[# | ]]<br />
[[# | ]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Producing competent cells ==<br />
<br />
Starting from a single colony on a plate:<br />
*Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight<br />
*Take 10ml of the culture and innoculate into one litre LB and grow in shaking incubator until OD600 of 0.2-0.3 (4 hours?)<br />
*Put culture on ice for 30 minutes <br />
*Centrifuge at 4000g for 6 minutes<br />
*Remove supernatant and resuspend cells in an equal volume of ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in 0.5 volume ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in ice-cold 10% glycerol (20ml)<br />
*Combine to form two tubes of 40ml glycerol<br />
*Repeat centrifugation<br />
*Resuspend in ice-cold glycerol (3ml)<br />
*Divide cells into 100ul aliquots and store at -80<br />
<br />
(Cells should be at a final volume of ~3 x 10^10 cells.ml^-1)<br />
<br />
== Competent cells Transformation ==<br />
<br />
*Electrocompetent cells thawed on ice<br />
*Prepare vector DNA on ice<br />
*Biobricks<br />
:*With pipette tip, punch hole through foil cover into designated well<br />
:*Add 20uL DIW<br />
:*We will be removing about 5uL; the rest needs to go in an eppendorf, labeled with biobrick number, and stored at -20°C <br />
*Violacein and melanin need to be thawed<br />
*Vector DNA pipetted into chilled 1mm separation electrocuvette = 4 total<br />
*5uL of biobricks<br />
*0.5uL of melanin and violacein plasmid<br />
*Add 45 uL Competent cells <br />
*Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles<br />
*Thoroughly dry the cuvette<br />
*1.68 kV passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 uF<br />
*Add 0.25 mL SOC liquid medium to electrocuvette <br />
*Incubate electrocuvettes at 37 degrees C for 60 minutes <br />
*Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader<br />
*Orange genes biobrick: ampicillin<br />
*Promoter for orange genes biobrick: ampicillin<br />
*Melanin: ampicillin, copper, and tyrosine<br />
*Violacin: trimethoprim <br />
*Do 1:10 dilution with SDW into a new eppendorf<br />
*Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums<br />
<br />
== Carotene extraction with acetone ==<br />
<br />
Adopted from ''Yuan et al. (2006)'':<br />
<br />
1. Incubate ''E.coli'' in 5ml LB with antibiotics at 37 oC for 20 hours.<br />
<br />
2. Harvest cells using centrifugation at 4000 rpm for 10 minutes.<br />
<br />
3. Re-suspend cells in 300 ul acetone and '''''vortex for 5 minutes'''***'' ''(***: original protocol recommended "Homogenise cells with glass beads in Bead-Beater for 30s (Biospec products)", but we did not have the equipment. Vortex is used for homogenisation instead)''. <br />
<br />
4. Centrifuge sample at 14,000 rpm for 1 minute. Collect supernatant.<br />
<br />
5. Measure absorption using spectrophotometer at 450 nm. Normalise data to cell density (OD 600 nm). This is performed using '''Omega Microplate Readers''' from BMG-Labtech.<br />
<br />
'''CAUTION: Acetone may corrode plastic microplates and cause severe damage to the equipment if left in the plate reader for too long. With microplate readers, dilute acetone extract 10 times with water (to give 200ul) before loading into plastic wells.'''<br />
<br />
== Agarose gel electrophoresis ==<br />
=== Gel preparation ===<br />
To prepare 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1X TAE buffer (obtained by diluting 10X TAE stock buffer with water) and heat in microwave until all powder desolves. Gel stains should be added when the agarose becomes cool enough to touch.<br />
<br />
For ethidium bromide gels, add 2 uL EtBr to 20 mL of agarose (makes 1 gel).<br />
<br />
=== Electrophoresis setting ===<br />
For electrophoresis, set constant voltage at 80V (with current at approximately 3 mA) and run for 30--60 minutes (or until sufficient separation of DNA).<br />
<br />
<br />
==Finnzymes Phusion (TM) PCR==<br />
<br />
===Shuna's protocol===<br />
<br />
====PCR Master Mix====<br />
*25ul 2x Phusion Master Mix<br />
*0.5uM of each primer<br />
*~5ng of DNA<br />
*Make up to 50ul with SDW<br />
<br />
====PCR Cycle====<br />
Thirty seconds at 98 degrees<br />
<br />
Thirty cycles:<br />
*98 degrees for 5-10 seconds<br />
*67 degrees for 10-30 seconds (depending on primer)<br />
*72 degrees for 30 seconds/kb<br />
Final extension at 72 degrees for 5-10 minutes<br />
Hold at four degrees.<br />
<br />
===James's protocols:===<br />
<br />
====Phusion master mix====<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
25 uL 2x Phusion master mix<br />
<br />
optional 1.5 uL DMSO<br />
<br />
make up to 50 uL with SDW<br />
<br />
====Using Phusion enzyme====<br />
<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
1 uL dNTPs<br />
<br />
0.5 uL Phusion polymerase<br />
<br />
10 uL x5 buffer (use buffer GC for high GC content, has shown to give higher product yeilds)<br />
<br />
make up to 50 uL with SDW<br />
<br />
<br />
==PCR procedures==<br />
<br />
[[Image:NANODROP.JPG | G-Storm and Finnzymes PCR Machines |thumb| right| 200px]]<br />
[[Image:FLUOstar.jpg | A FLUOstar Omega Platereader | thumb| right| 200px]]<br />
<br />
'''For high accuracy sequence PCR'''<br />
*Use the Phusion set from Finnzymes<br />
*50ul solution<br />
*Can alter conditions for optimisation<br />
<br />
'''For verification of plasmid presence/length'''<br />
*Use TAQ polymerase and buffer from stores<br />
*20ul solution<br />
*Run with standard procedure as follows (from James) for Colony PCR<br />
<br />
*'''Reaction Mixture'''<br />
:*Template: 1 uL from O/N culture (1-2 uL if colony picked straight from plate into water)<br />
:*VF2: 1uL<br />
:*VR: 1uL<br />
:*Eco-Taq: 0.2 uL<br />
:*10X buffer: 5 uL<br />
:*dNTPs: 0.4 uL (stock is 10 mM)<br />
:*make up to 20 uL volume with H20<br />
*'''Reaction procedure'''<br />
:*95 degrees C for 2 minutes<br />
:*33 cycles of:<br />
::*95 degrees C for 30 seconds (denaturation)<br />
::*65 degrees C for 30 seconds (annealing)<br />
::*72 degrees C, 1000bp/min (elongation)<br />
:*72 degrees C for 5 minutes<br />
:*hold at 4 degrees C<br />
<br />
'''Gels'''<br />
*Run on EtBr for good quality viewing (make agarose gel). This is viewed in the red-room downstairs.<br />
*Run on SYBR-safe gel if the DNA is required. This can be viewed under blue light in the covered dark-area.<br />
<br />
==Glycerol Stocks==<br />
<br />
===For -80===<br />
<br />
Cells in 10-15% glycerol, 1:4 ratio of glycerol:culture<br />
*0.5mL 80% glycerol<br />
*2mL fresh culture<br />
<br />
===For -20===<br />
<br />
Cells in 20-40% glycerol, 1:1 ratio of glycerol:culture<br />
*1mL 80% glycerol<br />
*1mL fresh culture<br />
<br />
Add cells to glycerol, vortex, put on ice immediately, then store.<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/ProtocolsTeam:Cambridge/Protocols2009-09-17T15:29:06Z<p>Awalbridge: /* Protocols */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Protocols =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop}}<br />
[[#Producing competent cells | Competent Cells]]<br />
[[#Competent cells Transformation | Transformation ]]<br />
[[#Carotene extraction with acetone | Carotene Extraction ]]<br />
[[#Agarose gel electrophoresis | Electrophoresis ]]<br />
[[#Finnzymes Phusion (TM) PCR | Finnzymes Phusion PCR]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
{{Template:Cambridgetemplatetop}}<br />
[[#PCR procedures | PCR Procedures]]<br />
[[#Glycerol Stocks | Glycerol Stocks]]<br />
[[# | ]]<br />
[[# | ]]<br />
[[# | ]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Producing competent cells ==<br />
<br />
Starting from a single colony on a plate:<br />
*Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight<br />
*Take 10ml of the culture and innoculate into one litre LB and grow in shaking incubator until OD600 of 0.2-0.3 (4 hours?)<br />
*Put culture on ice for 30 minutes <br />
*Centrifuge at 4000g for 6 minutes<br />
*Remove supernatant and resuspend cells in an equal volume of ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in 0.5 volume ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in ice-cold 10% glycerol (20ml)<br />
*Combine to form two tubes of 40ml glycerol<br />
*Repeat centrifugation<br />
*Resuspend in ice-cold glycerol (3ml)<br />
*Divide cells into 100ul aliquots and store at -80<br />
<br />
(Cells should be at a final volume of ~3 x 10^10 cells.ml^-1)<br />
<br />
== Competent cells Transformation ==<br />
<br />
*Electrocompetent cells thawed on ice<br />
*Prepare vector DNA on ice<br />
*Biobricks<br />
:*With pipette tip, punch hole through foil cover into designated well<br />
:*Add 20uL DIW<br />
:*We will be removing about 5uL; the rest needs to go in an eppendorf, labeled with biobrick number, and stored at -20°C <br />
*Violacein and melanin need to be thawed<br />
*Vector DNA pipetted into chilled 1mm separation electrocuvette = 4 total<br />
*5uL of biobricks<br />
*0.5uL of melanin and violacein plasmid<br />
*Add 45 uL Competent cells <br />
*Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles<br />
*Thoroughly dry the cuvette<br />
*1.68 kV passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 uF<br />
*Add 0.25 mL SOC liquid medium to electrocuvette <br />
*Incubate electrocuvettes at 37 degrees C for 60 minutes <br />
*Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader<br />
*Orange genes biobrick: ampicillin<br />
*Promoter for orange genes biobrick: ampicillin<br />
*Melanin: ampicillin, copper, and tyrosine<br />
*Violacin: trimethoprim <br />
*Do 1:10 dilution with SDW into a new eppendorf<br />
*Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums<br />
<br />
== Carotene extraction with acetone ==<br />
<br />
Adopted from ''Yuan et al. (2006)'':<br />
<br />
1. Incubate ''E.coli'' in 5ml LB with antibiotics at 37 oC for 20 hours.<br />
<br />
2. Harvest cells using centrifugation at 4000 rpm for 10 minutes.<br />
<br />
3. Re-suspend cells in 300 ul acetone and '''''vortex for 5 minutes'''***'' ''(***: original protocol recommended "Homogenise cells with glass beads in Bead-Beater for 30s (Biospec products)", but we did not have the equipment. Vortex is used for homogenisation instead)''. <br />
<br />
4. Centrifuge sample at 14,000 rpm for 1 minute. Collect supernatant.<br />
<br />
5. Measure absorption using spectrophotometer at 450 nm. Normalise data to cell density (OD 600 nm). This is performed using '''Omega Microplate Readers''' from BMG-Labtech.<br />
<br />
'''CAUTION: Acetone may corrode plastic microplates and cause severe damage to the equipment if left in the plate reader for too long. With microplate readers, dilute acetone extract 10 times with water (to give 200ul) before loading into plastic wells.'''<br />
<br />
== Agarose gel electrophoresis ==<br />
=== Gel preparation ===<br />
To prepare 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1X TAE buffer (obtained by diluting 10X TAE stock buffer with water) and heat in microwave until all powder desolves. Gel stains should be added when the agarose becomes cool enough to touch.<br />
<br />
For ethidium bromide gels, add 2 uL EtBr to 20 mL of agarose (makes 1 gel).<br />
<br />
=== Electrophoresis setting ===<br />
For electrophoresis, set constant voltage at 80V (with current at approximately 3 mA) and run for 30--60 minutes (or until sufficient separation of DNA).<br />
<br />
<br />
==Finnzymes Phusion (TM) PCR==<br />
<br />
===Shuna's protocol===<br />
<br />
====PCR Master Mix====<br />
*25ul 2x Phusion Master Mix<br />
*0.5uM of each primer<br />
*~5ng of DNA<br />
*Make up to 50ul with SDW<br />
<br />
====PCR Cycle====<br />
Thirty seconds at 98 degrees<br />
<br />
Thirty cycles:<br />
*98 degrees for 5-10 seconds<br />
*67 degrees for 10-30 seconds (depending on primer)<br />
*72 degrees for 30 seconds/kb<br />
Final extension at 72 degrees for 5-10 minutes<br />
Hold at four degrees.<br />
<br />
===James's protocols:===<br />
<br />
====Phusion master mix====<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
25 uL 2x Phusion master mix<br />
<br />
optional 1.5 uL DMSO<br />
<br />
make up to 50 uL with SDW<br />
<br />
====Using Phusion enzyme====<br />
<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
1 uL dNTPs<br />
<br />
0.5 uL Phusion polymerase<br />
<br />
10 uL x5 buffer (use buffer GC for high GC content, has shown to give higher product yeilds)<br />
<br />
make up to 50 uL with SDW<br />
<br />
<br />
==PCR procedures==<br />
<br />
[[Image:NANODROP.JPG | G-Storm and Finnzymes PCR Machines |thumb| right| 200px]]<br />
[[Image:FLUOstar.jpg | A FLUOstar Omega Platereader | thumb| right| 200px]]<br />
<br />
'''For high accuracy sequence PCR'''<br />
*Use the Phusion set from Finnzymes<br />
*50ul solution<br />
*Can alter conditions for optimisation<br />
<br />
'''For verification of plasmid presence/length'''<br />
*Use TAQ polymerase and buffer from stores<br />
*20ul solution<br />
*Run with standard procedure as follows (from James) for Colony PCR<br />
<br />
*'''Reaction Mixture'''<br />
:*Template: 1 uL from O/N culture (1-2 uL if colony picked straight from plate into water)<br />
:*VF2: 1uL<br />
:*VR: 1uL<br />
:*Eco-Taq: 0.2 uL<br />
:*10X buffer: 5 uL<br />
:*dNTPs: 0.4 uL (stock is 10 mM)<br />
:*make up to 20 uL volume with H20<br />
*'''Reaction procedure'''<br />
:*95 degrees C for 2 minutes<br />
:*33 cycles of:<br />
::*95 degrees C for 30 seconds (denaturation)<br />
::*65 degrees C for 30 seconds (annealing)<br />
::*72 degrees C, 1000bp/min (elongation)<br />
:*72 degrees C for 5 minutes<br />
:*hold at 4 degrees C<br />
<br />
'''Gels'''<br />
*Run on EtBr for good quality viewing (make agarose gel). This is viewed in the red-room downstairs.<br />
*Run on SYBR-safe gel if the DNA is required. This can be viewed under blue light in the covered dark-area.<br />
<br />
==Glycerol Stocks==<br />
<br />
===For -80===<br />
<br />
Cells in 10-15% glycerol, 1:4 ratio of glycerol:culture<br />
*0.5mL 80% glycerol<br />
*2mL fresh culture<br />
<br />
===For -20===<br />
<br />
Cells in 20-40% glycerol, 1:1 ratio of glycerol:culture<br />
*1mL 80% glycerol<br />
*1mL fresh culture<br />
<br />
Add cells to glycerol, vortex, put on ice immediately, then store.<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/ProtocolsTeam:Cambridge/Protocols2009-09-17T15:28:45Z<p>Awalbridge: /* Protocols */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Protocols =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop}}<br />
[[#Producing competent cells | Competent Cells]]<br />
[[#Competent cells Transformation | Transformation ]]<br />
[[#Carotene extraction with acetone | Carotene Extraction ]]<br />
[[#Agarose gel electrophoresis | Electrophoresis ]]<br />
[[#Finnzymes Phusion (TM) PCR | Finnzymes Phusion (TM) PCR]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
{{Template:Cambridgetemplatetop}}<br />
[[#PCR procedures | PCR Procedures]]<br />
[[#Glycerol Stocks | Glycerol Stocks]]<br />
[[# | ]]<br />
[[# | ]]<br />
[[# | ]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Producing competent cells ==<br />
<br />
Starting from a single colony on a plate:<br />
*Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight<br />
*Take 10ml of the culture and innoculate into one litre LB and grow in shaking incubator until OD600 of 0.2-0.3 (4 hours?)<br />
*Put culture on ice for 30 minutes <br />
*Centrifuge at 4000g for 6 minutes<br />
*Remove supernatant and resuspend cells in an equal volume of ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in 0.5 volume ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in ice-cold 10% glycerol (20ml)<br />
*Combine to form two tubes of 40ml glycerol<br />
*Repeat centrifugation<br />
*Resuspend in ice-cold glycerol (3ml)<br />
*Divide cells into 100ul aliquots and store at -80<br />
<br />
(Cells should be at a final volume of ~3 x 10^10 cells.ml^-1)<br />
<br />
== Competent cells Transformation ==<br />
<br />
*Electrocompetent cells thawed on ice<br />
*Prepare vector DNA on ice<br />
*Biobricks<br />
:*With pipette tip, punch hole through foil cover into designated well<br />
:*Add 20uL DIW<br />
:*We will be removing about 5uL; the rest needs to go in an eppendorf, labeled with biobrick number, and stored at -20°C <br />
*Violacein and melanin need to be thawed<br />
*Vector DNA pipetted into chilled 1mm separation electrocuvette = 4 total<br />
*5uL of biobricks<br />
*0.5uL of melanin and violacein plasmid<br />
*Add 45 uL Competent cells <br />
*Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles<br />
*Thoroughly dry the cuvette<br />
*1.68 kV passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 uF<br />
*Add 0.25 mL SOC liquid medium to electrocuvette <br />
*Incubate electrocuvettes at 37 degrees C for 60 minutes <br />
*Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader<br />
*Orange genes biobrick: ampicillin<br />
*Promoter for orange genes biobrick: ampicillin<br />
*Melanin: ampicillin, copper, and tyrosine<br />
*Violacin: trimethoprim <br />
*Do 1:10 dilution with SDW into a new eppendorf<br />
*Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums<br />
<br />
== Carotene extraction with acetone ==<br />
<br />
Adopted from ''Yuan et al. (2006)'':<br />
<br />
1. Incubate ''E.coli'' in 5ml LB with antibiotics at 37 oC for 20 hours.<br />
<br />
2. Harvest cells using centrifugation at 4000 rpm for 10 minutes.<br />
<br />
3. Re-suspend cells in 300 ul acetone and '''''vortex for 5 minutes'''***'' ''(***: original protocol recommended "Homogenise cells with glass beads in Bead-Beater for 30s (Biospec products)", but we did not have the equipment. Vortex is used for homogenisation instead)''. <br />
<br />
4. Centrifuge sample at 14,000 rpm for 1 minute. Collect supernatant.<br />
<br />
5. Measure absorption using spectrophotometer at 450 nm. Normalise data to cell density (OD 600 nm). This is performed using '''Omega Microplate Readers''' from BMG-Labtech.<br />
<br />
'''CAUTION: Acetone may corrode plastic microplates and cause severe damage to the equipment if left in the plate reader for too long. With microplate readers, dilute acetone extract 10 times with water (to give 200ul) before loading into plastic wells.'''<br />
<br />
== Agarose gel electrophoresis ==<br />
=== Gel preparation ===<br />
To prepare 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1X TAE buffer (obtained by diluting 10X TAE stock buffer with water) and heat in microwave until all powder desolves. Gel stains should be added when the agarose becomes cool enough to touch.<br />
<br />
For ethidium bromide gels, add 2 uL EtBr to 20 mL of agarose (makes 1 gel).<br />
<br />
=== Electrophoresis setting ===<br />
For electrophoresis, set constant voltage at 80V (with current at approximately 3 mA) and run for 30--60 minutes (or until sufficient separation of DNA).<br />
<br />
<br />
==Finnzymes Phusion (TM) PCR==<br />
<br />
===Shuna's protocol===<br />
<br />
====PCR Master Mix====<br />
*25ul 2x Phusion Master Mix<br />
*0.5uM of each primer<br />
*~5ng of DNA<br />
*Make up to 50ul with SDW<br />
<br />
====PCR Cycle====<br />
Thirty seconds at 98 degrees<br />
<br />
Thirty cycles:<br />
*98 degrees for 5-10 seconds<br />
*67 degrees for 10-30 seconds (depending on primer)<br />
*72 degrees for 30 seconds/kb<br />
Final extension at 72 degrees for 5-10 minutes<br />
Hold at four degrees.<br />
<br />
===James's protocols:===<br />
<br />
====Phusion master mix====<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
25 uL 2x Phusion master mix<br />
<br />
optional 1.5 uL DMSO<br />
<br />
make up to 50 uL with SDW<br />
<br />
====Using Phusion enzyme====<br />
<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
1 uL dNTPs<br />
<br />
0.5 uL Phusion polymerase<br />
<br />
10 uL x5 buffer (use buffer GC for high GC content, has shown to give higher product yeilds)<br />
<br />
make up to 50 uL with SDW<br />
<br />
<br />
==PCR procedures==<br />
<br />
[[Image:NANODROP.JPG | G-Storm and Finnzymes PCR Machines |thumb| right| 200px]]<br />
[[Image:FLUOstar.jpg | A FLUOstar Omega Platereader | thumb| right| 200px]]<br />
<br />
'''For high accuracy sequence PCR'''<br />
*Use the Phusion set from Finnzymes<br />
*50ul solution<br />
*Can alter conditions for optimisation<br />
<br />
'''For verification of plasmid presence/length'''<br />
*Use TAQ polymerase and buffer from stores<br />
*20ul solution<br />
*Run with standard procedure as follows (from James) for Colony PCR<br />
<br />
*'''Reaction Mixture'''<br />
:*Template: 1 uL from O/N culture (1-2 uL if colony picked straight from plate into water)<br />
:*VF2: 1uL<br />
:*VR: 1uL<br />
:*Eco-Taq: 0.2 uL<br />
:*10X buffer: 5 uL<br />
:*dNTPs: 0.4 uL (stock is 10 mM)<br />
:*make up to 20 uL volume with H20<br />
*'''Reaction procedure'''<br />
:*95 degrees C for 2 minutes<br />
:*33 cycles of:<br />
::*95 degrees C for 30 seconds (denaturation)<br />
::*65 degrees C for 30 seconds (annealing)<br />
::*72 degrees C, 1000bp/min (elongation)<br />
:*72 degrees C for 5 minutes<br />
:*hold at 4 degrees C<br />
<br />
'''Gels'''<br />
*Run on EtBr for good quality viewing (make agarose gel). This is viewed in the red-room downstairs.<br />
*Run on SYBR-safe gel if the DNA is required. This can be viewed under blue light in the covered dark-area.<br />
<br />
==Glycerol Stocks==<br />
<br />
===For -80===<br />
<br />
Cells in 10-15% glycerol, 1:4 ratio of glycerol:culture<br />
*0.5mL 80% glycerol<br />
*2mL fresh culture<br />
<br />
===For -20===<br />
<br />
Cells in 20-40% glycerol, 1:1 ratio of glycerol:culture<br />
*1mL 80% glycerol<br />
*1mL fresh culture<br />
<br />
Add cells to glycerol, vortex, put on ice immediately, then store.<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/ProtocolsTeam:Cambridge/Protocols2009-09-17T15:27:34Z<p>Awalbridge: /* Protocols */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Protocols =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop}}<br />
[[#Producing competent cells | Competent Cells]]<br />
[[#Competent cells Transformation | Transformation ]]<br />
[[#Carotene extraction with acetone | Carotene Extraction ]]<br />
[[#Agarose gel electrophoresis | Electrophoresis ]]<br />
[[#Finnzymes Phusion (TM) PCR | Finnzymes Phusion (TM) PCR]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
{{Template:Cambridgetemplatetop}}<br />
[[#PCR procedures | PCR Procedures]]<br />
[[#Glycerol Stocks | Glycerol Stocks]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Producing competent cells ==<br />
<br />
Starting from a single colony on a plate:<br />
*Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight<br />
*Take 10ml of the culture and innoculate into one litre LB and grow in shaking incubator until OD600 of 0.2-0.3 (4 hours?)<br />
*Put culture on ice for 30 minutes <br />
*Centrifuge at 4000g for 6 minutes<br />
*Remove supernatant and resuspend cells in an equal volume of ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in 0.5 volume ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in ice-cold 10% glycerol (20ml)<br />
*Combine to form two tubes of 40ml glycerol<br />
*Repeat centrifugation<br />
*Resuspend in ice-cold glycerol (3ml)<br />
*Divide cells into 100ul aliquots and store at -80<br />
<br />
(Cells should be at a final volume of ~3 x 10^10 cells.ml^-1)<br />
<br />
== Competent cells Transformation ==<br />
<br />
*Electrocompetent cells thawed on ice<br />
*Prepare vector DNA on ice<br />
*Biobricks<br />
:*With pipette tip, punch hole through foil cover into designated well<br />
:*Add 20uL DIW<br />
:*We will be removing about 5uL; the rest needs to go in an eppendorf, labeled with biobrick number, and stored at -20°C <br />
*Violacein and melanin need to be thawed<br />
*Vector DNA pipetted into chilled 1mm separation electrocuvette = 4 total<br />
*5uL of biobricks<br />
*0.5uL of melanin and violacein plasmid<br />
*Add 45 uL Competent cells <br />
*Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles<br />
*Thoroughly dry the cuvette<br />
*1.68 kV passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 uF<br />
*Add 0.25 mL SOC liquid medium to electrocuvette <br />
*Incubate electrocuvettes at 37 degrees C for 60 minutes <br />
*Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader<br />
*Orange genes biobrick: ampicillin<br />
*Promoter for orange genes biobrick: ampicillin<br />
*Melanin: ampicillin, copper, and tyrosine<br />
*Violacin: trimethoprim <br />
*Do 1:10 dilution with SDW into a new eppendorf<br />
*Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums<br />
<br />
== Carotene extraction with acetone ==<br />
<br />
Adopted from ''Yuan et al. (2006)'':<br />
<br />
1. Incubate ''E.coli'' in 5ml LB with antibiotics at 37 oC for 20 hours.<br />
<br />
2. Harvest cells using centrifugation at 4000 rpm for 10 minutes.<br />
<br />
3. Re-suspend cells in 300 ul acetone and '''''vortex for 5 minutes'''***'' ''(***: original protocol recommended "Homogenise cells with glass beads in Bead-Beater for 30s (Biospec products)", but we did not have the equipment. Vortex is used for homogenisation instead)''. <br />
<br />
4. Centrifuge sample at 14,000 rpm for 1 minute. Collect supernatant.<br />
<br />
5. Measure absorption using spectrophotometer at 450 nm. Normalise data to cell density (OD 600 nm). This is performed using '''Omega Microplate Readers''' from BMG-Labtech.<br />
<br />
'''CAUTION: Acetone may corrode plastic microplates and cause severe damage to the equipment if left in the plate reader for too long. With microplate readers, dilute acetone extract 10 times with water (to give 200ul) before loading into plastic wells.'''<br />
<br />
== Agarose gel electrophoresis ==<br />
=== Gel preparation ===<br />
To prepare 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1X TAE buffer (obtained by diluting 10X TAE stock buffer with water) and heat in microwave until all powder desolves. Gel stains should be added when the agarose becomes cool enough to touch.<br />
<br />
For ethidium bromide gels, add 2 uL EtBr to 20 mL of agarose (makes 1 gel).<br />
<br />
=== Electrophoresis setting ===<br />
For electrophoresis, set constant voltage at 80V (with current at approximately 3 mA) and run for 30--60 minutes (or until sufficient separation of DNA).<br />
<br />
<br />
==Finnzymes Phusion (TM) PCR==<br />
<br />
===Shuna's protocol===<br />
<br />
====PCR Master Mix====<br />
*25ul 2x Phusion Master Mix<br />
*0.5uM of each primer<br />
*~5ng of DNA<br />
*Make up to 50ul with SDW<br />
<br />
====PCR Cycle====<br />
Thirty seconds at 98 degrees<br />
<br />
Thirty cycles:<br />
*98 degrees for 5-10 seconds<br />
*67 degrees for 10-30 seconds (depending on primer)<br />
*72 degrees for 30 seconds/kb<br />
Final extension at 72 degrees for 5-10 minutes<br />
Hold at four degrees.<br />
<br />
===James's protocols:===<br />
<br />
====Phusion master mix====<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
25 uL 2x Phusion master mix<br />
<br />
optional 1.5 uL DMSO<br />
<br />
make up to 50 uL with SDW<br />
<br />
====Using Phusion enzyme====<br />
<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
1 uL dNTPs<br />
<br />
0.5 uL Phusion polymerase<br />
<br />
10 uL x5 buffer (use buffer GC for high GC content, has shown to give higher product yeilds)<br />
<br />
make up to 50 uL with SDW<br />
<br />
<br />
==PCR procedures==<br />
<br />
[[Image:NANODROP.JPG | G-Storm and Finnzymes PCR Machines |thumb| right| 200px]]<br />
[[Image:FLUOstar.jpg | A FLUOstar Omega Platereader | thumb| right| 200px]]<br />
<br />
'''For high accuracy sequence PCR'''<br />
*Use the Phusion set from Finnzymes<br />
*50ul solution<br />
*Can alter conditions for optimisation<br />
<br />
'''For verification of plasmid presence/length'''<br />
*Use TAQ polymerase and buffer from stores<br />
*20ul solution<br />
*Run with standard procedure as follows (from James) for Colony PCR<br />
<br />
*'''Reaction Mixture'''<br />
:*Template: 1 uL from O/N culture (1-2 uL if colony picked straight from plate into water)<br />
:*VF2: 1uL<br />
:*VR: 1uL<br />
:*Eco-Taq: 0.2 uL<br />
:*10X buffer: 5 uL<br />
:*dNTPs: 0.4 uL (stock is 10 mM)<br />
:*make up to 20 uL volume with H20<br />
*'''Reaction procedure'''<br />
:*95 degrees C for 2 minutes<br />
:*33 cycles of:<br />
::*95 degrees C for 30 seconds (denaturation)<br />
::*65 degrees C for 30 seconds (annealing)<br />
::*72 degrees C, 1000bp/min (elongation)<br />
:*72 degrees C for 5 minutes<br />
:*hold at 4 degrees C<br />
<br />
'''Gels'''<br />
*Run on EtBr for good quality viewing (make agarose gel). This is viewed in the red-room downstairs.<br />
*Run on SYBR-safe gel if the DNA is required. This can be viewed under blue light in the covered dark-area.<br />
<br />
==Glycerol Stocks==<br />
<br />
===For -80===<br />
<br />
Cells in 10-15% glycerol, 1:4 ratio of glycerol:culture<br />
*0.5mL 80% glycerol<br />
*2mL fresh culture<br />
<br />
===For -20===<br />
<br />
Cells in 20-40% glycerol, 1:1 ratio of glycerol:culture<br />
*1mL 80% glycerol<br />
*1mL fresh culture<br />
<br />
Add cells to glycerol, vortex, put on ice immediately, then store.<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/ProtocolsTeam:Cambridge/Protocols2009-09-17T15:26:41Z<p>Awalbridge: /* Protocols */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Protocols =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop}}<br />
[[#Producing competent cells | Competent Cells]]<br />
[[#Competent cells Transformation | Transformation ]]<br />
[[#Carotene extraction with acetone | Carotene Extraction ]]<br />
[[#Agarose gel electrophoresis | Electrophoresis ]]<br />
[[#Finnzymes Phusion (TM) PCR | PCR]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
{{Template:Cambridgetemplatetop}}<br />
[[#PCR procedures | PCR Procedures]]<br />
[[#Glycerol Stock | Glycerol<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Producing competent cells ==<br />
<br />
Starting from a single colony on a plate:<br />
*Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight<br />
*Take 10ml of the culture and innoculate into one litre LB and grow in shaking incubator until OD600 of 0.2-0.3 (4 hours?)<br />
*Put culture on ice for 30 minutes <br />
*Centrifuge at 4000g for 6 minutes<br />
*Remove supernatant and resuspend cells in an equal volume of ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in 0.5 volume ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in ice-cold 10% glycerol (20ml)<br />
*Combine to form two tubes of 40ml glycerol<br />
*Repeat centrifugation<br />
*Resuspend in ice-cold glycerol (3ml)<br />
*Divide cells into 100ul aliquots and store at -80<br />
<br />
(Cells should be at a final volume of ~3 x 10^10 cells.ml^-1)<br />
<br />
== Competent cells Transformation ==<br />
<br />
*Electrocompetent cells thawed on ice<br />
*Prepare vector DNA on ice<br />
*Biobricks<br />
:*With pipette tip, punch hole through foil cover into designated well<br />
:*Add 20uL DIW<br />
:*We will be removing about 5uL; the rest needs to go in an eppendorf, labeled with biobrick number, and stored at -20°C <br />
*Violacein and melanin need to be thawed<br />
*Vector DNA pipetted into chilled 1mm separation electrocuvette = 4 total<br />
*5uL of biobricks<br />
*0.5uL of melanin and violacein plasmid<br />
*Add 45 uL Competent cells <br />
*Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles<br />
*Thoroughly dry the cuvette<br />
*1.68 kV passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 uF<br />
*Add 0.25 mL SOC liquid medium to electrocuvette <br />
*Incubate electrocuvettes at 37 degrees C for 60 minutes <br />
*Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader<br />
*Orange genes biobrick: ampicillin<br />
*Promoter for orange genes biobrick: ampicillin<br />
*Melanin: ampicillin, copper, and tyrosine<br />
*Violacin: trimethoprim <br />
*Do 1:10 dilution with SDW into a new eppendorf<br />
*Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums<br />
<br />
== Carotene extraction with acetone ==<br />
<br />
Adopted from ''Yuan et al. (2006)'':<br />
<br />
1. Incubate ''E.coli'' in 5ml LB with antibiotics at 37 oC for 20 hours.<br />
<br />
2. Harvest cells using centrifugation at 4000 rpm for 10 minutes.<br />
<br />
3. Re-suspend cells in 300 ul acetone and '''''vortex for 5 minutes'''***'' ''(***: original protocol recommended "Homogenise cells with glass beads in Bead-Beater for 30s (Biospec products)", but we did not have the equipment. Vortex is used for homogenisation instead)''. <br />
<br />
4. Centrifuge sample at 14,000 rpm for 1 minute. Collect supernatant.<br />
<br />
5. Measure absorption using spectrophotometer at 450 nm. Normalise data to cell density (OD 600 nm). This is performed using '''Omega Microplate Readers''' from BMG-Labtech.<br />
<br />
'''CAUTION: Acetone may corrode plastic microplates and cause severe damage to the equipment if left in the plate reader for too long. With microplate readers, dilute acetone extract 10 times with water (to give 200ul) before loading into plastic wells.'''<br />
<br />
== Agarose gel electrophoresis ==<br />
=== Gel preparation ===<br />
To prepare 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1X TAE buffer (obtained by diluting 10X TAE stock buffer with water) and heat in microwave until all powder desolves. Gel stains should be added when the agarose becomes cool enough to touch.<br />
<br />
For ethidium bromide gels, add 2 uL EtBr to 20 mL of agarose (makes 1 gel).<br />
<br />
=== Electrophoresis setting ===<br />
For electrophoresis, set constant voltage at 80V (with current at approximately 3 mA) and run for 30--60 minutes (or until sufficient separation of DNA).<br />
<br />
<br />
==Finnzymes Phusion (TM) PCR==<br />
<br />
===Shuna's protocol===<br />
<br />
====PCR Master Mix====<br />
*25ul 2x Phusion Master Mix<br />
*0.5uM of each primer<br />
*~5ng of DNA<br />
*Make up to 50ul with SDW<br />
<br />
====PCR Cycle====<br />
Thirty seconds at 98 degrees<br />
<br />
Thirty cycles:<br />
*98 degrees for 5-10 seconds<br />
*67 degrees for 10-30 seconds (depending on primer)<br />
*72 degrees for 30 seconds/kb<br />
Final extension at 72 degrees for 5-10 minutes<br />
Hold at four degrees.<br />
<br />
===James's protocols:===<br />
<br />
====Phusion master mix====<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
25 uL 2x Phusion master mix<br />
<br />
optional 1.5 uL DMSO<br />
<br />
make up to 50 uL with SDW<br />
<br />
====Using Phusion enzyme====<br />
<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
1 uL dNTPs<br />
<br />
0.5 uL Phusion polymerase<br />
<br />
10 uL x5 buffer (use buffer GC for high GC content, has shown to give higher product yeilds)<br />
<br />
make up to 50 uL with SDW<br />
<br />
<br />
==PCR procedures==<br />
<br />
[[Image:NANODROP.JPG | G-Storm and Finnzymes PCR Machines |thumb| right| 200px]]<br />
[[Image:FLUOstar.jpg | A FLUOstar Omega Platereader | thumb| right| 200px]]<br />
<br />
'''For high accuracy sequence PCR'''<br />
*Use the Phusion set from Finnzymes<br />
*50ul solution<br />
*Can alter conditions for optimisation<br />
<br />
'''For verification of plasmid presence/length'''<br />
*Use TAQ polymerase and buffer from stores<br />
*20ul solution<br />
*Run with standard procedure as follows (from James) for Colony PCR<br />
<br />
*'''Reaction Mixture'''<br />
:*Template: 1 uL from O/N culture (1-2 uL if colony picked straight from plate into water)<br />
:*VF2: 1uL<br />
:*VR: 1uL<br />
:*Eco-Taq: 0.2 uL<br />
:*10X buffer: 5 uL<br />
:*dNTPs: 0.4 uL (stock is 10 mM)<br />
:*make up to 20 uL volume with H20<br />
*'''Reaction procedure'''<br />
:*95 degrees C for 2 minutes<br />
:*33 cycles of:<br />
::*95 degrees C for 30 seconds (denaturation)<br />
::*65 degrees C for 30 seconds (annealing)<br />
::*72 degrees C, 1000bp/min (elongation)<br />
:*72 degrees C for 5 minutes<br />
:*hold at 4 degrees C<br />
<br />
'''Gels'''<br />
*Run on EtBr for good quality viewing (make agarose gel). This is viewed in the red-room downstairs.<br />
*Run on SYBR-safe gel if the DNA is required. This can be viewed under blue light in the covered dark-area.<br />
<br />
==Glycerol Stocks==<br />
<br />
===For -80===<br />
<br />
Cells in 10-15% glycerol, 1:4 ratio of glycerol:culture<br />
*0.5mL 80% glycerol<br />
*2mL fresh culture<br />
<br />
===For -20===<br />
<br />
Cells in 20-40% glycerol, 1:1 ratio of glycerol:culture<br />
*1mL 80% glycerol<br />
*1mL fresh culture<br />
<br />
Add cells to glycerol, vortex, put on ice immediately, then store.<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/ProtocolsTeam:Cambridge/Protocols2009-09-15T09:42:17Z<p>Awalbridge: </p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Protocols =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop}}<br />
[[#Producing competent cells | Competent Cells]]<br />
[[#Competent cells Transformation | Transformation ]]<br />
[[#Carotene extraction with acetone | Carotene Extraction ]]<br />
[[#Agarose gel electrophoresis | Electrophoresis ]]<br />
[[#Finnzymes Phusion (TM) PCR | PCR]]<br />
[[#PCR procedures | PCR Procedures]]<br />
[[#Glycerol Stock | Glycerol<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Producing competent cells ==<br />
<br />
Starting from a single colony on a plate:<br />
*Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight<br />
*Take 10ml of the culture and innoculate into one litre LB and grow in shaking incubator until OD600 of 0.2-0.3 (4 hours?)<br />
*Put culture on ice for 30 minutes <br />
*Centrifuge at 4000g for 6 minutes<br />
*Remove supernatant and resuspend cells in an equal volume of ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in 0.5 volume ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in ice-cold 10% glycerol (20ml)<br />
*Combine to form two tubes of 40ml glycerol<br />
*Repeat centrifugation<br />
*Resuspend in ice-cold glycerol (3ml)<br />
*Divide cells into 100ul aliquots and store at -80<br />
<br />
(Cells should be at a final volume of ~3 x 10^10 cells.ml^-1)<br />
<br />
== Competent cells Transformation ==<br />
<br />
*Electrocompetent cells thawed on ice<br />
*Prepare vector DNA on ice<br />
*Biobricks<br />
:*With pipette tip, punch hole through foil cover into designated well<br />
:*Add 20uL DIW<br />
:*We will be removing about 5uL; the rest needs to go in an eppendorf, labeled with biobrick number, and stored at -20°C <br />
*Violacein and melanin need to be thawed<br />
*Vector DNA pipetted into chilled 1mm separation electrocuvette = 4 total<br />
*5uL of biobricks<br />
*0.5uL of melanin and violacein plasmid<br />
*Add 45 uL Competent cells <br />
*Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles<br />
*Thoroughly dry the cuvette<br />
*1.68 kV passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 uF<br />
*Add 0.25 mL SOC liquid medium to electrocuvette <br />
*Incubate electrocuvettes at 37 degrees C for 60 minutes <br />
*Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader<br />
*Orange genes biobrick: ampicillin<br />
*Promoter for orange genes biobrick: ampicillin<br />
*Melanin: ampicillin, copper, and tyrosine<br />
*Violacin: trimethoprim <br />
*Do 1:10 dilution with SDW into a new eppendorf<br />
*Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums<br />
<br />
== Carotene extraction with acetone ==<br />
<br />
Adopted from ''Yuan et al. (2006)'':<br />
<br />
1. Incubate ''E.coli'' in 5ml LB with antibiotics at 37 oC for 20 hours.<br />
<br />
2. Harvest cells using centrifugation at 4000 rpm for 10 minutes.<br />
<br />
3. Re-suspend cells in 300 ul acetone and '''''vortex for 5 minutes'''***'' ''(***: original protocol recommended "Homogenise cells with glass beads in Bead-Beater for 30s (Biospec products)", but we did not have the equipment. Vortex is used for homogenisation instead)''. <br />
<br />
4. Centrifuge sample at 14,000 rpm for 1 minute. Collect supernatant.<br />
<br />
5. Measure absorption using spectrophotometer at 450 nm. Normalise data to cell density (OD 600 nm). This is performed using '''Omega Microplate Readers''' from BMG-Labtech.<br />
<br />
'''CAUTION: Acetone may corrode plastic microplates and cause severe damage to the equipment if left in the plate reader for too long. With microplate readers, dilute acetone extract 10 times with water (to give 200ul) before loading into plastic wells.'''<br />
<br />
== Agarose gel electrophoresis ==<br />
=== Gel preparation ===<br />
To prepare 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1X TAE buffer (obtained by diluting 10X TAE stock buffer with water) and heat in microwave until all powder desolves. Gel stains should be added when the agarose becomes cool enough to touch.<br />
<br />
For ethidium bromide gels, add 2 uL EtBr to 20 mL of agarose (makes 1 gel).<br />
<br />
=== Electrophoresis setting ===<br />
For electrophoresis, set constant voltage at 80V (with current at approximately 3 mA) and run for 30--60 minutes (or until sufficient separation of DNA).<br />
<br />
<br />
==Finnzymes Phusion (TM) PCR==<br />
<br />
===Shuna's protocol===<br />
<br />
====PCR Master Mix====<br />
*25ul 2x Phusion Master Mix<br />
*0.5uM of each primer<br />
*~5ng of DNA<br />
*Make up to 50ul with SDW<br />
<br />
====PCR Cycle====<br />
Thirty seconds at 98 degrees<br />
<br />
Thirty cycles:<br />
*98 degrees for 5-10 seconds<br />
*67 degrees for 10-30 seconds (depending on primer)<br />
*72 degrees for 30 seconds/kb<br />
Final extension at 72 degrees for 5-10 minutes<br />
Hold at four degrees.<br />
<br />
===James's protocols:===<br />
<br />
====Phusion master mix====<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
25 uL 2x Phusion master mix<br />
<br />
optional 1.5 uL DMSO<br />
<br />
make up to 50 uL with SDW<br />
<br />
====Using Phusion enzyme====<br />
<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
1 uL dNTPs<br />
<br />
0.5 uL Phusion polymerase<br />
<br />
10 uL x5 buffer (use buffer GC for high GC content, has shown to give higher product yeilds)<br />
<br />
make up to 50 uL with SDW<br />
<br />
<br />
==PCR procedures==<br />
<br />
[[Image:NANODROP.JPG | G-Storm and Finnzymes PCR Machines |thumb| right| 200px]]<br />
[[Image:FLUOstar.jpg | A FLUOstar Omega Platereader | thumb| right| 200px]]<br />
<br />
'''For high accuracy sequence PCR'''<br />
*Use the Phusion set from Finnzymes<br />
*50ul solution<br />
*Can alter conditions for optimisation<br />
<br />
'''For verification of plasmid presence/length'''<br />
*Use TAQ polymerase and buffer from stores<br />
*20ul solution<br />
*Run with standard procedure as follows (from James) for Colony PCR<br />
<br />
*'''Reaction Mixture'''<br />
:*Template: 1 uL from O/N culture (1-2 uL if colony picked straight from plate into water)<br />
:*VF2: 1uL<br />
:*VR: 1uL<br />
:*Eco-Taq: 0.2 uL<br />
:*10X buffer: 5 uL<br />
:*dNTPs: 0.4 uL (stock is 10 mM)<br />
:*make up to 20 uL volume with H20<br />
*'''Reaction procedure'''<br />
:*95 degrees C for 2 minutes<br />
:*33 cycles of:<br />
::*95 degrees C for 30 seconds (denaturation)<br />
::*65 degrees C for 30 seconds (annealing)<br />
::*72 degrees C, 1000bp/min (elongation)<br />
:*72 degrees C for 5 minutes<br />
:*hold at 4 degrees C<br />
<br />
'''Gels'''<br />
*Run on EtBr for good quality viewing (make agarose gel). This is viewed in the red-room downstairs.<br />
*Run on SYBR-safe gel if the DNA is required. This can be viewed under blue light in the covered dark-area.<br />
<br />
==Glycerol Stocks==<br />
<br />
===For -80===<br />
<br />
Cells in 10-15% glycerol, 1:4 ratio of glycerol:culture<br />
*0.5mL 80% glycerol<br />
*2mL fresh culture<br />
<br />
===For -20===<br />
<br />
Cells in 20-40% glycerol, 1:1 ratio of glycerol:culture<br />
*1mL 80% glycerol<br />
*1mL fresh culture<br />
<br />
Add cells to glycerol, vortex, put on ice immediately, then store.<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/ProtocolsTeam:Cambridge/Protocols2009-09-15T09:41:43Z<p>Awalbridge: </p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop}}<br />
[[#Producing competent cells | Competent Cells]]<br />
[[#Competent cells Transformation | Transformation ]]<br />
[[#Carotene extraction with acetone | Carotene Extraction ]]<br />
[[#Agarose gel electrophoresis | Electrophoresis ]]<br />
[[#Finnzymes Phusion (TM) PCR | PCR]]<br />
[[#PCR procedures | PCR Procedures]]<br />
[[#Glycerol Stock | Glycerol<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
= Protocols =<br />
<br />
== Producing competent cells ==<br />
<br />
Starting from a single colony on a plate:<br />
*Transfer colony into 50ml liquid LB media and leave in a 200rpm shaking incubator overnight<br />
*Take 10ml of the culture and innoculate into one litre LB and grow in shaking incubator until OD600 of 0.2-0.3 (4 hours?)<br />
*Put culture on ice for 30 minutes <br />
*Centrifuge at 4000g for 6 minutes<br />
*Remove supernatant and resuspend cells in an equal volume of ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in 0.5 volume ice-cold 0.1mM HEPES<br />
*Repeat centrifugation<br />
*Resuspend cells in ice-cold 10% glycerol (20ml)<br />
*Combine to form two tubes of 40ml glycerol<br />
*Repeat centrifugation<br />
*Resuspend in ice-cold glycerol (3ml)<br />
*Divide cells into 100ul aliquots and store at -80<br />
<br />
(Cells should be at a final volume of ~3 x 10^10 cells.ml^-1)<br />
<br />
== Competent cells Transformation ==<br />
<br />
*Electrocompetent cells thawed on ice<br />
*Prepare vector DNA on ice<br />
*Biobricks<br />
:*With pipette tip, punch hole through foil cover into designated well<br />
:*Add 20uL DIW<br />
:*We will be removing about 5uL; the rest needs to go in an eppendorf, labeled with biobrick number, and stored at -20°C <br />
*Violacein and melanin need to be thawed<br />
*Vector DNA pipetted into chilled 1mm separation electrocuvette = 4 total<br />
*5uL of biobricks<br />
*0.5uL of melanin and violacein plasmid<br />
*Add 45 uL Competent cells <br />
*Tap electrocuvette gently to evenly spread mixture in the electrocuvette gap with no air bubbles<br />
*Thoroughly dry the cuvette<br />
*1.68 kV passed across cuvette, 5.1-5.4 time constant at 200 ohms and 25 uF<br />
*Add 0.25 mL SOC liquid medium to electrocuvette <br />
*Incubate electrocuvettes at 37 degrees C for 60 minutes <br />
*Pipette 150uL onto a (warmed) selective LB agar plate, spread with blue spreader<br />
*Orange genes biobrick: ampicillin<br />
*Promoter for orange genes biobrick: ampicillin<br />
*Melanin: ampicillin, copper, and tyrosine<br />
*Violacin: trimethoprim <br />
*Do 1:10 dilution with SDW into a new eppendorf<br />
*Pipette 150uL onto a selective LB agar plate, spread with blue spreader, 4 separate inoculums<br />
<br />
== Carotene extraction with acetone ==<br />
<br />
Adopted from ''Yuan et al. (2006)'':<br />
<br />
1. Incubate ''E.coli'' in 5ml LB with antibiotics at 37 oC for 20 hours.<br />
<br />
2. Harvest cells using centrifugation at 4000 rpm for 10 minutes.<br />
<br />
3. Re-suspend cells in 300 ul acetone and '''''vortex for 5 minutes'''***'' ''(***: original protocol recommended "Homogenise cells with glass beads in Bead-Beater for 30s (Biospec products)", but we did not have the equipment. Vortex is used for homogenisation instead)''. <br />
<br />
4. Centrifuge sample at 14,000 rpm for 1 minute. Collect supernatant.<br />
<br />
5. Measure absorption using spectrophotometer at 450 nm. Normalise data to cell density (OD 600 nm). This is performed using '''Omega Microplate Readers''' from BMG-Labtech.<br />
<br />
'''CAUTION: Acetone may corrode plastic microplates and cause severe damage to the equipment if left in the plate reader for too long. With microplate readers, dilute acetone extract 10 times with water (to give 200ul) before loading into plastic wells.'''<br />
<br />
== Agarose gel electrophoresis ==<br />
=== Gel preparation ===<br />
To prepare 1% agarose gel (say 200ml), add 2g of agarose powder to 200 ml of 1X TAE buffer (obtained by diluting 10X TAE stock buffer with water) and heat in microwave until all powder desolves. Gel stains should be added when the agarose becomes cool enough to touch.<br />
<br />
For ethidium bromide gels, add 2 uL EtBr to 20 mL of agarose (makes 1 gel).<br />
<br />
=== Electrophoresis setting ===<br />
For electrophoresis, set constant voltage at 80V (with current at approximately 3 mA) and run for 30--60 minutes (or until sufficient separation of DNA).<br />
<br />
<br />
==Finnzymes Phusion (TM) PCR==<br />
<br />
===Shuna's protocol===<br />
<br />
====PCR Master Mix====<br />
*25ul 2x Phusion Master Mix<br />
*0.5uM of each primer<br />
*~5ng of DNA<br />
*Make up to 50ul with SDW<br />
<br />
====PCR Cycle====<br />
Thirty seconds at 98 degrees<br />
<br />
Thirty cycles:<br />
*98 degrees for 5-10 seconds<br />
*67 degrees for 10-30 seconds (depending on primer)<br />
*72 degrees for 30 seconds/kb<br />
Final extension at 72 degrees for 5-10 minutes<br />
Hold at four degrees.<br />
<br />
===James's protocols:===<br />
<br />
====Phusion master mix====<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
25 uL 2x Phusion master mix<br />
<br />
optional 1.5 uL DMSO<br />
<br />
make up to 50 uL with SDW<br />
<br />
====Using Phusion enzyme====<br />
<br />
2.5 uL forward primter<br />
<br />
2.5 uL reverse primer<br />
<br />
0.5 uL template<br />
<br />
1 uL dNTPs<br />
<br />
0.5 uL Phusion polymerase<br />
<br />
10 uL x5 buffer (use buffer GC for high GC content, has shown to give higher product yeilds)<br />
<br />
make up to 50 uL with SDW<br />
<br />
<br />
==PCR procedures==<br />
<br />
[[Image:NANODROP.JPG | G-Storm and Finnzymes PCR Machines |thumb| right| 200px]]<br />
[[Image:FLUOstar.jpg | A FLUOstar Omega Platereader | thumb| right| 200px]]<br />
<br />
'''For high accuracy sequence PCR'''<br />
*Use the Phusion set from Finnzymes<br />
*50ul solution<br />
*Can alter conditions for optimisation<br />
<br />
'''For verification of plasmid presence/length'''<br />
*Use TAQ polymerase and buffer from stores<br />
*20ul solution<br />
*Run with standard procedure as follows (from James) for Colony PCR<br />
<br />
*'''Reaction Mixture'''<br />
:*Template: 1 uL from O/N culture (1-2 uL if colony picked straight from plate into water)<br />
:*VF2: 1uL<br />
:*VR: 1uL<br />
:*Eco-Taq: 0.2 uL<br />
:*10X buffer: 5 uL<br />
:*dNTPs: 0.4 uL (stock is 10 mM)<br />
:*make up to 20 uL volume with H20<br />
*'''Reaction procedure'''<br />
:*95 degrees C for 2 minutes<br />
:*33 cycles of:<br />
::*95 degrees C for 30 seconds (denaturation)<br />
::*65 degrees C for 30 seconds (annealing)<br />
::*72 degrees C, 1000bp/min (elongation)<br />
:*72 degrees C for 5 minutes<br />
:*hold at 4 degrees C<br />
<br />
'''Gels'''<br />
*Run on EtBr for good quality viewing (make agarose gel). This is viewed in the red-room downstairs.<br />
*Run on SYBR-safe gel if the DNA is required. This can be viewed under blue light in the covered dark-area.<br />
<br />
==Glycerol Stocks==<br />
<br />
===For -80===<br />
<br />
Cells in 10-15% glycerol, 1:4 ratio of glycerol:culture<br />
*0.5mL 80% glycerol<br />
*2mL fresh culture<br />
<br />
===For -20===<br />
<br />
Cells in 20-40% glycerol, 1:1 ratio of glycerol:culture<br />
*1mL 80% glycerol<br />
*1mL fresh culture<br />
<br />
Add cells to glycerol, vortex, put on ice immediately, then store.<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Project/AmplificationTeam:Cambridge/Project/Amplification2009-09-11T18:29:12Z<p>Awalbridge: /* Characterisation */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
= The Threshold Device =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop}}<br />
[[#Introduction | Introduction ]]<br />
[[#Recreating Previous Work | Previous Work]]<br />
[[#Characterisation | Characterisation ]]<br />
[[# | ]]<br />
[[# | ]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Introduction ==<br />
===Cambridge 2007===<br />
<br />
The Cambridge 2007 iGEM team developed a PoPS amplifier system using phage activators and promoters. The system works by using a PoPS input to make an activator protein, as shown in the diagram from their wiki below, which then binds to a promoter and generates a PoPS output. <br />
<br />
[[Image:amplifier07.jpg]]<br />
<br />
<br />
In order to quantify the ratio between PoPS in and PoPS out, the team built the following construction on the high copy plasmid pSB1A2, with mRFP and GFP as PoPS reporter. They genenerated 15 total combinations of different activators and promoters.<br />
<br />
[[Image:construction07.jpg]]<br />
<br />
<br />
They successfully quantified the PoPS amplification factors for each activator/promoter combination after arabinose induction. <br />
<br />
===Further Work - Cambridge 2009===<br />
<br />
We have two major goals - characterisation and reconstruction. <br />
<br />
'''Characterisation'''<br />
<br />
First, we hope to characterise the Cambridge 2007 activator constructs with RFP and GFP reporters on low copy plasmids, looking at three major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.<br />
<br />
[[Image:characterization.jpg]]<br />
<br />
'''Reconstruction'''<br />
<br />
Our second goal is to build a library of devices following the pattern in the figure below, which can also be abstracted as a PoPS converter:<br />
<br />
[[Image:thresholddevice3.jpg]] = [[Image:converter.jpg]] <br />
<br />
Our hope is that this well characterised library can be used by future iGEM teams to fit the needs of their projects.<br />
<br />
== Recreating Previous Work ==<br />
<br />
We began by recreating the 2007 team's data with some select amplifier constructs. We have the advantage over the 2007 team in that we have a better plate reader that is able to take OD600 absorbance readings at the same time as taking RFP and GFP output readings. For our transformations, we used the ''E. coli'' host strain BW27783. This host strain constitutively expresses arabinose transporters and is unable to metabolise arabinose, making it an ideal host for arabinose titration experiments.<br />
<br />
'''Results''': The 2007 team's hopes for future work included investigating a problem they attributed to the toxicity of high levels of activator in the cell. Overnight OD600 readings of cells transformed with their amplifier constructs indicated cell death. However, these OD readings were conducted separately from their RFP and GFP output measurements. The 2009 team gathered data on the plate reader capable of taking OD600 absorbance readings as well as RFP and GFP output readings; no OD600 readings suggested cell death due to toxicity.<br />
<br />
*graphs*<br />
<br />
== Characterisation ==<br />
<br />
We moved all 15 activator constructs onto pSB3K3, a low copy plasmid. The standard promoter for 1 RPU, J69591, is also on pSB3K3 and has a GFP reporter, so we can make meaningful comparisons on the plate reader.<br />
<br />
=== Maximum Rates against Arabinose Concentrations ===<br />
<br />
==== 80 ====<br />
[[Image:Cambridge_maxrates1.jpg | 600px]]<br />
<br />
==== 81 ====<br />
[[Image:Cambridge_maxrates2.jpg | 600px]]<br />
<br />
==== 82 ====<br />
[[Image:Cambridge_maxrates3.jpg | 600px]]<br />
<br />
==== 84 ====<br />
[[Image:Cambridge_maxrates4.jpg | 600px]]<br />
<br />
==== 85 ====<br />
[[Image:Cambridge_maxrates5.jpg | 600px]]<br />
<br />
==== 90 ====<br />
[[Image:Cambridge_maxrates6.jpg | 600px]]<br />
<br />
==== 92 ====<br />
[[Image:Cambridge_maxrates7.jpg | 600px]]<br />
<br />
==== 94 ====<br />
[[Image:Cambridge_maxrates8.jpg | 600px]]<br />
<br />
==== 95 ====<br />
[[Image:Cambridge_maxrates9.jpg | 600px]]<br />
<br />
== Results ==<br />
<br />
<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates9.jpgFile:Cambridge maxrates9.jpg2009-09-11T18:26:36Z<p>Awalbridge: uploaded a new version of "Image:Cambridge maxrates9.jpg"</p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates8.jpgFile:Cambridge maxrates8.jpg2009-09-11T18:26:13Z<p>Awalbridge: uploaded a new version of "Image:Cambridge maxrates8.jpg"</p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates7.jpgFile:Cambridge maxrates7.jpg2009-09-11T18:25:45Z<p>Awalbridge: uploaded a new version of "Image:Cambridge maxrates7.jpg"</p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates6.jpgFile:Cambridge maxrates6.jpg2009-09-11T18:25:26Z<p>Awalbridge: uploaded a new version of "Image:Cambridge maxrates6.jpg"</p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates5.jpgFile:Cambridge maxrates5.jpg2009-09-11T18:25:07Z<p>Awalbridge: uploaded a new version of "Image:Cambridge maxrates5.jpg"</p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates4.jpgFile:Cambridge maxrates4.jpg2009-09-11T18:24:45Z<p>Awalbridge: uploaded a new version of "Image:Cambridge maxrates4.jpg"</p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates3.jpgFile:Cambridge maxrates3.jpg2009-09-11T18:24:22Z<p>Awalbridge: uploaded a new version of "Image:Cambridge maxrates3.jpg"</p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates2.jpgFile:Cambridge maxrates2.jpg2009-09-11T18:23:56Z<p>Awalbridge: uploaded a new version of "Image:Cambridge maxrates2.jpg"</p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates1.jpgFile:Cambridge maxrates1.jpg2009-09-11T17:46:55Z<p>Awalbridge: uploaded a new version of "Image:Cambridge maxrates1.jpg"</p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates9.jpgFile:Cambridge maxrates9.jpg2009-09-11T17:37:21Z<p>Awalbridge: </p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates8.jpgFile:Cambridge maxrates8.jpg2009-09-11T17:37:09Z<p>Awalbridge: </p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates7.jpgFile:Cambridge maxrates7.jpg2009-09-11T17:36:57Z<p>Awalbridge: </p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates6.jpgFile:Cambridge maxrates6.jpg2009-09-11T17:36:45Z<p>Awalbridge: </p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates5.jpgFile:Cambridge maxrates5.jpg2009-09-11T17:36:33Z<p>Awalbridge: </p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates4.jpgFile:Cambridge maxrates4.jpg2009-09-11T17:36:21Z<p>Awalbridge: </p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates3.jpgFile:Cambridge maxrates3.jpg2009-09-11T17:36:05Z<p>Awalbridge: </p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates2.jpgFile:Cambridge maxrates2.jpg2009-09-11T17:35:55Z<p>Awalbridge: </p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/File:Cambridge_maxrates1.jpgFile:Cambridge maxrates1.jpg2009-09-11T17:35:10Z<p>Awalbridge: </p>
<hr />
<div></div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Project/AmplificationTeam:Cambridge/Project/Amplification2009-09-11T14:32:34Z<p>Awalbridge: /* The Threshold Device */ ize -> ise</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
= The Threshold Device =<br />
<br />
<!-- This is for the top grey / blue links bar !--><br />
{{Template:Cambridgetemplatetop}}<br />
[[#Introduction | Introduction ]]<br />
[[#Recreating Previous Work | Previous Work]]<br />
[[#Characterisation | Characterisation ]]<br />
[[# | ]]<br />
[[# | ]]<br />
{{Template:Cambridgetemplatebottom}}<br />
<br />
== Introduction ==<br />
===Cambridge 2007===<br />
<br />
The Cambridge 2007 iGEM team developed a PoPS amplifier system using phage activators and promoters. The system works by using a PoPS input to make an activator protein, as shown in the diagram from their wiki below, which then binds to a promoter and generates a PoPS output. <br />
<br />
[[Image:amplifier07.jpg]]<br />
<br />
<br />
In order to quantify the ratio between PoPS in and PoPS out, the team built the following construction on the high copy plasmid pSB1A2, with mRFP and GFP as PoPS reporter. They genenerated 15 total combinations of different activators and promoters.<br />
<br />
[[Image:construction07.jpg]]<br />
<br />
<br />
They successfully quantified the PoPS amplification factors for each activator/promoter combination after arabinose induction. <br />
<br />
===Further Work - Cambridge 2009===<br />
<br />
We have two major goals - characterisation and reconstruction. <br />
<br />
'''Characterisation'''<br />
<br />
First, we hope to characterise the Cambridge 2007 activator constructs with RFP and GFP reporters on low copy plasmids, looking at three major characteristics relating input (arabinose) to output (GFP) and how they are modified compared to pBad/AraC on its own.<br />
<br />
[[Image:characterization.jpg]]<br />
<br />
'''Reconstruction'''<br />
<br />
Our second goal is to build a library of devices following the pattern in the figure below, which can also be abstracted as a PoPS converter:<br />
<br />
[[Image:thresholddevice3.jpg]] = [[Image:converter.jpg]] <br />
<br />
Our hope is that this well characterised library can be used by future iGEM teams to fit the needs of their projects.<br />
<br />
== Recreating Previous Work ==<br />
<br />
We began by recreating the 2007 team's data with some select amplifier constructs. We have the advantage over the 2007 team in that we have a better plate reader that is able to take OD600 absorbance readings at the same time as taking RFP and GFP output readings. For our transformations, we used the ''E. coli'' host strain BW27783. This host strain constitutively expresses arabinose transporters and is unable to metabolise arabinose, making it an ideal host for arabinose titration experiments.<br />
<br />
'''Results''': The 2007 team's hopes for future work included investigating a problem they attributed to the toxicity of high levels of activator in the cell. Overnight OD600 readings of cells transformed with their amplifier constructs indicated cell death. However, these OD readings were conducted separately from their RFP and GFP output measurements. The 2009 team gathered data on the plate reader capable of taking OD600 absorbance readings as well as RFP and GFP output readings; no OD600 readings suggested cell death due to toxicity.<br />
<br />
*graphs*<br />
<br />
== Characterisation ==<br />
<br />
We moved all 15 activator constructs onto pSB3K3, a low copy plasmid. The standard promoter for 1 RPU, J69591, is also on pSB3K3 and has a GFP reporter, so we can make meaningful comparisons on the plate reader.<br />
<br />
== Results ==<br />
<br />
<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Notebook/Week9Team:Cambridge/Notebook/Week92009-09-11T14:23:56Z<p>Awalbridge: /* Week 9 */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Week 9 =<br />
<br />
{{Template:CambridgeNotepad}}<br />
<br />
== Monday ==<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Overnight cultures of 70, 71, 72, 74, 91 in pSB3K3 in arabinose strain in order to make glycerol stocks and streak single colonies for the plate reader.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Tuesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
All activator constructs are now ready for analysis on the plate reader. <br />
<br />
82 run on plate reader during day<br />
<br />
85 overnight<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Wednesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Confirmed successful ligation of pBad and I746350, I746351, I746352. <br />
<br />
90 run during day<br />
<br />
92 run overnight<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Thursday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
94 run during day<br />
<br />
95 overnight<br />
<br />
Attempted the following standard assemblies:<br />
<br />
*pBad + I746350 to B0015<br />
*pBad + I746351 to B0015<br />
*pBad + I746352 to B0015<br />
*I746351 to B0015<br />
*I746352 to B0015<br />
*I746352 to B0015<br />
<br />
The first three will be used to construct a complete device, with pBad as the sensor promoter and a pigment operon as the pigment-generating device. The pigment we chose for our proof of concept will be placed downstream of each of the 5 phage promoters, and then combined to give 15 combinations of activators and promoters, giving the construction below. We still need to decide which pigment to use for our proof of concept.<br />
<br />
<br />
[[Image:proofofconcept.jpg]]<br />
<br />
<br />
The second three will be used to construct a library of threshold devices which can be abstracted as a PoPS converter (below).<br />
<br />
[[Image:thresholddeviceabstraction.jpg]]<br />
<br />
The next step will be to attach the phage promoter downstream to create a catalogue of 15 different devices of the form:<br />
<br />
[[Image:completedevice.jpg]]<br />
<br />
<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Friday ==<br />
<br />
75 run during day<br />
<br />
91 overnight<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Notebook/Week9Team:Cambridge/Notebook/Week92009-09-11T14:22:28Z<p>Awalbridge: /* Threshold Devices */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Week 9 =<br />
<br />
{{Template:CambridgeNotepad}}<br />
<br />
== Monday ==<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Overnight cultures of 70, 71, 72, 74, 91 in pSB3K3 in arabinose strain in order to make glycerol stocks and streak single colonies for the plate reader.<br />
<br />
82 run on plate reader during day<br />
<br />
85 overnight<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Tuesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
All activator constructs are now ready for analysis on the plate reader. <br />
<br />
90 run during day<br />
<br />
92 run overnight<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Wednesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Confirmed successful ligation of pBad and I746350, I746351, I746352. <br />
<br />
94 run during day<br />
<br />
95 overnight<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Thursday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Attempted the following standard assemblies:<br />
<br />
*pBad + I746350 to B0015<br />
*pBad + I746351 to B0015<br />
*pBad + I746352 to B0015<br />
*I746351 to B0015<br />
*I746352 to B0015<br />
*I746352 to B0015<br />
<br />
The first three will be used to construct a complete device, with pBad as the sensor promoter and a pigment operon as the pigment-generating device. The pigment we chose for our proof of concept will be placed downstream of each of the 5 phage promoters, and then combined to give 15 combinations of activators and promoters, giving the construction below. We still need to decide which pigment to use for our proof of concept.<br />
<br />
<br />
[[Image:proofofconcept.jpg]]<br />
<br />
<br />
The second three will be used to construct a library of threshold devices which can be abstracted as a PoPS converter (below).<br />
<br />
[[Image:thresholddeviceabstraction.jpg]]<br />
<br />
The next step will be to attach the phage promoter downstream to create a catalogue of 15 different devices of the form:<br />
<br />
[[Image:completedevice.jpg]]<br />
<br />
<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Friday ==<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Notebook/Week9Team:Cambridge/Notebook/Week92009-09-11T14:22:01Z<p>Awalbridge: /* Threshold Devices */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Week 9 =<br />
<br />
{{Template:CambridgeNotepad}}<br />
<br />
== Monday ==<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Overnight cultures of 70, 71, 72, 74, 91 in pSB3K3 in arabinose strain in order to make glycerol stocks and streak single colonies for the plate reader.<br />
<br />
82 run on plate reader during day<br />
<br />
85 overnight<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Tuesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
All activator constructs are now ready for analysis on the plate reader. <br />
<br />
90 run during day<br />
<br />
92 run overnight<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Wednesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Confirmed successful ligation of pBad and I746350, I746351, I746352. <br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Thursday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Attempted the following standard assemblies:<br />
<br />
*pBad + I746350 to B0015<br />
*pBad + I746351 to B0015<br />
*pBad + I746352 to B0015<br />
*I746351 to B0015<br />
*I746352 to B0015<br />
*I746352 to B0015<br />
<br />
The first three will be used to construct a complete device, with pBad as the sensor promoter and a pigment operon as the pigment-generating device. The pigment we chose for our proof of concept will be placed downstream of each of the 5 phage promoters, and then combined to give 15 combinations of activators and promoters, giving the construction below. We still need to decide which pigment to use for our proof of concept.<br />
<br />
<br />
[[Image:proofofconcept.jpg]]<br />
<br />
<br />
The second three will be used to construct a library of threshold devices which can be abstracted as a PoPS converter (below).<br />
<br />
[[Image:thresholddeviceabstraction.jpg]]<br />
<br />
The next step will be to attach the phage promoter downstream to create a catalogue of 15 different devices of the form:<br />
<br />
[[Image:completedevice.jpg]]<br />
<br />
<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Friday ==<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Notebook/Week9Team:Cambridge/Notebook/Week92009-09-11T14:21:47Z<p>Awalbridge: /* Wet Work */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Week 9 =<br />
<br />
{{Template:CambridgeNotepad}}<br />
<br />
== Monday ==<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Overnight cultures of 70, 71, 72, 74, 91 in pSB3K3 in arabinose strain in order to make glycerol stocks and streak single colonies for the plate reader.<br />
<br />
82 run on plate reader during day<br />
85 overnight<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Tuesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
All activator constructs are now ready for analysis on the plate reader. <br />
<br />
90 run during day<br />
<br />
92 run overnight<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Wednesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Confirmed successful ligation of pBad and I746350, I746351, I746352. <br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Thursday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Attempted the following standard assemblies:<br />
<br />
*pBad + I746350 to B0015<br />
*pBad + I746351 to B0015<br />
*pBad + I746352 to B0015<br />
*I746351 to B0015<br />
*I746352 to B0015<br />
*I746352 to B0015<br />
<br />
The first three will be used to construct a complete device, with pBad as the sensor promoter and a pigment operon as the pigment-generating device. The pigment we chose for our proof of concept will be placed downstream of each of the 5 phage promoters, and then combined to give 15 combinations of activators and promoters, giving the construction below. We still need to decide which pigment to use for our proof of concept.<br />
<br />
<br />
[[Image:proofofconcept.jpg]]<br />
<br />
<br />
The second three will be used to construct a library of threshold devices which can be abstracted as a PoPS converter (below).<br />
<br />
[[Image:thresholddeviceabstraction.jpg]]<br />
<br />
The next step will be to attach the phage promoter downstream to create a catalogue of 15 different devices of the form:<br />
<br />
[[Image:completedevice.jpg]]<br />
<br />
<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Friday ==<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Notebook/Week9Team:Cambridge/Notebook/Week92009-09-11T14:21:03Z<p>Awalbridge: /* Wet Work */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Week 9 =<br />
<br />
{{Template:CambridgeNotepad}}<br />
<br />
== Monday ==<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Overnight cultures of 70, 71, 72, 74, 91 in pSB3K3 in arabinose strain in order to make glycerol stocks and streak single colonies for the plate reader.<br />
<br />
82 run on plate reader during day<br />
85 overnight<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Tuesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
All activator constructs are now ready for analysis on the plate reader. <br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Wednesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Confirmed successful ligation of pBad and I746350, I746351, I746352. <br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Thursday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold Devices====<br />
<br />
Attempted the following standard assemblies:<br />
<br />
*pBad + I746350 to B0015<br />
*pBad + I746351 to B0015<br />
*pBad + I746352 to B0015<br />
*I746351 to B0015<br />
*I746352 to B0015<br />
*I746352 to B0015<br />
<br />
The first three will be used to construct a complete device, with pBad as the sensor promoter and a pigment operon as the pigment-generating device. The pigment we chose for our proof of concept will be placed downstream of each of the 5 phage promoters, and then combined to give 15 combinations of activators and promoters, giving the construction below. We still need to decide which pigment to use for our proof of concept.<br />
<br />
<br />
[[Image:proofofconcept.jpg]]<br />
<br />
<br />
The second three will be used to construct a library of threshold devices which can be abstracted as a PoPS converter (below).<br />
<br />
[[Image:thresholddeviceabstraction.jpg]]<br />
<br />
The next step will be to attach the phage promoter downstream to create a catalogue of 15 different devices of the form:<br />
<br />
[[Image:completedevice.jpg]]<br />
<br />
<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Friday ==<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Notebook/Week8Team:Cambridge/Notebook/Week82009-09-11T14:20:02Z<p>Awalbridge: /* Wet Work */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Week 8 =<br />
<br />
{{Template:CambridgeNotepad}}<br />
<br />
== Monday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold device====<br />
<br />
Transformed 72, 92, 94, and 95 in pSB3K3 into arabinose strain.<br />
<br />
====Melanin Biobrick====<br />
<br />
Did the last stage of PCR for the Melanin Biobrick creation. Fragments AB and CF were added with primers A and F and the Finnzymes enzyme, then run on a CYBR-safe gel. The results showed smears rather than bands. As the CF fragment was a little dodgy on the first extraction it was decided to re-extract and try again.<br />
<br />
The CF band was reproduced and extracted in the afternoon, creating more CF fragment stock.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Tuesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold device====<br />
<br />
Successfully moved 70, 71, 72, 74, 91 into pSB3K3. That makes all 15 activator constructs yay!<br />
<br />
81 was run overnight in the plate reader.<br />
<br />
====Melanin Biobrick====<br />
<br />
Re-tried the PRC using the new CF fragment under the same PCR conditions. Still getting smeared bands.<br />
<br />
===Dry Work===<br />
<br />
Work on data analysis: make sure the scripts are working, and can read the standard plate layout.<br />
Then plot graphs: raw data over time, rate, normalised rate, max rate v concentration.<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Wednesday ==<br />
<br />
===Wet Work===<br />
<br />
====Melanin Biobrick====<br />
<br />
Looked up the possible reasons for smeared bands after PCR, three main ones discovered:<br />
:*Too much template DNA<br />
:*Incorrect salt concentration<br />
:*Comtaminated PCR mix<br />
Nanodropped the fragments and diluted AB 1/10 to get the recommended DNA concentration, and used new pipettes and Primer aliquots. Two tubes were made up using a new Finnzymes mix, and two using the old one.<br />
<br />
The gel still showed smears! For all the samples.<br />
<br />
====Violacein Characterisation====<br />
<br />
Incubated 20ml LB with violacein and 20ml LB with Top10 control cells in 50ml falcons for characterisation using the plate reader.<br />
<br />
====Threshold Device====<br />
<br />
84 run overnight in plate reader<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Thursday ==<br />
<br />
===Wet Work===<br />
<br />
====Melanin Biobrick====<br />
<br />
Talked with Jim Ajioka about the gel smears after the Melanin PCR. he suggested re-ordering Primers B and C with a longer overlap region, which would make the fragments easier to stick together. In view of this, new primers were designed (shown below):<br />
<br />
[[Image:Longer_primers.PNG]]<br />
<br />
====Threshold Device====<br />
<br />
80 was run overnight in plate reader<br />
<br />
====Final Product Design====<br />
<br />
Thinking about out final product, we decided that for long term product use and transport, it would be best to have the bacteria, and the media, in dry form, activated by adding liquid. We previously looked into anhydrobiotics which, while not feasible for us in the short span of the iGEM competition, would nevertheless be a viable way of storing and transporting the bacteria. In order to test whether the media could also be used in this way we added 2.5g of ''E. coli'' FastMedia (TM) from InvivoGen to 250ml of cold sterilised water, added bacteria and left at 37 degrees in a shaking incubator overnight.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Friday ==<br />
<br />
====Threshold device====<br />
<br />
Moved I13540 (pBad followed by arabinose) into pSB3K3, will confirm if it was successful next week. <br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Notebook/Week8Team:Cambridge/Notebook/Week82009-09-11T14:19:21Z<p>Awalbridge: /* Wet Work */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Week 8 =<br />
<br />
{{Template:CambridgeNotepad}}<br />
<br />
== Monday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold device====<br />
<br />
Transformed 72, 92, 94, and 95 in pSB3K3 into arabinose strain.<br />
<br />
====Melanin Biobrick====<br />
<br />
Did the last stage of PCR for the Melanin Biobrick creation. Fragments AB and CF were added with primers A and F and the Finnzymes enzyme, then run on a CYBR-safe gel. The results showed smears rather than bands. As the CF fragment was a little dodgy on the first extraction it was decided to re-extract and try again.<br />
<br />
The CF band was reproduced and extracted in the afternoon, creating more CF fragment stock.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Tuesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold device====<br />
<br />
Successfully moved 70, 71, 72, 74, 91 into pSB3K3. That makes all 15 activator constructs yay!<br />
<br />
81 was run overnight in the plate reader.<br />
<br />
====Melanin Biobrick====<br />
<br />
Re-tried the PRC using the new CF fragment under the same PCR conditions. Still getting smeared bands.<br />
<br />
===Dry Work===<br />
<br />
Work on data analysis: make sure the scripts are working, and can read the standard plate layout.<br />
Then plot graphs: raw data over time, rate, normalised rate, max rate v concentration.<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Wednesday ==<br />
<br />
===Wet Work===<br />
<br />
====Melanin Biobrick====<br />
<br />
Looked up the possible reasons for smeared bands after PCR, three main ones discovered:<br />
:*Too much template DNA<br />
:*Incorrect salt concentration<br />
:*Comtaminated PCR mix<br />
Nanodropped the fragments and diluted AB 1/10 to get the recommended DNA concentration, and used new pipettes and Primer aliquots. Two tubes were made up using a new Finnzymes mix, and two using the old one.<br />
<br />
The gel still showed smears! For all the samples.<br />
<br />
====Violacein Characterisation====<br />
<br />
Incubated 20ml LB with violacein and 20ml LB with Top10 control cells in 50ml falcons for characterisation using the plate reader.<br />
<br />
====Threshold Device====<br />
<br />
84 run overnight in plate reader<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Thursday ==<br />
<br />
===Wet Work===<br />
<br />
====Melanin Biobrick====<br />
<br />
Talked with Jim Ajioka about the gel smears after the Melanin PCR. he suggested re-ordering Primers B and C with a longer overlap region, which would make the fragments easier to stick together. In view of this, new primers were designed (shown below):<br />
<br />
[[Image:Longer_primers.PNG]]<br />
<br />
====Final Product Design====<br />
<br />
Thinking about out final product, we decided that for long term product use and transport, it would be best to have the bacteria, and the media, in dry form, activated by adding liquid. We previously looked into anhydrobiotics which, while not feasible for us in the short span of the iGEM competition, would nevertheless be a viable way of storing and transporting the bacteria. In order to test whether the media could also be used in this way we added 2.5g of ''E. coli'' FastMedia (TM) from InvivoGen to 250ml of cold sterilised water, added bacteria and left at 37 degrees in a shaking incubator overnight.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Friday ==<br />
<br />
====Threshold device====<br />
<br />
Moved I13540 (pBad followed by arabinose) into pSB3K3, will confirm if it was successful next week. <br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Notebook/Week8Team:Cambridge/Notebook/Week82009-09-11T14:18:38Z<p>Awalbridge: /* Threshold device */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Week 8 =<br />
<br />
{{Template:CambridgeNotepad}}<br />
<br />
== Monday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold device====<br />
<br />
Transformed 72, 92, 94, and 95 in pSB3K3 into arabinose strain.<br />
<br />
====Melanin Biobrick====<br />
<br />
Did the last stage of PCR for the Melanin Biobrick creation. Fragments AB and CF were added with primers A and F and the Finnzymes enzyme, then run on a CYBR-safe gel. The results showed smears rather than bands. As the CF fragment was a little dodgy on the first extraction it was decided to re-extract and try again.<br />
<br />
The CF band was reproduced and extracted in the afternoon, creating more CF fragment stock.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Tuesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold device====<br />
<br />
Successfully moved 70, 71, 72, 74, 91 into pSB3K3. That makes all 15 activator constructs yay!<br />
<br />
81 was run overnight in the plate reader.<br />
<br />
====Melanin Biobrick====<br />
<br />
Re-tried the PRC using the new CF fragment under the same PCR conditions. Still getting smeared bands.<br />
<br />
===Dry Work===<br />
<br />
Work on data analysis: make sure the scripts are working, and can read the standard plate layout.<br />
Then plot graphs: raw data over time, rate, normalised rate, max rate v concentration.<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Wednesday ==<br />
<br />
===Wet Work===<br />
<br />
====Melanin Biobrick====<br />
<br />
Looked up the possible reasons for smeared bands after PCR, three main ones discovered:<br />
:*Too much template DNA<br />
:*Incorrect salt concentration<br />
:*Comtaminated PCR mix<br />
Nanodropped the fragments and diluted AB 1/10 to get the recommended DNA concentration, and used new pipettes and Primer aliquots. Two tubes were made up using a new Finnzymes mix, and two using the old one.<br />
<br />
The gel still showed smears! For all the samples.<br />
<br />
====Violacein Characterisation====<br />
<br />
Incubated 20ml LB with violacein and 20ml LB with Top10 control cells in 50ml falcons for characterisation using the plate reader.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Thursday ==<br />
<br />
===Wet Work===<br />
<br />
====Melanin Biobrick====<br />
<br />
Talked with Jim Ajioka about the gel smears after the Melanin PCR. he suggested re-ordering Primers B and C with a longer overlap region, which would make the fragments easier to stick together. In view of this, new primers were designed (shown below):<br />
<br />
[[Image:Longer_primers.PNG]]<br />
<br />
====Final Product Design====<br />
<br />
Thinking about out final product, we decided that for long term product use and transport, it would be best to have the bacteria, and the media, in dry form, activated by adding liquid. We previously looked into anhydrobiotics which, while not feasible for us in the short span of the iGEM competition, would nevertheless be a viable way of storing and transporting the bacteria. In order to test whether the media could also be used in this way we added 2.5g of ''E. coli'' FastMedia (TM) from InvivoGen to 250ml of cold sterilised water, added bacteria and left at 37 degrees in a shaking incubator overnight.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Friday ==<br />
<br />
====Threshold device====<br />
<br />
Moved I13540 (pBad followed by arabinose) into pSB3K3, will confirm if it was successful next week. <br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Notebook/Week8Team:Cambridge/Notebook/Week82009-09-11T14:08:28Z<p>Awalbridge: /* Dry Work */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Week 8 =<br />
<br />
{{Template:CambridgeNotepad}}<br />
<br />
== Monday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold device====<br />
<br />
Transformed 72, 92, 94, and 95 in pSB3K3 into arabinose strain.<br />
<br />
====Melanin Biobrick====<br />
<br />
Did the last stage of PCR for the Melanin Biobrick creation. Fragments AB and CF were added with primers A and F and the Finnzymes enzyme, then run on a CYBR-safe gel. The results showed smears rather than bands. As the CF fragment was a little dodgy on the first extraction it was decided to re-extract and try again.<br />
<br />
The CF band was reproduced and extracted in the afternoon, creating more CF fragment stock.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Tuesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold device====<br />
<br />
Successfully moved 70, 71, 72, 74, 91 into pSB3K3. That makes all 15 activator constructs yay!<br />
<br />
====Melanin Biobrick====<br />
<br />
Re-tried the PRC using the new CF fragment under the same PCR conditions. Still getting smeared bands.<br />
<br />
===Dry Work===<br />
<br />
Work on data analysis: make sure the scripts are working, and can read the standard plate layout.<br />
Then plot graphs: raw data over time, rate, normalised rate, max rate v concentration.<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Wednesday ==<br />
<br />
===Wet Work===<br />
<br />
====Melanin Biobrick====<br />
<br />
Looked up the possible reasons for smeared bands after PCR, three main ones discovered:<br />
:*Too much template DNA<br />
:*Incorrect salt concentration<br />
:*Comtaminated PCR mix<br />
Nanodropped the fragments and diluted AB 1/10 to get the recommended DNA concentration, and used new pipettes and Primer aliquots. Two tubes were made up using a new Finnzymes mix, and two using the old one.<br />
<br />
The gel still showed smears! For all the samples.<br />
<br />
====Violacein Characterisation====<br />
<br />
Incubated 20ml LB with violacein and 20ml LB with Top10 control cells in 50ml falcons for characterisation using the plate reader.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Thursday ==<br />
<br />
===Wet Work===<br />
<br />
====Melanin Biobrick====<br />
<br />
Talked with Jim Ajioka about the gel smears after the Melanin PCR. he suggested re-ordering Primers B and C with a longer overlap region, which would make the fragments easier to stick together. In view of this, new primers were designed (shown below):<br />
<br />
[[Image:Longer_primers.PNG]]<br />
<br />
====Final Product Design====<br />
<br />
Thinking about out final product, we decided that for long term product use and transport, it would be best to have the bacteria, and the media, in dry form, activated by adding liquid. We previously looked into anhydrobiotics which, while not feasible for us in the short span of the iGEM competition, would nevertheless be a viable way of storing and transporting the bacteria. In order to test whether the media could also be used in this way we added 2.5g of ''E. coli'' FastMedia (TM) from InvivoGen to 250ml of cold sterilised water, added bacteria and left at 37 degrees in a shaking incubator overnight.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Friday ==<br />
<br />
====Threshold device====<br />
<br />
Moved I13540 (pBad followed by arabinose) into pSB3K3, will confirm if it was successful next week. <br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:CambridgeTeam:Cambridge2009-09-10T14:08:47Z<p>Awalbridge: /* Overview */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
=Overview=<br />
[[Image:Cambridge_Frontpage2.png|400px|right]]<br />
<br />
Previous iGEM teams have focused on genetically engineering bacterial biosensors by enabling bacteria to respond to novel inputs, especially biologically significant compounds. There is an unmistakable need to also develop devices that can 1) manipulate the input by changing the behaviour of the response of the input-sensitive promoter, and that can 2) report using clear, user-friendly outputs. The most popular output is the expression of a fluorescent protein, detectable using fluorescence microscopy. But, what if we could simply see the output with our own eyes? <br />
<br />
The Cambridge [https://2009.igem.org/Main_Page 2009 iGEM] team is engineering ''E. coli'' to produce different pigments in response to different concentrations of an inducer. <br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Notebook/Week8Team:Cambridge/Notebook/Week82009-09-04T10:08:20Z<p>Awalbridge: /* Monday */</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Week 8 =<br />
<br />
{{Template:CambridgeNotepad}}<br />
<br />
== Monday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold device====<br />
<br />
Transformed 72, 92, 94, and 95 in pSB3K3 into arabinose strain.<br />
<br />
====Melanin Biobrick====<br />
<br />
Did the last stage of PCR for the Melanin Biobrick creation. Fragments AB and CF were added with primers A and F and the Finnzymes enzyme, then run on a CYBR-safe gel. The results showed smears rather than bands. As the CF fragment was a little dodgy on the first extraction it was decided to re-extract and try again.<br />
<br />
The CF band was reproduced and extracted in the afternoon, creating more CF fragment stock.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Tuesday ==<br />
<br />
===Wet Work===<br />
<br />
====Threshold device====<br />
<br />
Successfully moved 70, 71, 72, 74, 91 into pSB3K3. That makes all 15 activator constructs yay!<br />
<br />
====Melanin Biobrick====<br />
<br />
Re-tried the PRC using the new CF fragment under the same PCR conditions. Still getting smeared bands.<br />
<br />
===Dry Work===<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Wednesday ==<br />
<br />
===Wet Work===<br />
<br />
====Melanin Biobrick====<br />
<br />
Looked up the possible reasons for smeared bands after PCR, three main ones discovered:<br />
:*Too much template DNA<br />
:*Incorrect salt concentration<br />
:*Comtaminated PCR mix<br />
Nanodropped the fragments and diluted AB 1/10 to get the recommended DNA concentration, and used new pipettes and Primer aliquots. Two tubes were made up using a new Finnzymes mix, and two using the old one.<br />
<br />
The gel still showed smears! For all the samples.<br />
<br />
====Violacein Characterisation====<br />
<br />
Incubated 20ml LB with violacein and 20ml LB with Top10 control cells in 50ml falcons for characterisation using the plate reader.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Thursday ==<br />
<br />
===Wet Work===<br />
<br />
====Melanin Biobrick====<br />
<br />
Talked with Jim Ajioka about the gel smears after the Melanin PCR. he suggested re-ordering Primers B and C with a longer overlap region, which would make the fragments easier to stick together. In view of this, new primers were designed (shown below):<br />
<br />
[[Image:Longer_primers.PNG]]<br />
<br />
====Final Product Design====<br />
<br />
Thinking about out final product, we decided that for long term product use and transport, it would be best to have the bacteria, and the media, in dry form, activated by adding liquid. We previously looked into anhydrobiotics which, while not feasible for us in the short span of the iGEM competition, would nevertheless be a viable way of storing and transporting the bacteria. In order to test whether the media could also be used in this way we added 2.5g of ''E. coli'' FastMedia (TM) from InvivoGen to 250ml of cold sterilised water, added bacteria and left at 37 degrees in a shaking incubator overnight.<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Friday ==<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridgehttp://2009.igem.org/Template:Cambridge2Template:Cambridge22009-09-01T12:37:33Z<p>Awalbridge: </p>
<hr />
<div><html><br />
<br />
<style type="text/css"><br />
<br />
body {<br />
-x-system-font:none;<br />
/*<br />
https://static.igem.org/mediawiki/2009/b/b8/Cambridge_Tiledback3.png<br />
*/<br />
background:url(https://static.igem.org/mediawiki/2009/7/75/Cambridge_Background1.png) repeat top left;<br />
color:#FFE2EE;<br />
font-family:Arial,Sans-Serif;<br />
/*font-size:0.6em;*/<br />
font-size-adjust:none;<br />
font-stretch:normal;<br />
font-style:normal;<br />
font-variant:normal;<br />
font-weight:normal;<br />
line-height:1.8em;<br />
}<br />
a {<br />
color:#6587E0;<br />
text-decoration:none;<br />
margin:0 0px 0;<br />
}<br />
a:visited {<br />
color:#6587E0;<br />
text-decoration:none;<br />
}<br />
a:hover {<br />
color:#333333;<br />
text-decoration:underline;<br />
}<br />
h1 {<br />
font-size:32px;<br />
margin:-18px 10px 0px;<br />
}<br />
h2 {<br />
font-size:26px;<br />
margin:0 0 10px;<br />
padding:10px 10px 3px;<br />
}<br />
h3 {<br />
font-size:20px;<br />
font-weight:normal;<br />
padding-left:10px;<br />
}<br />
<br />
ul {<br />
margin:0 0 10px;<br />
padding:0;<br />
}<br />
li {<br />
list-style-type:none;<br />
}<br />
img {<br />
border:medium none;<br />
}<br />
#header {<br />
background: url(https://static.igem.org/mediawiki/2009/f/f9/Cambridge_topbanner5.png) repeat-x scroll 0 0;<br />
height:150px;<br />
width: 965px;<br />
position: relative;<br />
margin-top:-42px;<br />
}<br />
#header h1 {<br />
font-size:90px;<br />
color:#FFF;<br />
font-weight:300;<br />
letter-spacing:-1px;<br />
padding:22px 0 10px 10px;<br />
}<br />
#header h1 a {<br />
color:#FFFFFF;<br />
text-decoration:none;<br />
}<br />
#header h1 a:hover {<br />
color:#000000;<br />
text-decoration:none;<br />
}<br />
#header h2 {<br />
color:#EEEEEE;<br />
font-size:19px;<br />
font-weight:100;<br />
letter-spacing:-1px;<br />
line-height:12px;<br />
padding:0 0 0 11px;<br />
}<br />
<br />
#left {<br />
background:#727072 none repeat scroll 0 0;<br />
color:#FFE2EE;<br />
float:left;<br />
margin-top:4px;<br />
margin-bottom:0px;<br />
width:180px;<br />
}<br />
#left ul {<br />
margin:0;<br />
padding:1px 1px 1px;<br />
}<br />
#left a {<br />
display: block;<br />
width: 170px;<br />
height: 16px;<br />
margin: 1px -1px;<br />
/*background: url(https://static.igem.org/mediawiki/2009/b/ba/Cambridge_Butt1.png) no-repeat left top;*/<br />
background:#A3C3FF no-repeat left top;<br />
padding: 2px 2px;<br />
text-decoration: none;<br />
color: #555;<br />
}<br />
#left a:hover {<br />
display: block;<br />
width: 170px;<br />
height: 16px;<br />
margin-top: 1px;<br />
/*background: url(https://static.igem.org/mediawiki/2009/6/60/Cambridge_Buttpink.png) no-repeat left top;*/<br />
background:#E5EDFF no-repeat left top; https://static.igem.org/mediawiki/2009/6/6b/Cambridge_Buttdark.png<br />
padding: 2px 4px;<br />
text-decoration: none;<br />
color: #888;<br />
}<br />
<br />
#left h4 {<br />
margin: -24px -1px;<br />
margin-bottom: -2px;<br />
background: url(https://static.igem.org/mediawiki/2009/4/48/Cambridge_Roundedleftheader4.png) no-repeat left top;<br />
/*background:#3D5089 no-repeat left top;*/<br />
color: #CCC;<br />
font-size:19px;<br />
padding: 2px 2px;<br />
}<br />
<br />
#left .selflink {<br />
display: block;<br />
width: 170px;<br />
height: 16px;<br />
margin: 1px -1px;<br />
/*background: url(https://static.igem.org/mediawiki/2009/6/6b/Cambridge_Buttdark.png) no-repeat left top;*/<br />
background:#6D7DBC no-repeat left top;<br />
padding: 2px 2px;<br />
text-decoration: none;<br />
color: #BBB;<br />
font-weight:normal;<br />
}<br />
/*<br />
#left a.external {<br />
margin: 0;<br />
padding: 0;<br />
width: 170px;<br />
}<br />
*/<br />
#right {<br />
float:right;<br />
margin-bottom:10px;<br />
margin-top:10px;<br />
padding:0 10px 10px;<br />
width:150px;<br />
}<br />
<br />
<br />
#sponsorbox {<br />
width: 752px;<br />
padding:5px;<br />
position: relative;<br />
float: right;<br />
margin: 21px 10px 0px;<br />
background:#b2b0b2 none repeat scroll 0 0;<br />
text-align: justify;<br />
}<br />
<br />
<br />
#contentbox {<br />
width: 755px;<br />
padding:20px;<br />
position: relative;<br />
float: right;<br />
margin: 21px -12px 0px;<br />
background:url(https://static.igem.org/mediawiki/2009/4/44/Cambridge_Lines2.png) repeat-y top left;<br />
/*color:#C4FFD4;*/<br />
text-align: justify;<br />
}<br />
<br />
<br />
#contentbox_top {<br />
width: 785px;<br />
height: 19px;<br />
margin: -39px -20px 0px;<br />
background:url(https://static.igem.org/mediawiki/2009/6/6b/Camrbidge_Linestop4.png) no-repeat bottom left;<br />
position: relative;<br />
/* float: left; */<br />
}<br />
<br />
#contentbox_bottom {<br />
width: 785px;<br />
height: 18px;<br />
margin: 10px -20px -21px;<br />
background:url(https://static.igem.org/mediawiki/2009/5/59/Camrbidge_Linebottom5.png) no-repeat top left;<br />
position: relative;<br />
/* float: left; */ <br />
}<br />
<br />
#pb {<br />
margin:10px -20px 10px;<br />
background:url(https://static.igem.org/mediawiki/2009/5/54/Cambridge_Pagebreak.png) no-repeat scroll 0 0;<br />
height:60px;<br />
width: 785px;<br />
position: relative;<br />
}<br />
<br />
#content {<br />
padding: 5px;<br />
border-left: 1px solid #727072;<br />
border-right: 1px solid #727072;<br />
background:#727072 none repeat scroll 0 0;<br />
position: relative;<br />
/* float: left; /* <br />
font-size: 9pt;<br />
text-align: justify;<br />
}<br />
<br />
<br />
<br />
<br />
#footer {<br />
border-top:1px solid #CCCCCC;<br />
font-size:11px;<br />
padding:10px 5px;<br />
text-align:center;<br />
}<br />
<br />
<br />
<br />
p {<br />
margin:5px 0px 10px;<br />
/* margin:0; */<br />
/* margin-bottom: 10px; */<br />
}<br />
.editsection {<br />
font-size: 75%;<br />
font-weight: normal;<br />
}<br />
/* This hides the Table of Contents. <br />
#toc {<br />
display: none;<br />
}<br />
Not good as disables wikis from all pages.<br />
*/<br />
<br />
</style><br />
</html><br />
<div id="header"></div><br />
<div id="left"><br />
<h4>Categories :</h4><br />
<ul><br />
[[Team:Cambridge | Home]]<br />
[[Team:Cambridge/Team | Team]]<br />
[[Team:Cambridge/Sponsors | Sponsors]]<br />
[[Team:Cambridge/Parts | Parts Submitted to Registry]]<br />
[[Team:Cambridge/ImageGallery | Image Gallery]]<br />
[[Team:Cambridge/Contact | Leave a Message!]]<br />
</ul><br />
<h4>Project :</h4><br />
<ul><br />
[[Team:Cambridge/Project | Overview]]<br />
[[Team:Cambridge/Project/Carotenoids | Carotenoids]]<br />
[[Team:Cambridge/Project/Melanin | Melanin]]<br />
[[Team:Cambridge/Project/Violacein | Violacein]]<br />
[[Team:Cambridge/Project/Amplification | Amplification]]<br />
[[Team:Cambridge/Modelling | Modelling]]<br />
[[Team:Cambridge/Protocols | Protocols]]<br />
</ul><br />
<h4>Notebook :</h4><br />
<ul><br />
[[Team:Cambridge/Notebook/Week1 | Week 1]] <br />
[[Team:Cambridge/Notebook/Week2 | Week 2]] <br />
[[Team:Cambridge/Notebook/Week3 | Week 3]] <br />
[[Team:Cambridge/Notebook/Week4 | Week 4]]<br />
[[Team:Cambridge/Notebook/Week5 | Week 5]] <br />
[[Team:Cambridge/Notebook/Week6 | Week 6]]<br />
[[Team:Cambridge/Notebook/Week7 | Week 7]]<br />
[[Team:Cambridge/Notebook/Week8 | Week 8]]<br />
[[Team:Cambridge/Notebook/Week9 | Week 9]]<br />
[[Team:Cambridge/Notebook/Week10 | Week 10]]<br />
</ul><br />
<h4>Team Logistics :</h4><br />
<ul><br />
[[Team:Cambridge/Calender | Calendar]]<br />
[[Team:Cambridge/Stock | Stock List]] <br />
[[Team:Cambridge/Research | Research]]<br />
[[Team:Cambridge/Paste | Shared Links and Help]] <br />
</ul></div><br />
<br />
<div id="contentbox"><br />
<div id="contentbox_top"></div><br />
<br />
__NOTOC__</div>Awalbridgehttp://2009.igem.org/Template:CambridgeTemplate:Cambridge2009-09-01T12:36:01Z<p>Awalbridge: </p>
<hr />
<div><html><br />
<br />
<style type="text/css"><br />
<br />
body {<br />
-x-system-font:none;<br />
background:#C4FFD4 none repeat scroll 0 0;<br />
color:#FFE2EE;<br />
font-family:Arial,Sans-Serif;<br />
/*font-size:0.6em;*/<br />
font-size-adjust:none;<br />
font-stretch:normal;<br />
font-style:normal;<br />
font-variant:normal;<br />
font-weight:normal;<br />
line-height:1.8em;<br />
}<br />
a {<br />
color:#6587E0;<br />
text-decoration:none;<br />
margin:0 5px 0;<br />
font-weight:100;<br />
}<br />
a:visited {<br />
color:#6587E0;<br />
text-decoration:none;<br />
}<br />
a:hover {<br />
color:#333333;<br />
text-decoration:underline;<br />
}<br />
h1 {<br />
/*color:#FFF;<br />
letter-spacing:-1px;<br />
padding:22px 0 5px 10px; */<br />
text-decoration:none;<br />
margin:0 10px 0;<br />
}<br />
h2 {<br />
margin:0 0 10px;<br />
padding:10px 0 3px;<br />
text-decoration:none;<br />
}<br />
h3 {<br />
background:#FFFFFF repeat-x scroll 0 0;<br />
color:#000000;<br />
font-size:1.6em;<br />
height:0px;<br />
line-height:0px;<br />
padding-left:10px;<br />
}<br />
ul {<br />
margin:0 0 10px;<br />
padding:0;<br />
}<br />
li {<br />
list-style-type:none;<br />
}<br />
img {<br />
border:medium none;<br />
}<br />
#header {<br />
background: url(https://static.igem.org/mediawiki/2009/2/26/Cambridge_header.png) repeat-x scroll 0 0;<br />
height:150px;<br />
width: 965px;<br />
position: relative;<br />
}<br />
#header h1 {<br />
font-size:90px;<br />
color:#FFF;<br />
font-weight:200;<br />
letter-spacing:-1px;<br />
padding:22px 0 10px 10px;<br />
}<br />
#header h1 a {<br />
color:#FFFFFF;<br />
text-decoration:none;<br />
}<br />
#header h1 a:hover {<br />
color:#000000;<br />
text-decoration:none;<br />
}<br />
#header h2 {<br />
color:#EEEEEE;<br />
font-size:19px;<br />
font-weight:100;<br />
letter-spacing:-1px;<br />
line-height:12px;<br />
padding:0 0 0 11px;<br />
}<br />
<br />
#left {<br />
background:#727072 none repeat scroll 0 0;<br />
color:#FFE2EE;<br />
float:left;<br />
margin-top:3px;<br />
margin-bottom:0px;<br />
width:180px;<br />
}<br />
#left ul {<br />
margin:0;<br />
padding:1px 1px 1px;<br />
}<br />
#left a {<br />
display: block;<br />
width: 170px;<br />
height: 16px;<br />
margin: 1px -1px;<br />
/*background: url(whatever.png) no-repeat left top;*/<br />
background:#A3C3FF no-repeat left top;<br />
padding: 2px 2px;<br />
text-decoration: none;<br />
color: #555;<br />
}<br />
#left a:hover {<br />
display: block;<br />
width: 170px;<br />
height: 16px;<br />
margin-top: 1px;<br />
/*background: url(whatever.png) no-repeat left top;*/<br />
background:#E5EDFF no-repeat left top;<br />
padding: 2px 4px;<br />
text-decoration: none;<br />
color: #888;<br />
}<br />
<br />
#left h4 {<br />
margin: -25px -1px;<br />
margin-bottom: -4px;<br />
background:#3D5089 no-repeat left top;<br />
color: #CCC;<br />
font-size:19px;<br />
padding: 2px 2px;<br />
}<br />
<br />
#left .selflink {<br />
display: block;<br />
width: 170px;<br />
height: 16px;<br />
margin: 1px -1px;<br />
/*background: url(whatever.png) no-repeat left top;*/<br />
background:#435989 no-repeat left top;<br />
padding: 2px 2px;<br />
text-decoration: none;<br />
color: #BBB;<br />
}<br />
/*<br />
#left a.external {<br />
margin: 0;<br />
padding: 0;<br />
width: 170px;<br />
}<br />
*/<br />
#right {<br />
float:right;<br />
margin-bottom:10px;<br />
margin-top:10px;<br />
padding:0 10px 10px;<br />
width:150px;<br />
}<br />
<br />
<br />
<br />
#contentbox {<br />
width: 765px;<br />
padding:10px;<br />
position: relative;<br />
float: right;<br />
margin: 19px 0px 0px;<br />
background:url(https://static.igem.org/mediawiki/2009/4/44/Cambridge_Lines2.png) repeat-y top left;<br />
/*color:#C4FFD4;*/<br />
}<br />
<br />
<br />
#contentbox_top {<br />
width: 785px;<br />
height: 19px;<br />
margin: -29px -10px 0px;<br />
background:url(https://static.igem.org/mediawiki/2009/c/c9/Cambridge_Linestop.png) no-repeat bottom left;<br />
position: relative;<br />
/* float: left; */<br />
}<br />
<br />
#contentbox_bottom {<br />
width: 785px;<br />
height: 19px;<br />
margin: 0px -10px -21px;<br />
background:url(https://static.igem.org/mediawiki/2009/2/2d/Cambridge_Linesbottom.png) no-repeat top left;<br />
position: relative;<br />
/* float: left; */ <br />
}<br />
<br />
#content {<br />
padding: 5px;<br />
border-left: 1px solid #CFDDFF;<br />
border-right: 1px solid #CFDDFF;<br />
background:#727072 none repeat scroll 0 0;<br />
position: relative;<br />
/* float: left; /* <br />
font-size: 9pt;<br />
text-align: justify;<br />
}<br />
<br />
<br />
<br />
<br />
#footer {<br />
border-top:1px solid #CCCCCC;<br />
font-size:11px;<br />
padding:10px 5px;<br />
text-align:center;<br />
}<br />
<br />
<br />
<br />
p {<br />
margin:5px 0 10px;<br />
/* margin:0; */<br />
/* margin-bottom: 10px; */<br />
}<br />
.editsection {<br />
font-size: 75%;<br />
font-weight: normal;<br />
}<br />
/* This hides the Table of Contents. */<br />
#toc {<br />
display: none;<br />
}<br />
<br />
<br />
<br />
</style><br />
</html><br />
<div id="header"></div><br />
<div id="left"><br />
<ul><br />
<h4>Categories :</h4><br />
[[Team:Cambridge | Home]]<br />
[[Team:Cambridge/Team | Team]]<br />
[[Team:Cambridge/Project | Project]]<br />
[[Team:Cambridge/Parts | Parts Submitted to Registry]]<br />
[[Team:Cambridge/Modelling | Modelling]]<br />
[[Team:Cambridge/Protocols | Research]]<br />
[[Team:Cambridge/ImageGallery | Image Gallery]]<br />
[[Team:Cambridge/Contact | Contact Us]]<br />
<h4>Team Stuff :</h4><br />
[[Team:Cambridge/Calender | Calendar]]<br />
[[Team:Cambridge/Stock | Stock List]] <br />
[[Team:Cambridge/Protocols | Protocols]] <br />
[[Team:Cambridge/Paste | Shared Links]] <br />
<h4>Notebook :</h4><br />
[[Team:Cambridge/Notebook/Week1 | Week 1]] <br />
[[Team:Cambridge/Notebook/Week2 | Week 2]] <br />
[[Team:Cambridge/Notebook/Week3 | Week 3]] <br />
[[Team:Cambridge/Notebook/Week4 | Week 4]]<br />
[[Team:Cambridge/Notebook/Week5 | Week 5]] <br />
[[Team:Cambridge/Notebook/Week6 | Week 6]]<br />
[[Team:Cambridge/Notebook/Week7 | Week 7]]<br />
[[Team:Cambridge/Notebook/Week8 | Week 8]]<br />
[[Team:Cambridge/Notebook/Week9 | Week 9]]<br />
[[Team:Cambridge/Notebook/Week10 | Week 10]]<br />
</ul><br />
<br />
</div><br />
<br />
<div id="contentbox"><br />
<div id="contentbox_top"></div></div>Awalbridgehttp://2009.igem.org/Template:Cambridge2Template:Cambridge22009-09-01T12:35:29Z<p>Awalbridge: </p>
<hr />
<div><html><br />
<br />
<style type="text/css"><br />
<br />
body {<br />
-x-system-font:none;<br />
/*<br />
https://static.igem.org/mediawiki/2009/b/b8/Cambridge_Tiledback3.png<br />
*/<br />
background:url(https://static.igem.org/mediawiki/2009/7/75/Cambridge_Background1.png) repeat top left;<br />
color:#FFE2EE;<br />
font-family:Arial,Sans-Serif;<br />
/*font-size:0.6em;*/<br />
font-size-adjust:none;<br />
font-stretch:normal;<br />
font-style:normal;<br />
font-variant:normal;<br />
font-weight:normal;<br />
line-height:1.8em;<br />
}<br />
a {<br />
color:#6587E0;<br />
text-decoration:none;<br />
margin:0 0px 0;<br />
}<br />
a:visited {<br />
color:#6587E0;<br />
text-decoration:none;<br />
}<br />
a:hover {<br />
color:#333333;<br />
text-decoration:underline;<br />
}<br />
h1 {<br />
font-size:32px;<br />
margin:-18px 10px 0px;<br />
}<br />
h2 {<br />
font-size:26px;<br />
margin:0 0 10px;<br />
padding:10px 10px 3px;<br />
}<br />
h3 {<br />
font-size:20px;<br />
font-weight:normal;<br />
padding-left:10px;<br />
}<br />
<br />
ul {<br />
margin:0 0 10px;<br />
padding:0;<br />
}<br />
li {<br />
list-style-type:none;<br />
}<br />
img {<br />
border:medium none;<br />
}<br />
#header {<br />
background: url(https://static.igem.org/mediawiki/2009/f/f9/Cambridge_topbanner5.png) repeat-x scroll 0 0;<br />
height:150px;<br />
width: 965px;<br />
position: relative;<br />
margin-top:-42px;<br />
}<br />
#header h1 {<br />
font-size:90px;<br />
color:#FFF;<br />
font-weight:300;<br />
letter-spacing:-1px;<br />
padding:22px 0 10px 10px;<br />
}<br />
#header h1 a {<br />
color:#FFFFFF;<br />
text-decoration:none;<br />
}<br />
#header h1 a:hover {<br />
color:#000000;<br />
text-decoration:none;<br />
}<br />
#header h2 {<br />
color:#EEEEEE;<br />
font-size:19px;<br />
font-weight:100;<br />
letter-spacing:-1px;<br />
line-height:12px;<br />
padding:0 0 0 11px;<br />
}<br />
<br />
#left {<br />
background:#727072 none repeat scroll 0 0;<br />
color:#FFE2EE;<br />
float:left;<br />
margin-top:4px;<br />
margin-bottom:0px;<br />
width:180px;<br />
}<br />
#left ul {<br />
margin:0;<br />
padding:1px 1px 1px;<br />
}<br />
#left a {<br />
display: block;<br />
width: 170px;<br />
height: 16px;<br />
margin: 1px -1px;<br />
/*background: url(https://static.igem.org/mediawiki/2009/b/ba/Cambridge_Butt1.png) no-repeat left top;*/<br />
background:#A3C3FF no-repeat left top;<br />
padding: 2px 2px;<br />
text-decoration: none;<br />
color: #555;<br />
}<br />
#left a:hover {<br />
display: block;<br />
width: 170px;<br />
height: 16px;<br />
margin-top: 1px;<br />
/*background: url(https://static.igem.org/mediawiki/2009/6/60/Cambridge_Buttpink.png) no-repeat left top;*/<br />
background:#E5EDFF no-repeat left top; https://static.igem.org/mediawiki/2009/6/6b/Cambridge_Buttdark.png<br />
padding: 2px 4px;<br />
text-decoration: none;<br />
color: #888;<br />
}<br />
<br />
#left h4 {<br />
margin: -24px -1px;<br />
margin-bottom: -2px;<br />
background: url(https://static.igem.org/mediawiki/2009/4/48/Cambridge_Roundedleftheader4.png) no-repeat left top;<br />
/*background:#3D5089 no-repeat left top;*/<br />
color: #CCC;<br />
font-size:19px;<br />
padding: 2px 2px;<br />
}<br />
<br />
#left .selflink {<br />
display: block;<br />
width: 170px;<br />
height: 16px;<br />
margin: 1px -1px;<br />
/*background: url(https://static.igem.org/mediawiki/2009/6/6b/Cambridge_Buttdark.png) no-repeat left top;*/<br />
background:#6D7DBC no-repeat left top;<br />
padding: 2px 2px;<br />
text-decoration: none;<br />
color: #BBB;<br />
font-weight:normal;<br />
}<br />
/*<br />
#left a.external {<br />
margin: 0;<br />
padding: 0;<br />
width: 170px;<br />
}<br />
*/<br />
#right {<br />
float:right;<br />
margin-bottom:10px;<br />
margin-top:10px;<br />
padding:0 10px 10px;<br />
width:150px;<br />
}<br />
<br />
<br />
#sponsorbox {<br />
width: 752px;<br />
padding:5px;<br />
position: relative;<br />
float: right;<br />
margin: 21px 10px 0px;<br />
background:#b2b0b2 none repeat scroll 0 0;<br />
text-align: justify;<br />
}<br />
<br />
<br />
#contentbox {<br />
width: 755px;<br />
padding:20px;<br />
position: relative;<br />
float: right;<br />
margin: 21px -12px 0px;<br />
background:url(https://static.igem.org/mediawiki/2009/4/44/Cambridge_Lines2.png) repeat-y top left;<br />
/*color:#C4FFD4;*/<br />
text-align: justify;<br />
}<br />
<br />
<br />
#contentbox_top {<br />
width: 785px;<br />
height: 19px;<br />
margin: -39px -20px 0px;<br />
background:url(https://static.igem.org/mediawiki/2009/6/6b/Camrbidge_Linestop4.png) no-repeat bottom left;<br />
position: relative;<br />
/* float: left; */<br />
}<br />
<br />
#contentbox_bottom {<br />
width: 785px;<br />
height: 18px;<br />
margin: 10px -20px -21px;<br />
background:url(https://static.igem.org/mediawiki/2009/5/59/Camrbidge_Linebottom5.png) no-repeat top left;<br />
position: relative;<br />
/* float: left; */ <br />
}<br />
<br />
#pb {<br />
margin:10px -20px 10px;<br />
background:url(https://static.igem.org/mediawiki/2009/5/54/Cambridge_Pagebreak.png) no-repeat scroll 0 0;<br />
height:60px;<br />
width: 785px;<br />
position: relative;<br />
}<br />
<br />
#content {<br />
padding: 5px;<br />
border-left: 1px solid #727072;<br />
border-right: 1px solid #727072;<br />
background:#727072 none repeat scroll 0 0;<br />
position: relative;<br />
/* float: left; /* <br />
font-size: 9pt;<br />
text-align: justify;<br />
}<br />
<br />
<br />
<br />
<br />
#footer {<br />
border-top:1px solid #CCCCCC;<br />
font-size:11px;<br />
padding:10px 5px;<br />
text-align:center;<br />
}<br />
<br />
<br />
<br />
p {<br />
margin:5px 0px 10px;<br />
/* margin:0; */<br />
/* margin-bottom: 10px; */<br />
}<br />
.editsection {<br />
font-size: 75%;<br />
font-weight: normal;<br />
}<br />
/* This hides the Table of Contents. <br />
#toc {<br />
display: none;<br />
}<br />
Not good as disables wikis from all pages.<br />
*/<br />
<br />
</style><br />
</html><br />
<div id="header"></div><br />
<div id="left"><br />
<h4>Categories :</h4><br />
<ul><br />
[[Team:Cambridge | Home]]<br />
[[Team:Cambridge/Team | Team]]<br />
[[Team:Cambridge/Sponsors | Sponsors]]<br />
[[Team:Cambridge/Parts | Parts Submitted to Registry]]<br />
[[Team:Cambridge/ImageGallery | Image Gallery]]<br />
[[Team:Cambridge/Contact | Leave a Message!]]<br />
</ul><br />
<h4>Project :</h4><br />
<ul><br />
[[Team:Cambridge/Project | Overview]]<br />
[[Team:Cambridge/Project/Carotenoids | Carotenoids]]<br />
[[Team:Cambridge/Project/Melanin | Melanin]]<br />
[[Team:Cambridge/Project/Violacein | Violacein]]<br />
[[Team:Cambridge/Project/Amplification | Amplification]]<br />
[[Team:Cambridge/Modelling | Modelling]]<br />
[[Team:Cambridge/Protocols | Protocols]]<br />
</ul><br />
<h4>Notebook :</h4><br />
<ul><br />
[[Team:Cambridge/Notebook/Week1 | Week 1]] <br />
[[Team:Cambridge/Notebook/Week2 | Week 2]] <br />
[[Team:Cambridge/Notebook/Week3 | Week 3]] <br />
[[Team:Cambridge/Notebook/Week4 | Week 4]]<br />
[[Team:Cambridge/Notebook/Week5 | Week 5]] <br />
[[Team:Cambridge/Notebook/Week6 | Week 6]]<br />
[[Team:Cambridge/Notebook/Week7 | Week 7]]<br />
[[Team:Cambridge/Notebook/Week8 | Week 8]]<br />
[[Team:Cambridge/Notebook/Week9 | Week 9]]<br />
[[Team:Cambridge/Notebook/Week10 | Week 10]]<br />
</ul><br />
<h4>Team Stuff :</h4><br />
<ul><br />
[[Team:Cambridge/Calender | Calendar]]<br />
[[Team:Cambridge/Stock | Stock List]] <br />
[[Team:Cambridge/Research | Research]]<br />
[[Team:Cambridge/Paste | Shared Links and Help]] <br />
</ul></div><br />
<br />
<div id="contentbox"><br />
<div id="contentbox_top"></div><br />
<br />
__NOTOC__</div>Awalbridgehttp://2009.igem.org/Team:Cambridge/Notebook/Week10Team:Cambridge/Notebook/Week102009-09-01T12:33:26Z<p>Awalbridge: New page: {{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--> = Week 10 = {{Template:CambridgeNotepad}} == Monday == {{Template:CambridgeNewPage}} == Tuesday == ...</p>
<hr />
<div>{{Template:Cambridge2}}<!--Do not remove the first and last lines in this page!--><br />
<br />
= Week 10 =<br />
<br />
{{Template:CambridgeNotepad}}<br />
<br />
== Monday ==<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Tuesday ==<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Wednesday ==<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Thursday ==<br />
<br />
{{Template:CambridgeNewPage}}<br />
<br />
== Friday ==<br />
<br />
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}</div>Awalbridge