Team:Cambridge/Project/Amplification/Characterisation

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[[#Introduction | Introduction ]]
[[#Introduction | Introduction ]]
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[[#Recreating Previous Work | Previous Work]]
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[[#Characterisation | Characterisation]]
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[[#Characterisation | Characterisation ]]
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[[#Data| Data]]
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[[# | ]]
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[[#Discussion | Discussion]]
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[[# | ]]
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== Introduction ==
== Introduction ==
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===Cambridge 2007===
 
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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. 
+
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:
[[Image:amplifier07.jpg]]
[[Image:amplifier07.jpg]]
 +
We re-designed these constructs to be PoPS converters as follows...
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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.
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[[Image:thresholddevice3.jpg]]  
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[[Image:construction07.jpg]]
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...and generated our own set of Sensitivity Tuners:
 +
{| border="1"
 +
|+
 +
!  !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator
 +
|-
 +
! PF promoter
 +
| <partinfo>BBa_K274370</partinfo> || <partinfo>BBa_K274380</partinfo>||
 +
|-
 +
! PO promoter
 +
|<partinfo>BBa_K274371</partinfo>
 +
|<partinfo>BBa_K274381</partinfo>
 +
|<partinfo>BBa_K274391</partinfo>
 +
|-
 +
! PP promoter
 +
|
 +
|<partinfo>BBa_K274382</partinfo>
 +
|<partinfo>BBa_K274392</partinfo>
 +
|-
 +
! Psid promoter
 +
|<partinfo>BBa_K274374</partinfo>
 +
|<partinfo>BBa_K274384</partinfo>
 +
|<partinfo>BBa_K274394</partinfo>
 +
|-
 +
! PLL promoter
 +
|<partinfo>BBa_K274375</partinfo>
 +
|
 +
|<partinfo>BBa_K274395</partinfo>
 +
|}
-
They successfully quantified the PoPS amplification factors for each activator/promoter combination after arabinose induction.   
+
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.
-
===Further Work - Cambridge 2009===
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== Characterisation ==
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====Re-design====
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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.
-
+
[[Image:Cambridge_hillfunction.gif]]
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'''Characterisation'''
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[[Image:characterization3.jpg]]
-
 
+
-
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.
+
-
 
+
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[[Image:characterization1.jpg]]
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-
 
+
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'''Reconstruction'''
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-
 
+
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Our second goal is to build a kif of parts following the pattern in the figure below, which can also be abstracted as a PoPS converter:
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-
 
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[[Image:thresholddevice3.jpg]] = [[Image:converter.jpg]]
+
-
 
+
-
his kit of sensitivity tuners, characterised using the Cambridge 2007 contructs, can be used by future iGEM teams to fit the needs of their projects. Further, this kit of parts can be expanded in the future to include more phage activators and promoters that have yet to be added to the registry.
+
-
 
+
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== Recreating Previous Work ==
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-
 
+
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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.
+
-
 
+
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'''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.
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-
 
+
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*graphs*
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-
 
+
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== Characterisation ==
+
-
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.
+
For our characterisation assay, we transformed the ''E. coli'' strain BW27783 with the Cambridge 2007 amplifiers (biobricks summarised in the table below):
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=== Maximum Rates against Arabinose Concentrations ===
+
{| border="1"
 +
|+
 +
!  !! P2 ogr activator !! PSP3 pag activator !! phiR73 delta activator
 +
|-
 +
! PF promoter
 +
| <partinfo>BBa_I746370</partinfo> || <partinfo>BBa_I746380</partinfo>||<partinfo>BBa_I746390</partinfo>
 +
|-
 +
! PO promoter
 +
|<partinfo>BBa_I746371</partinfo>
 +
|<partinfo>BBa_I746381</partinfo>
 +
|<partinfo>BBa_I746391</partinfo>
 +
|-
 +
! PP promoter
 +
|<partinfo>BBa_I746372</partinfo>
 +
|<partinfo>BBa_I746382</partinfo>
 +
|<partinfo>BBa_I746392</partinfo>
 +
|-
 +
! Psid promoter
 +
|<partinfo>BBa_I746374</partinfo>
 +
|<partinfo>BBa_I746384</partinfo>
 +
|<partinfo>BBa_I746394</partinfo>
 +
|-
 +
! PLL promoter
 +
|<partinfo>BBa_I746375</partinfo>
 +
|<partinfo>BBa_I746385</partinfo>
 +
|<partinfo>BBa_I746395</partinfo>
 +
|}
-
==== 80 ====
+
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.
-
[[Image:Cambridge_maxrates1.jpg | 600px]]
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-
==== 81 ====
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==Data==
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[[Image:Cambridge_maxrates2.jpg | 600px]]
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==== 82 ====
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=== Parameters of Fitted Curves ===
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[[Image:Cambridge_maxrates3.jpg | 600px]]
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==== 84 ====
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{| border="1"
-
[[Image:Cambridge_maxrates4.jpg | 600px]]
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|+
 +
! Construct !! Increase in Rate, a (RPU) !! Basal Rate, c (RPU) !! Switch Point, k (uM) !! Hill Coefficient, n
 +
|-
 +
!<partinfo>BBa_I746370</partinfo>
 +
| 0.205 || 0.022 || 0.85 || 2.72
 +
|-
 +
!<partinfo>BBa_I746371</partinfo>
 +
| 0.323 || 0.039 || 0.92 || 2.89
 +
|-
 +
!<partinfo>BBa_I746374</partinfo>
 +
| 0.137 || 0.021 || 0.88 || 2.51
 +
|-
 +
!<partinfo>BBa_I746375</partinfo>
 +
| 0.196 || 0.032 || 0.95 || 2.71
 +
|-
 +
!<partinfo>BBa_I746380</partinfo>
 +
| 0.129 || 0.018 || 2.20 || 4.00
 +
|-
 +
!<partinfo>BBa_I746381</partinfo>
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| 0.452 || 0.118 || 1.05 || 2.45
 +
|-
 +
!<partinfo>BBa_I746382</partinfo>
 +
| 0.496 || 0.029 || 3.98 || 2.37
 +
|-
 +
!<partinfo>BBa_I746384</partinfo>
 +
| 0.194 || 0.018 || 1.46 || 3.96
 +
|-
 +
!<partinfo>BBa_I746391</partinfo>
 +
| 1.312 || 0.058 || 1.11 || 2.78
 +
|-
 +
!<partinfo>BBa_I746392</partinfo>
 +
| 1.271 || 0.040 || 0.35 || 2.26
 +
|-
 +
!<partinfo>BBa_I746394</partinfo>
 +
| 1.105 || 0.046 || 1.81 || 4.00
 +
|-
 +
!<partinfo>BBa_I746395</partinfo>
 +
| 1.366 || 0.040 || 1.91 || 4.00
 +
|}
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==== 85 ====
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=== Sample Graph ===
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[[Image:Cambridge_maxrates5.jpg | 600px]]
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-
==== 90 ====
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[[Image:Cambridge_characterisation82.png]]
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[[Image:Cambridge_maxrates6.jpg | 600px]]
+
-
==== 92 ====
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== Discussion ==
-
[[Image:Cambridge_maxrates7.jpg | 600px]]
+
-
==== 94 ====
+
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.
-
[[Image:Cambridge_maxrates8.jpg | 600px]]
+
-
==== 95 ====
+
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.
-
[[Image:Cambridge_maxrates9.jpg | 600px]]
+
-
== Reconstruction ==
+
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.
-
Below is a table listing the sensitivity tuners as they appear in the registry as biobricks:
+
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.
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}
<!--Do not remove the first and last lines in this page!-->{{Template:CambridgeBottom}}

Latest revision as of 03:08, 22 October 2009


The Sensitivity Tuner

Introduction

The Cambridge 2007 iGEM team build 15 "amplifiers," constructs with RFP and GFP reporters that amplified the PoPS output of the promoter pBad/AraC (), as described below:

Amplifier07.jpg

We re-designed these constructs to be PoPS converters as follows...

Thresholddevice3.jpg

...and generated our own set of Sensitivity Tuners:

P2 ogr activator PSP3 pag activator phiR73 delta activator
PF promoter
PO promoter
PP promoter
Psid promoter
PLL promoter

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.

Characterisation

For characterisation, we moved the Cambridge 2007 amplifiers onto a low copy plasmid in order to make meaningful comparisons with , 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.

Cambridge hillfunction.gif

Characterization3.jpg

For our characterisation assay, we transformed the E. coli strain BW27783 with the Cambridge 2007 amplifiers (biobricks summarised in the table below):

P2 ogr activator PSP3 pag activator phiR73 delta activator
PF promoter
PO promoter
PP promoter
Psid promoter
PLL promoter

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.

Data

Parameters of Fitted Curves

Construct Increase in Rate, a (RPU) Basal Rate, c (RPU) Switch Point, k (uM) Hill Coefficient, n
0.205 0.022 0.85 2.72
0.323 0.039 0.92 2.89
0.137 0.021 0.88 2.51
0.196 0.032 0.95 2.71
0.129 0.018 2.20 4.00
0.452 0.118 1.05 2.45
0.496 0.029 3.98 2.37
0.194 0.018 1.46 3.96
1.312 0.058 1.11 2.78
1.271 0.040 0.35 2.26
1.105 0.046 1.81 4.00
1.366 0.040 1.91 4.00

Sample Graph

Cambridge characterisation82.png

Discussion

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.

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.

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.

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.

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Retrieved from "http://2009.igem.org/Team:Cambridge/Project/Amplification/Characterisation"