July 6

From 2007.igem.org

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* Open Loop [Ee]
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i) Cells were transferred to 50 mL Glu M9 in 250 mL flask with [aTc]=50 ng/mL and inoculum=1,0.1 uL/mL
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ii) K12Z1 was inoculated in 5 mL Glu M9 in 50 mL tube with [aTc]=50 ng/mL and inoculum=1,0.1 uL/mL
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iii) pL.LuxR.Y.pR.C, K12z1, pL.Y were inoculated in LB
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iv) The contents of the flask with OD=0.217 were filtered out.
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v) 7 dilutions were made (full range of IPTG conc) for pL.LuxR.Y.pR.C with AI from 50 ng/mL aTc and new 2xM9 in 1:1 ratio.
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* Open Loop [Cc]
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i) Cells were transferred to 50 mL Glu M9 in 250 mL flask with [aTc]=5 ng/mL and inoculum=1,0.1 uL/mL
 +
 +
ii) K12z1 was inoculated in 5 mL Glu M9 in 50 mL tube with [aTc]=5 ng/mL and inoculum=1,0.1 uL/mL
 +
 +
iii) pL.LuxR.Y.pR.C, K12z1, pL.Y were inoculated in LB.
 +
 +
iv) The contents of the flask with OD=0.103 were filtered out.
 +
 +
v) 7 dilutions over the full range of IPTG were made for pL.LuxR.Y.pR.C with AI from 5 ng/mL aTc and new 2xM9 in 1:1 ratio
 +
 +
 +
* Control experiments:
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** -ve control: K12z1
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**Inoculated positive control:
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***pL.YFP; 1xGlu M9; 500 uM IPTG
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***I726081; K12Z1 in aTc (50 ng/mL) medium and new 2xGlu M9 in 1:1 ratio; 500 uM IPTG
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***I726081; only new 1xGlu M9; 500 uM IPTG
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'''Microscopy'''
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* The open loop samples [Ee] were imaged and FACS was done.
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* The open loop samples [Cc] were imaged and FACS was done.
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* The open loop samples [Dd] were imaged and FACS was done.
==Analysis==
==Analysis==
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|align="justify"|These values, obtained from the microscopy experiments, give us a clear idea of the equivalence between luxI.CFP and luxI.YFP.
|align="justify"|These values, obtained from the microscopy experiments, give us a clear idea of the equivalence between luxI.CFP and luxI.YFP.

Latest revision as of 15:22, 18 August 2007

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Experiments

  • Open Loop [Ee]

i) Cells were transferred to 50 mL Glu M9 in 250 mL flask with [aTc]=50 ng/mL and inoculum=1,0.1 uL/mL

ii) K12Z1 was inoculated in 5 mL Glu M9 in 50 mL tube with [aTc]=50 ng/mL and inoculum=1,0.1 uL/mL

iii) pL.LuxR.Y.pR.C, K12z1, pL.Y were inoculated in LB

iv) The contents of the flask with OD=0.217 were filtered out.

v) 7 dilutions were made (full range of IPTG conc) for pL.LuxR.Y.pR.C with AI from 50 ng/mL aTc and new 2xM9 in 1:1 ratio.


  • Open Loop [Cc]

i) Cells were transferred to 50 mL Glu M9 in 250 mL flask with [aTc]=5 ng/mL and inoculum=1,0.1 uL/mL

ii) K12z1 was inoculated in 5 mL Glu M9 in 50 mL tube with [aTc]=5 ng/mL and inoculum=1,0.1 uL/mL

iii) pL.LuxR.Y.pR.C, K12z1, pL.Y were inoculated in LB.

iv) The contents of the flask with OD=0.103 were filtered out.

v) 7 dilutions over the full range of IPTG were made for pL.LuxR.Y.pR.C with AI from 5 ng/mL aTc and new 2xM9 in 1:1 ratio


  • Control experiments:
    • -ve control: K12z1
    • Inoculated positive control:
      • pL.YFP; 1xGlu M9; 500 uM IPTG
      • I726081; K12Z1 in aTc (50 ng/mL) medium and new 2xGlu M9 in 1:1 ratio; 500 uM IPTG
      • I726081; only new 1xGlu M9; 500 uM IPTG


Microscopy

  • The open loop samples [Ee] were imaged and FACS was done.
  • The open loop samples [Cc] were imaged and FACS was done.
  • The open loop samples [Dd] were imaged and FACS was done.

Analysis

Today we compiled the data collected from all our open-loop experiments performed at various concentrations of aTc (ng/ml). The following matrix of information was obtained:

CFP mean

values

IPTG (uM) or luxR.YFP

0 5 10 50 100 500 1000

aTc (ng/ml)

or

luxI.CFP

0
1
5
10
20
50

YFP mean

values

IPTG (uM) or luxR.YFP

0 5 10 50 100 500 1000

aTc (ng/ml)

or

luxI.CFP

0
1
5
10
20
50
These values, obtained from the microscopy experiments, give us a clear idea of the equivalence between luxI.CFP and luxI.YFP.

A scatter plot with each dot representing a cell was also plotted to see the variation of fluorescence with the change in aTc and IPTG concentrations.

  • Red : 0 ng/ml aTc
  • Green : 1 ng/ml aTc
  • Blue : 5 ng/ml aTc
  • Cyan : 10 ng/ml aTc
  • Magenta : 20 ng/ml aTc
  • Yellow : 50 ng/ml aTc


CFP Fluorescence YFP Fluorescence
CFP fluorescence
YFP fluorescence
CFP fluorescence
YFP fluorescence

Surface plots : The increasing values along the luxI.CFP axis are the increasing concentrations of aTc whereas the increasing values along the luxR.YFP axis are the increasing concentrations of IPTG.

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