IGEM:IMPERIAL/2007/Projects/Experimental Design/Phase1: Difference between revisions

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===1.2 Initial testing for Infector Detector===
===1.2 Initial testing for Infector Detector===
'''Construct:''' pTet - LuxR - pLux - GFP <br>
'''Construct:''' pTet - LuxR - pLux - GFP [[IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 1.2 |Protocol]] <br>
Test to see if construct will express in vivo. Experiments carried out in incubator at 30<sup>o</sup>C at a range of inducer concentrations. To do this, we induce E. coli cells transfected with the construct with known concentrations of AHL. We then record the change in GFP, such that we can calculate the rate of GFP production relative to concentration of AHL in solution.
Test to see if construct will express in vivo. Experiments carried out in incubator at 30<sup>o</sup>C at a range of inducer concentrations. To do this, we induce E. coli cells transfected with the construct with known concentrations of AHL. We then record the change in GFP, such that we can calculate the rate of GFP production relative to concentration of AHL in solution.
<br>
<br>
Line 162: Line 162:
*Positive Control: In vitro system with purified GFP added
*Positive Control: In vitro system with purified GFP added
}}
}}
<br>
===Protocol===
===Equipment===
*Incubator 30<sup>o</sup>C
*Fluorometer
*Spectrometer
*Plate x1
*Plate Centrifuge
*6x1.5ml tubes
*Gilson Pipettes
*Stop watch
'''DNA Concentration'''
#We need to be able to quantify the DNA from the mini preps and midi preps. To do this we measure the A<sub>260nm</sub> where one unit corresponds to 50ug/ml of double stranded DNA.
#First remove 5ul of the DNA sample and add to 1ml of MiiA/Nuclease Free water to a quatz curvette.
#Place an empty curvette and zero the machine.
#Place the curvette containing the DNA in the spectrometer and measure the A<sub>260nm</sub>.
#To calculate the A<sub>260nm</sub>,
<p align="center"> '''DNA ug/ml = 50 x A<sub>260nm</sub>'''
#In addition we can measure A<sub>280nm</sub> and calculate the purity of the DNA sample. To do this measure, A<sub>280nm</sub> and calculate A<sub>260nm</sub>/A<sub>280nm</sub>
#A value between 1.8 - 2.0 indicates pure  DNA
<br>
'''Preparations of Chemicals and Reagents'''<br>
After preparing all solutions preheat to 30<sup>o</sup>C
#Remove .... of our cell extract solution from storage solution
#Now we need to prepare 50μl of the commercial E.coli and T7 cell extract solution. First we need to prepare complete amino acid mixture for both extract solutions: Add the 7.5μl volume of two amino acid minus mixtures for both kits''
#Take 3x1.5ml tube and add 5µl of the E.coli complete amino acid mixture
#Take 3x1.5ml tube and add 5µl of the T7 complete amino acid mixture
#To each 1.5ml tube add 20µl of ''S30 Premix Without Amino Acid''
#Add 15µl of ''S30 Extract Circular''
#Add ...''nuclease-Free Water''
<br>
'''Loading Plate'''
#Follow the schematic for the plate adding everything but the DNA sample.
#Place the top on the plate and place in the incubator. Leave for a few minutes to heat to 30<sup>o</sup>C
#Remove from incubator and centrifuge for 1 minute
#Remove lid and Measure in the flourometer.
#Then to begin the reaction add .... purified DNA sample.
#Place lid back on and place back in the incubator at 30<sup>o</sup>C
<br>
'''Collecting Data'''
#After 5 minutes of incubation measure the fluorescence by repeating procedure 3-4 above.
#Repeat measurements after every 5 minutes until the Fluorescence is constant
#Before every measurement in the fluorometer spin the plates in a plate centrifuge
'''Schematic'''
{| border="1" cellpadding="1"
|
{| border="1" cellpadding="2"
!<u>Well </u> || <u>Test Construct</u> !! <u>In vitro chassis</u>
|-
|<font color=blue> A1
|<font color=blue> pTet
|<font color=blue> Commercial E.coli extract
|-
|<font color=blue>A2
|<font color=blue>pTet
|<font color=blue>Commercial E.coli extract
|-
|<font color=blue>A3
|<font color=blue>pTet
|<font color=blue>Commercial E.coli extract
|-
|<font color=blue>A4
|<font color=blue>pTet
|<font color=blue>Our S30 Cell extract
|-
|<font color=blue>A5
|<font color=blue>pTet
|<font color=blue>Our S30 Cell extract
|-
|<font color=blue>A6
|<font color=blue>pTet
|<font color=blue>Our S30 Cell extract
|-
|<font color=green>A7
|<font color=green>None
|<font color=green>Commercial E.coli extract
|-
|<font color=green>A8
|<font color=green>None
|<font color=green>Our S30 Cell extract
|-
|<font color=purple>B1
|<font color=purple>pT7
|<font color=purple>Commercial T7 extract
|-
|<font color=purple>B2
|<font color=purple>pT7
|<font color=purple>Commercial T7 extract
|-
|<font color=purple>B3
|<font color=purple>pT7
|<font color=purple>Commercial T7 extract
|-
|<font color=purple>B4
|<font color=purple>pT7
|<font color=purple>Our S30 Cell extract
|-
|<font color=purple>B5
|<font color=purple>pT7
|<font color=purple>Our S30 Cell extract
|-
|<font color=purple>B6
|<font color=purple>pT7
|<font color=purple>Our S30 Cell extract
|-
|<font color=green>B7
|<font color=green>None
|<font color=green>Commercial T7 extract
|-
|<font color=green>B8
|<font color=green>None
|<font color=green>Our S30 Cell extract
|-
|}
|
[[Image:Intial testing.PNG|400px|T9002 96 well plate]]
|}
<br>
<br>

Revision as of 03:34, 14 August 2007



1. Initial Promoter Testing - In Vivo

1.1 Initial Testing for Cell by Date

Constructs: pTet-GFP, pT7-GFP, pcI-GFP
Test to see if construct will express in vivo. Experiments carried out at 30°C in an incubator.
Aims:

  • To determine if construct expresses in vivo
  • To get approximations of: life span, response time and rate of GFP synthesized.
  • To determine whether the constructs actually work before testing them in vitro.

Constant Conditions:

  • 50μl in vitro chassis
  • DNA concentration
  • Temperature stable at 30°C

Variables:

  • Rate of GFP synthesis
  • Life span of chassis
  • Response time

Sampling:

  • Every 5 minutes.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: E.Coli cultures without GFP expressing machinery
  • Positive Control: Diluted GFP in well



1.2 Initial testing for Infector Detector

Construct: pTet - LuxR - pLux - GFP Protocol
Test to see if construct will express in vivo. Experiments carried out in incubator at 30oC at a range of inducer concentrations. To do this, we induce E. coli cells transfected with the construct with known concentrations of AHL. We then record the change in GFP, such that we can calculate the rate of GFP production relative to concentration of AHL in solution.
Aims:

  • To determine if construct expresses GFP in vivo
  • To determine the rate of change of protein production relative to concentration of AHL
  • To determine whether the constructs need to be optimised before testing in vitro.

Constant Conditions:

  • Temperature stable at 30°C

Variables:

  • AHL concentrations
  • Rate of GFP synthesis
  • Life span of chassis
  • Response time

Sampling:

  • Every 5 minutes.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vivo system with no AHL added
  • Positive Control: In vivo system with a large amount of AHL added.


2. Initial Promoter Testing - In Vitro

2.1 Initial Testing for Cell by Date

Constructs: pTet-GFP, pT7-GFP, pcI-GFP
Test to see if construct will express in vitro. Experiments carried out at 30°C in an incubator.
Aims:

  • To determine if construct expresses in vitro
  • To get approximations of: life span, response time and rate of GFP synthesized.
  • To determine whether the constructs or the in vitro need to be optimised.

Constant Conditions:

  • 50μl in vitro chassis
  • DNA concentration
  • Temperature stable at 30°C

Variables:

  • Rate of GFP synthesis
  • Life span of chassis
  • Response time

Sampling:

  • Every 5 minutes.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system only
  • Positive Control: In vitro system with purified GFP added


2.2 Initial testing for Infector Detector

Construct: pTet - LuxR - pLux - GFP Protocol
Test to see if construct will express in vitro. Experiments carried out at incubator at 30oC and under inducer concentration that was shown to give a high induction in vivo.
Aims:

  • To determine if construct expresses in vitro
  • To get approximations of: life span, response time and rate of GFP
  • To determine whether the constructs or the in vitro need to be optimised.

Constant Conditions:

  • 25 oC
  • 50μl in vitro chassis
  • DNA added 2μg
  • AHL concentration 1000nM

Variables:

  • Independent variables :
  • Dependent variables:
    • Rate of GFP synthesis
    • Life span of chassis
    • Response time

Sampling:

  • Every 5 minutes.

Repetition:

  • 3 repeats

Controls:

  • Negative Control: In vitro system with no AHL added
  • Positive Control: In vitro system with purified GFP added


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