IGEM:IMPERIAL/2007/Projects/Cell by date/Implementation: Difference between revisions

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==Lab Notebook==
{|{| style="background:#F5FAFF; border: 1px solid #aabadd; color:Black"
|- style="background:#aabadd; color:Black"
|| '''Type of Promoter''' ||'''Test Constructs''' || '''Status'''
|- style="background:#d5dcef" 
| style="background:#aabadd" | Constitutive T7 Promoter
|[[image:ICGEMS_dvc_PT7_gfp.png|left|PT7 promoting GFP]]
|
*Entire construct is currently available in Biobricks Registry: [http://parts.mit.edu/registry/index.php/Part:BBa_E7104 BBa_E7104] (available), in [http://parts.mit.edu/registry/index.php/Part:pSB1A2 pSB1A2] (AmpR)
*pT7 can be used with T7 RNAP in vitro and in veso, and it gives constitutive expression.


*'''Week 5 (6 Aug):''' Phase 1<br>
|- style="background:#d5dcef"
*'''Week 6 (13 Aug):''' Phase 1<br>
| style="background:#aabadd" | Constitutive E.coli Promoter
*'''Week 7 (20 Aug):''' <br>
|[[image:ICGEMS_dvc_Ptet_gfp.png|left|P<sub>tet</sub promoting GFP]]
*'''Week 8 (27 Aug):''' <br>
|
*'''Week 9 (3 Sep):''' <br>
*Entire construct is currently in Biobricks Registry: [http://parts.mit.edu/registry/index.php/Part:BBa_I13522 BBa_I13522] (available), in [http://parts.mit.edu/registry/index.php/Part:pSB1A2 pSB1A2] (AmpR)
*'''Week 10 (10 Sep):''' <br>
*pTet can be used with E.coli RNAP in vitro and maybe in veso, and it gives constitutive expression.


|- style="background:#d5dcef"
| style="background:#aabadd" | Inducible E.coli Promoter
|[[image:ICGEMS_dvc_PBad_gfp.png|left|PBad promoting GFP]]
|
*pBad promoter and GFP available: [http://parts.mit.edu/registry/index.php/Part:BBa_J5528 BBa_J5528] (available), in [http://parts.mit.edu/registry/index.php/Part:pSB2K3 pSB2K3] (KanR)
*Need Ara C regulator protein in vitro and in veso. Ara C available with the RBS:[http://parts.mit.edu/registry/index.php/Part:BBa_S03550 BBa_S03550]
*pBad can be used with E.coli RNAP in vitro, and it is inducible by arabinose.
*To be used in veso, arabinose transport proteins have to be inserted in the phospholipid bilayer of the vesicles.


<calendar>
|- style="background:#d5dcef"
name=iGEM:IMPERIAL/2007/Notebook
| style="background:#aabadd" | Constitutive E.coli Promoter
date=2007/09/15
|[[image:PcI_construct.PNG|left|PcI promoting GFP]]
view=threemonths
|
format=%name/%year-%month-%day
*Entire construct is currently available in Biobricks Registry:  [http://parts.mit.edu/registry/index.php/Part:BBa_J07037 BBa_J07037] (avaiable), in [http://parts.mit.edu/registry/index.php/Part:pSB2K3 pSB2K3] (KanR)
weekstart=7
*pcI gives constitutive expression.
</calendar>


|- style="background:#d5dcef"
| style="background:#aabadd" | Constitutive E.coli Promoter
|[[image:ICGEMS_dvc_PBad2_gfp.PNG‎ |left|pBad promoting GFP and pcI promoting araC]]
|
*All parts are currently available in the Biobricks Registry.
*pcI promoter: [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 BBa_R0051] (available), in [http://parts.mit.edu/registry/index.php/Part:pSB1A2 pSB1A2] (AmpR)
*Arabinose C regulator protein with RBS: [http://parts.mit.edu/registry/index.php/Part:BBa_S03550 BBa_S03550] (available), in [http://parts.mit.edu/registry/index.php/Part:pSB1AC3 pSB1AC3] (A+CR)
*Stop codon: [http://parts.mit.edu/registry/index.php/Part:BBa_B0015 BBa_B0015], in [http://parts.mit.edu/registry/index.php/Part:pSB1AK3 pSB1AK3] (A+KR)
*pBAD with GFP: [http://parts.mit.edu/registry/index.php/Part:BBa_J5528 BBa_J5528], in [http://parts.mit.edu/registry/index.php/Part:pSB2K3 pSB2K3] (KanR)


 
|}
==Phase 1: Initial Testing==
===1) Initial Testing===
'''Constructs:''' pTet-GFP, pT7-GFP, pcI-GFP<br>
Test to see if construct will express ''in vitro''. Experiments carried out at 30&deg;C in an incubator.<br>
'''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.
'''Conditions:'''
<br>
{{hide|
*30&deg;C
*50μl in vitro chassis
*DNA concentration
}}
'''Variables:'''
<br>
{{hide|
*Rate of GFP synthesis
*Life span of chassis
*Response time
}}
'''Sampling:'''
<br>
{{hide|
*Every 15 minutes.
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
*Positive Control: In vitro system with purified GFP added
}}
<br>
 
==Phase 2: Characterizing Specific Construct==
===1) Calibration Curve for GFP===
Test to determine the relationship between fluorescence and in vitro concentration of GFP. To test this purified samples of known [GFP] are added to in vitro chassis and the fluorescence measured. From this a calibration curve of [GFP] vs Fluorescence can be made. This can be used for data analysis to convert fluorescence into a [GFP].<br>
'''Aims:'''
*To determine [GFP] vs Fluorescence
'''Conditions:'''<br>
{{hide|
*50μl in vitro chassis
*DNA added
**Do we need DNA, as it may absorb some of the fluorescence
*25<sup>o</sup>C
}}
'''Variables:'''
<br>
{{hide|
*[GFP] added
*Fluorescence
}}
'''Sampling:'''
<br>
{{hide|
*Measure after addition of GFP to minimize degradation of GFP
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
**No GFP is added to an in vitro chassis
}}
<br>
 
===2) Degradation Time of GFP===
Test the half life of GFP protein in an in vitro chassis. To test this a purified sample of known [GFP] are added to an in vitro chassis, then fluorescence will be measured at regular time intervals. The fluorescence will be converted into GFP molecules using the calibration curve. This will give; degradation of GFP as a function of time, from this the half life of GFP can be obtained. In addition, temperature may affect the half life of GFP and so the half life will be measured for an appropriate temperature range.<br>
'''Aims:'''
*To determine the half life of GFP for a range of temperatures
'''Conditions:'''
<br>
{{hide|
*50μl in vitro chassis
*[GFP] added
}}
'''Variables:'''
<br>
{{hide|
*Temperature range: 4<sup>o</sup>C, 15<sup>o</sup>C, 25<sup>o</sup>C, 30<sup>o</sup>C, 37<sup>o</sup>C, 50<sup>o</sup>C
*Range may change based upon the initial testing; will only test ranges that in vitro is stable at.
*Degradation of GFP
}}
'''Sampling:'''
<br>
{{hide|
*Every 15 minutes.
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
*Positive Control: In vitro chassis with high concentration of purified GFP added at high temperature
}}
<br>
 
===3) Operating Temperature Range===
'''Constructs:''' pTet-GFP, pT7-GFP or pcI-GFP<br>
Test to determine the operating range of the preferred construct ''in vitro''. Experiments carried out across various temperatures.<br>
'''Aims:'''
*To determine if construct expresses ''in vitro'' at temperatures of: 4<sup>o</sup>C, 15<sup>o</sup>C, 25<sup>o</sup>C, 30<sup>o</sup>C, 37<sup>o</sup>C, 50<sup>o</sup>C
*To determine specific life span at each temperature range.
*To determine the maximum rate of GFP produced at each temperature range.
'''Conditions:'''
<br>
{{hide|
*50μl in vitro chassis
*DNA concentration
}}
'''Variables:'''
<br>
{{hide|
*Rate of GFP synthesis
*Life span of chassis
*Response time
*Temperature: 4<sup>o</sup>C, 15<sup>o</sup>C, 25<sup>o</sup>C, 30<sup>o</sup>C, 37<sup>o</sup>C, 50<sup>o</sup>C
}}
'''Sampling:'''
<br>
{{hide|
*Every 15 minutes.
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
*Positive Control: In vitro system with purified GFP added
}}
<br>
 
===4) Varying Temperature Changes: Gentle Gradient===
'''Constructs:''' pTet-GFP, pT7-GFP or pcI-GFP<br>
'''Aims:'''
*To determine the effects of fluorescence with reference to a gentle change in temperature from 4&deg;C to 37&deg;C and vice versa over different time periods.
*Provide results for modelling.
**Investigate k constant as a function of temperature and time.
'''Conditions:'''
<br>
{{hide|
*50μl in vitro chassis
*DNA concentration
}}
'''Variables:'''
<br>
{{hide|
*Rate of GFP synthesis
*Life span of chassis
*Response time
*Temperature change 4<sup>o</sup>C, 25<sup>o</sup>C, 37<sup>o</sup>C
**Type of gradients: Gentle(1 hour)
**Temperature change : from 4°C to 25°C, 37°C and vice versa
**Time period before increment: 30 min, 1h, 2h (to be done in parallel)
}}
'''Sampling:'''
<br>
{{hide|
*Every 30 min interval.
*Every 15 minutes for 2 hours after change in temperature.
*Every 30 minutes thereafter.
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
*Positive Control: In vitro system with purified GFP added
}}
<br>
 
===5) Varying Temperature Changes: Steep Gradient===
'''Constructs:''' pTet-GFP, pT7-GFP or pcI-GFP<br>
'''Aims:'''
*To determine the effects of fluorescence with reference to a steep change in temperature from 4&deg;C to 37&deg;C and vice versa over different time periods.
*Provide results for modelling.
**Investigate k constant as a function of temperature and time.
'''Conditions:'''
<br>
{{hide|
*50μl in vitro chassis
*DNA concentration
}}
'''Variables:'''
<br>
{{hide|
*Rate of GFP synthesis
*Life span of chassis
*Response time
*Temperature change 4<sup>o</sup>C, 25<sup>o</sup>C, 37<sup>o</sup>C
**Type of gradients: Steep (5 min)
**Temperature change : from 4°C to 25<sup>o</sup>C, 37<sup>o</sup>C and vice versa
**Time period before increment: 30 min, 1h, 2h,
}}
'''Sampling:'''
<br>
{{hide|
*Every 30 min interval.
*Every 15 minutes for 2 hours after change in temperature.
*Every 30 minutes thereafter.
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
*Positive Control: In vitro system with purified GFP added
}}
<br>
 
==Phase 3: Testing/Validation of Modelling Analysis==
===1) Testing/Validation of Device===
'''Constructs:''' pTet-GFP, pT7-GFP or pcI-GFP<br>
'''Aims:'''
*To validate the predictions of modelling analysis.
*To fine-tune the ''in vitro'' system
'''Conditions:'''
<br>
{{hide|
*50μl in vitro chassis
*DNA concentration
}}
'''Variables:'''
<br>
{{hide|
*Rate of GFP synthesis
*Life span of chassis
*Response time
*Temperature change 4<sup>o</sup>C, 25<sup>o</sup>C, 37<sup>o</sup>C
**Type of gradients: Pulse(5 min, 1 hour)
**Temperature change : from 4°C to 25<sup>o</sup>C, 37<sup>o</sup>C and vice versa
**Time period before increment: 30 min, 1h, 2h (subject to change)
**Temperature period maintained: 5 min, 15 min, 30 min, 1h, 2h
}}
'''Sampling:'''
<br>
{{hide|
*Every 30 min interval.
*Every 15 minutes for 2 hours after change in temperature.
*Every 30 minutes thereafter.
Repetition:
*3 repeats
}}
'''Controls:'''
<br>
{{hide|
*Negative Control: In vitro system only
*Positive Control: In vitro system with purified GFP added
}}
<br>
<br>

Revision as of 08:43, 13 August 2007

Cell by Date: Implementation



Type of Promoter Test Constructs Status
Constitutive T7 Promoter
PT7 promoting GFP
PT7 promoting GFP
  • Entire construct is currently available in Biobricks Registry: BBa_E7104 (available), in pSB1A2 (AmpR)
  • pT7 can be used with T7 RNAP in vitro and in veso, and it gives constitutive expression.
Constitutive E.coli Promoter
Ptet</sub promoting GFP
Ptet</sub promoting GFP
  • Entire construct is currently in Biobricks Registry: BBa_I13522 (available), in pSB1A2 (AmpR)
  • pTet can be used with E.coli RNAP in vitro and maybe in veso, and it gives constitutive expression.
Inducible E.coli Promoter
PBad promoting GFP
PBad promoting GFP
  • pBad promoter and GFP available: BBa_J5528 (available), in pSB2K3 (KanR)
  • Need Ara C regulator protein in vitro and in veso. Ara C available with the RBS:BBa_S03550
  • pBad can be used with E.coli RNAP in vitro, and it is inducible by arabinose.
  • To be used in veso, arabinose transport proteins have to be inserted in the phospholipid bilayer of the vesicles.
Constitutive E.coli Promoter
PcI promoting GFP
PcI promoting GFP
  • Entire construct is currently available in Biobricks Registry: BBa_J07037 (avaiable), in pSB2K3 (KanR)
  • pcI gives constitutive expression.
Constitutive E.coli Promoter
pBad promoting GFP and pcI promoting araC
pBad promoting GFP and pcI promoting araC


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