Team:Imperial College London/Wetlab/Protocols

From 2009.igem.org

(Difference between revisions)
(→Protein production assays)
(→Calibrations)
 
(109 intermediate revisions not shown)
Line 1: Line 1:
{{Imperial/09/TemplateTop}}
{{Imperial/09/TemplateTop}}
{{Imperial/09/Tabs/Main/Wetlab/Protocols}}
{{Imperial/09/Tabs/Main/Wetlab/Protocols}}
 +
= Protocols =
-
=Autoinduction assays=
+
==[[Team:Imperial College London/Wetlab/Protocols/Cellculture| Cell culturing/cloning protocols]]==
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/Miniprep |<b>CC1</b>]]: Miniprep
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/Midiprep |<b>CC2</b>]]: Midiprep
 +
* <b>CC3</b>: Making cells competent
 +
* <b>CC4</b>: Ligation
 +
* <b>CC5</b>: Transformation into BL21 using electrical shock
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/Cellculture/TransTop10| <b>CC6</b>]]: Transformation into Top10 using chemical competence
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/LB_Plates |<b>CC7</b>]]: Making LB plates
 +
*  [[Team:Imperial_College_London/Wetlab/Protocols/Registry_Extraction |<b>CC8</b>]]: Extracting DNA from the registry
 +
* <b>[[Team:Team:Imperial College London/Wetlab/Protocols/Cellculture/CC9| CC9]]</b>: Preparation of XL1-Blue electrocompetent cells & XL1-Blue quality control
 +
* [[Team:Imperial College London/Wetlab/Protocols/PCR |<b>CC10</b>]]: PCR
 +
* <b>[[Team:Team:Imperial College London/Wetlab/Protocols/Cellculture/CC11| CC11]]</b>: SLIC
 +
*<b>[[Team:Team:Imperial College London/Wetlab/Protocols/Cellculture/CC12| CC12]]</b>: Biobricking parts from oligos
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/GenomePrep |<b>CC13</b>]]: Genome Prep for Restriction Assay
-
==Secondary Carbon Source and Diauxie growth assay==
+
==[[Team:Imperial College London/Wetlab/Protocols/Calibration| Calibrations]]==
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/Abs |<b>CA1</b>]]: Optical Density Calibration
 +
* GFP fluorescence calibration
 +
** [[Imperial_College_London/Wetlab/Protocols/FluorEx| <b>CA2</b>]]: External GFP fluorescence
 +
** [[Imperial_College_London/Wetlab/Protocols/FluorIn| <b>CA3</b>]]: Intracellular GFP fluorescence
-
===Aims===
+
==[[Team:Imperial College London/Wetlab/Protocols/PromoterCharacterisation| Promoter Characterisation]]==
-
* To generate the diauxie growth curves, as well as normal growth curves for Top-10 for modelling
+
* [[Team:Imperial_College_London/Wetlab/Protocols/IPTG-RFP |<b>PP1</b>]]: Lac Promoter Characterisation
-
* To determine the best secondary carbon source for optimal growth
+
-
===Assay===
+
==[[Team:Imperial College London/Wetlab/Protocols/Autoinduction| Autoinduction]]==
-
Cells are allowed to grow at 28°C, and their growth ie OD would be monitored using a plate reader, at 600nm, at regular intervals overnight. A plot of OD vs time will be generated. This result is to be fitted to a diauxie growth model. A control with only glucose and no secondary carbon source is used. This control will give us normal growth curves of Top-10 cells.
+
* [[Team:Imperial_College_London/Wetlab/Protocols/SecondaryCarbon |<b>AI1</b>]]: Secondary Carbon Source Experiment
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/Glucose_delay |<b>AI2</b>]]: Glucose Time Delay
 +
<!--[[Team:Imperial_College_London/Wetlab/Protocols/SecondaryCarbon| Secondary Carbon Source Experiment]]-->
-
[[Team:Imperial_College_London/Wetlab/Protocols/SecondaryCarbon|See protocol for details]]
+
==[[Team:Imperial College London/Wetlab/Protocols/M1| Protein Production]]==
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/IPTGgrowth |<b>PP1</b>]]: IPTG Toxicity
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/IPTG-RFP |<b>PP2</b>]]: IPTG characterisation
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/Cellulase |<b>PP3</b>]]: Cellulase
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/PAH |<b>PP4</b>]]: PAH
 +
<!--[[Team:Imperial_College_London/Wetlab/Protocols/Cellulase|IPTG Toxicity]]
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/Cellulase|Cellulase]]
 +
* [[Team:Imperial_College_London/Wetlab/Protocols/PAH|PAH]]
 +
-->
 +
==[[Team:Imperial College London/Wetlab/Protocols/M2| Encapsulation]]==
 +
* <b>EN1</b>: Colanic acid production amount
 +
* <b>EN2</b>: Colanic acid protection from pH
 +
* <b>[[Team:Imperial_College_London/Wetlab/Protocols/Trehalose | EN3]]</b>: Trehalose production
-
==Glucose time delay==
+
==[[Team:Imperial College London/Wetlab/Protocols/M3| Killing]]==
-
===Aims===
+
* [[Team:Imperial_College_London/Wetlab/Protocols/Thermoinduction |<b>KI1</b>]]: Thermoinduction
 +
* <b>KI2</b>: Restriction Enzyme activity
-
* Characterise the tunable time duration it takes before GFP expression (M2 activation)
+
<!--
 +
[[Team:Imperial_College_London/Wetlab/Protocols/ColanicAcid|Colanic Acid ]]
 +
# Trehalose Production
 +
#*  
 +
# Thermoinduction
 +
#* [[Team:Imperial_College_London/Wetlab/Protocols/Thermoinduction| Thermoinduction Assay]]<br>
 +
# Genome Restriction
 +
#* [[Team:Imperial_College_London/Wetlab/Protocols/Restriction|In Vitro Restriction Assay]]
 +
-->
-
===Assay===
+
<!--=Autoinduction assays=
-
 
+
-
*The cells will be grown until OD= 0.7. 
+
-
 
+
-
*The RFP value will be monitered, although it is the GFP values that are more critical in this assay.
+
-
 
+
-
*OD and fluorescence data for GFP which can be converted in [http://partsregistry.org/cgi/measurement/new_batch.cgi Specific Promoter Units (SPUs)].
+
-
 
+
-
*The secondary carbon source will be taken from the previous experiment (see Secondary carbon source selection for CRP promoter)
+
-
 
+
-
*The IPTG concentrations will be taken from the previous experiment (see Determining concentration of IPTG)
+
-
 
+
-
[[Team:Imperial_College_London/Wetlab/Protocols/Glucose delay|See Glucose time delay for details]]
+
-
 
+
-
=Protein production assays=
+
-
 
+
-
==[[Team:Imperial_College_London/Wetlab/Protocols/Cellulase| Cellulase Assay]]==
+
-
 
+
-
==[[Team:Imperial_College_London/Wetlab/Protocols/PAH| PAH Assay]]==
+
==IPTG effect on growth==
==IPTG effect on growth==
Line 46: Line 68:
===Aims===
===Aims===
* Measure growth rates at 28 degrees Celsius on minimal growth media so that subsequent testing timings etc. can be streamlined  
* Measure growth rates at 28 degrees Celsius on minimal growth media so that subsequent testing timings etc. can be streamlined  
-
* Determine the effect of IPTG toxicity on growth w/o any protein production complications  
+
* Determine the effect of IPTG toxicity on growth w/o any protein production complications
===Assay===
===Assay===
Line 88: Line 110:
[[Team:Imperial_College_London/Wetlab/Protocols/ColanicAcid| Click here for Colanic Acid assay details]]<br>
[[Team:Imperial_College_London/Wetlab/Protocols/ColanicAcid| Click here for Colanic Acid assay details]]<br>
-
=Genomic deletion assays=
+
 
-
[[Team:Imperial_College_London/Wetlab/Protocols/Restriction| Restriction Assay]]<br>
+
-->
-
[[Team:Imperial_College_London/Wetlab/Protocols/Thermoinduction| Thermoinduction Assay]]<br>
+
{{Imperial/09/TemplateBottom}}
{{Imperial/09/TemplateBottom}}

Latest revision as of 21:05, 20 October 2009



Contents

Protocols

Cell culturing/cloning protocols

  • CC1: Miniprep
  • CC2: Midiprep
  • CC3: Making cells competent
  • CC4: Ligation
  • CC5: Transformation into BL21 using electrical shock
  • CC6: Transformation into Top10 using chemical competence
  • CC7: Making LB plates
  • CC8: Extracting DNA from the registry
  • CC9: Preparation of XL1-Blue electrocompetent cells & XL1-Blue quality control
  • CC10: PCR
  • CC11: SLIC
  • CC12: Biobricking parts from oligos
  • CC13: Genome Prep for Restriction Assay

Calibrations

  • CA1: Optical Density Calibration
  • GFP fluorescence calibration
    • CA2: External GFP fluorescence
    • CA3: Intracellular GFP fluorescence

Promoter Characterisation

  • PP1: Lac Promoter Characterisation

Autoinduction

  • AI1: Secondary Carbon Source Experiment
  • AI2: Glucose Time Delay

Protein Production

  • PP1: IPTG Toxicity
  • PP2: IPTG characterisation
  • PP3: Cellulase
  • PP4: PAH

Encapsulation

  • EN1: Colanic acid production amount
  • EN2: Colanic acid protection from pH
  • EN3: Trehalose production

Killing

  • KI1: Thermoinduction
  • KI2: Restriction Enzyme activity


Mr. Gene   Geneart   Clontech   Giant Microbes

Retrieved from "http://2009.igem.org/Team:Imperial_College_London/Wetlab/Protocols"