Team:Imperial College London/Wetlab/Protocols

From 2009.igem.org

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= Protocols =
= Protocols =
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# Promoter Characterisation
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==[[Team:Imperial College London/Wetlab/Protocols/Cellculture| Cell culturing/cloning protocols]]==
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#* Lac Promoter
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* [[Team:Imperial_College_London/Wetlab/Protocols/Miniprep |<b>CC1</b>]]: Miniprep
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# Auto Induction
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* [[Team:Imperial_College_London/Wetlab/Protocols/Midiprep |<b>CC2</b>]]: Midiprep
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#*
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* <b>CC3</b>: Making cells competent
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#* [[Team:Imperial_College_London/Wetlab/Protocols/SecondaryCarbon| Secondary Carbon Source Experiment]]
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* <b>CC4</b>: Ligation
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# Protein Production
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* <b>CC5</b>: Transformation into BL21 using electrical shock
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#* [[Team:Imperial_College_London/Wetlab/Protocols/Cellulase|Cellulase]]
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* [[Team:Imperial_College_London/Wetlab/Protocols/Cellculture/TransTop10| <b>CC6</b>]]: Transformation into Top10 using chemical competence
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#* [[Team:Imperial_College_London/Wetlab/Protocols/PAH|PAH]]
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* [[Team:Imperial_College_London/Wetlab/Protocols/LB_Plates |<b>CC7</b>]]: Making LB plates
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# Colanic Acid Encapsulation
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* [[Team:Imperial_College_London/Wetlab/Protocols/Registry_Extraction |<b>CC8</b>]]: Extracting DNA from the registry
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#*  
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* <b>[[Team:Team:Imperial College London/Wetlab/Protocols/Cellculture/CC9| CC9]]</b>: Preparation of XL1-Blue electrocompetent cells & XL1-Blue quality control
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# Trehalose Production
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* [[Team:Imperial College London/Wetlab/Protocols/PCR |<b>CC10</b>]]: PCR
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#*  
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* <b>[[Team:Team:Imperial College London/Wetlab/Protocols/Cellculture/CC11| CC11]]</b>: SLIC
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# Thermoinduction
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*<b>[[Team:Team:Imperial College London/Wetlab/Protocols/Cellculture/CC12| CC12]]</b>: Biobricking parts from oligos
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#*  
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* [[Team:Imperial_College_London/Wetlab/Protocols/GenomePrep |<b>CC13</b>]]: Genome Prep for Restriction Assay
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# Genome Restriction
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#* [[Team:Imperial_College_London/Wetlab/Protocols/Restriction|In Vitro Restriction Assay]]
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=Promoter characterisation assays=
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==[[Team:Imperial College London/Wetlab/Protocols/Calibration| Calibrations]]==
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* [[Team:Imperial_College_London/Wetlab/Protocols/Abs |<b>CA1</b>]]: Optical Density Calibration
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* GFP fluorescence calibration
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** [[Imperial_College_London/Wetlab/Protocols/FluorEx| <b>CA2</b>]]: External GFP fluorescence
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** [[Imperial_College_London/Wetlab/Protocols/FluorIn| <b>CA3</b>]]: Intracellular GFP fluorescence
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==Absorbance calibration==
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==[[Team:Imperial College London/Wetlab/Protocols/PromoterCharacterisation| Promoter Characterisation]]==
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===Aim===
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* [[Team:Imperial_College_London/Wetlab/Protocols/IPTG-RFP |<b>PP1</b>]]: Lac Promoter Characterisation
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*To produce a calibration curve to aid in the normalising of absorbance values.  The relation of absorbance reading to number of cells varies with different cell strains. We are therefore doing one for Top-10. <br>
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===Assay===
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==[[Team:Imperial College London/Wetlab/Protocols/Autoinduction| Autoinduction]]==
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Cultures of the E.coli with the relevant vector are grown to various cell densities. A sample of these cultures are taken and a dilution plate is carried out to work out approximate colony forming units per ml of culture. This data set is then combined with absorbance readings to create a graph relating the number of colony forming cells per ml to their absorbance measurements. <br>
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* [[Team:Imperial_College_London/Wetlab/Protocols/SecondaryCarbon |<b>AI1</b>]]: Secondary Carbon Source Experiment
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* [[Team:Imperial_College_London/Wetlab/Protocols/Glucose_delay |<b>AI2</b>]]: Glucose Time Delay
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<!--[[Team:Imperial_College_London/Wetlab/Protocols/SecondaryCarbon| Secondary Carbon Source Experiment]]-->
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This curve then allows us to convert absorbance of a known volume of culture to colony forming units within the culture sample. <br>
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==[[Team:Imperial College London/Wetlab/Protocols/M1| Protein Production]]==
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* [[Team:Imperial_College_London/Wetlab/Protocols/IPTGgrowth |<b>PP1</b>]]: IPTG Toxicity
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* [[Team:Imperial_College_London/Wetlab/Protocols/IPTG-RFP |<b>PP2</b>]]: IPTG characterisation
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* [[Team:Imperial_College_London/Wetlab/Protocols/Cellulase |<b>PP3</b>]]: Cellulase
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* [[Team:Imperial_College_London/Wetlab/Protocols/PAH |<b>PP4</b>]]: PAH
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<!--[[Team:Imperial_College_London/Wetlab/Protocols/Cellulase|IPTG Toxicity]]
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* [[Team:Imperial_College_London/Wetlab/Protocols/Cellulase|Cellulase]]
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* [[Team:Imperial_College_London/Wetlab/Protocols/PAH|PAH]]
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-->
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[[Team:Imperial_College_London/Wetlab/Protocols/Abs| See the protocol for more details]]
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==[[Team:Imperial College London/Wetlab/Protocols/M2| Encapsulation]]==
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* <b>EN1</b>: Colanic acid production amount
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* <b>EN2</b>: Colanic acid protection from pH
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* <b>[[Team:Imperial_College_London/Wetlab/Protocols/Trehalose | EN3]]</b>: Trehalose production
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==[[Team:Imperial College London/Wetlab/Protocols/M3| Killing]]==
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* [[Team:Imperial_College_London/Wetlab/Protocols/Thermoinduction |<b>KI1</b>]]: Thermoinduction
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* <b>KI2</b>: Restriction Enzyme activity
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=Autoinduction assays=
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<!--
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[[Team:Imperial_College_London/Wetlab/Protocols/ColanicAcid|Colanic Acid ]]
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==Secondary Carbon Source and Diauxie growth assay==
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# Trehalose Production
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#*  
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===Aims===
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# Thermoinduction
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#* [[Team:Imperial_College_London/Wetlab/Protocols/Thermoinduction| Thermoinduction Assay]]<br>
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* To generate the diauxie growth curves, as well as normal growth curves for Top-10 for modelling
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# Genome Restriction
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* To determine the best secondary carbon source for optimal growth
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#* [[Team:Imperial_College_London/Wetlab/Protocols/Restriction|In Vitro Restriction Assay]]
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-->
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===Assay===
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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.
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[[Team:Imperial_College_London/Wetlab/Protocols/SecondaryCarbon|See protocol for details]]
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==Glucose time delay==
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===Aims===
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* Characterise the tunable time duration it takes before GFP expression (M2 activation)
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===Assay===
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*The cells will be grown until OD= 0.7. 
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*The RFP value will be monitered, although it is the GFP values that are more critical in this assay.
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*OD and fluorescence data for GFP which can be converted in [http://partsregistry.org/cgi/measurement/new_batch.cgi Specific Promoter Units (SPUs)].
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*The secondary carbon source will be taken from the previous experiment (see Secondary carbon source selection for CRP promoter)
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*The IPTG concentrations will be taken from the previous experiment (see Determining concentration of IPTG)
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[[Team:Imperial_College_London/Wetlab/Protocols/Glucose delay|See Glucose time delay for details]]
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<!--=Autoinduction assays=
==IPTG effect on growth==
==IPTG effect on growth==
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===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  
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* Determine the effect of IPTG toxicity on growth w/o any protein production complications  
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* Determine the effect of IPTG toxicity on growth w/o any protein production complications
===Assay===
===Assay===
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[[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>
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=Genomic deletion assays=
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[[Team:Imperial_College_London/Wetlab/Protocols/Restriction| Restriction Assay]]<br>
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-->
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[[Team:Imperial_College_London/Wetlab/Protocols/Thermoinduction| Thermoinduction Assay]]<br>
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{{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


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