Team:Imperial College London/Wetlab/Protocols

From 2009.igem.org

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= Protocols =
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=Autoinduction assays=
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==[[Team:Imperial College London/Wetlab/Protocols/Cellculture| Cell culturing/cloning protocols]]==
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* [[Team:Imperial_College_London/Wetlab/Protocols/Miniprep |<b>CC1</b>]]: Miniprep
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* [[Team:Imperial_College_London/Wetlab/Protocols/Midiprep |<b>CC2</b>]]: Midiprep
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* <b>CC3</b>: Making cells competent
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* <b>CC4</b>: Ligation
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* <b>CC5</b>: Transformation into BL21 using electrical shock
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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/LB_Plates |<b>CC7</b>]]: Making LB plates
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*  [[Team:Imperial_College_London/Wetlab/Protocols/Registry_Extraction |<b>CC8</b>]]: Extracting DNA from the registry
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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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* [[Team:Imperial College London/Wetlab/Protocols/PCR |<b>CC10</b>]]: PCR
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* <b>[[Team:Team:Imperial College London/Wetlab/Protocols/Cellculture/CC11| CC11]]</b>: SLIC
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*<b>[[Team:Team:Imperial College London/Wetlab/Protocols/Cellculture/CC12| CC12]]</b>: Biobricking parts from oligos
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* [[Team:Imperial_College_London/Wetlab/Protocols/GenomePrep |<b>CC13</b>]]: Genome Prep for Restriction Assay
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==Secondary Carbon Source and Diauxie growth assay==
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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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===Aims===
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==[[Team:Imperial College London/Wetlab/Protocols/PromoterCharacterisation| Promoter Characterisation]]==
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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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* [[Team:Imperial_College_London/Wetlab/Protocols/IPTG-RFP |<b>PP1</b>]]: Lac Promoter Characterisation
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* To determine the best secondary carbon source for optimal growth
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===Assay===
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==[[Team:Imperial College London/Wetlab/Protocols/Autoinduction| Autoinduction]]==
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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 |<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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[[Team:Imperial_College_London/Wetlab/Protocols/SecondaryCarbon|See protocol for details]]
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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/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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==Glucose time delay==
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==[[Team:Imperial College London/Wetlab/Protocols/M3| Killing]]==
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===Aims===
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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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* Characterise the tunable time duration it takes before GFP expression (M2 activation)
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<!--
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[[Team:Imperial_College_London/Wetlab/Protocols/ColanicAcid|Colanic Acid ]]
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# Trehalose Production
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#*  
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# Thermoinduction
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#* [[Team:Imperial_College_London/Wetlab/Protocols/Thermoinduction| Thermoinduction Assay]]<br>
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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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-->
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<!--=Autoinduction assays=
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==IPTG effect on growth==
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===Aims===
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* 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
===Assay===
===Assay===
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Normal cells (without any constructs) will be grown on M9 media until OD=0.7. <br>
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IPTG of various concentrations will then be added, and the OD of the cells will be followed over time <br>
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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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[[Team:Imperial_College_London/Wetlab/Protocols/IPTGgrowth| See the protocol for more details]]
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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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==Lac characterisation and IPTG effect on protein production==
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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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===Aims===
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*The IPTG concentrations will be taken from the previous experiment (see Determining concentration of IPTG)
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* Characterise Lac promoter by varying amounts of IPTG
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* Determine the IPTG concentration that allows for maximal protein production while still being non-toxic to the cell
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[[Team:Imperial_College_London/Wetlab/Protocols/Glucose delay|See Glucose time delay for details]]
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===Assay===
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=Protein production assays=
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The cells will be grown until OD=0.7. Now, IPTG of various concentrations will be added, and the RFP output will be measured. <br>
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[[Team:Imperial_College_London/Wetlab/Protocols/Cellulase| Cellulase Assay]]<br>
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The experiment will generate OD and fluoresence data for RFP <br>
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[[Team:Imperial_College_London/Wetlab/Protocols/PAH| PAH Assay]]<br>
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The secondary carbon source will be taken from the previous experiment (see Secondary carbon source selection)<br>
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The glucose concentration will be taken from commercial autoinduction media (0.05%) – takes about 7 hours to exhaust <br>
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[[Team:Imperial_College_London/Wetlab/Protocols/IPTG-RFP| See the protocol for more details]]
=Encapsulation assays=
=Encapsulation assays=
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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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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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