Team:Imperial College London/Wetlab/Protocols

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= Protocols =
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[[Team:Imperial_College_London/Wetlab/Protocols/Glucose delay|Glucose time delay]]
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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/ColanicAcid| Colanic Acid]]<br>
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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/Restriction| Restriction Assay]]
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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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==[[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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==[[Team:Imperial College London/Wetlab/Protocols/PromoterCharacterisation| Promoter Characterisation]]==
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* [[Team:Imperial_College_London/Wetlab/Protocols/IPTG-RFP |<b>PP1</b>]]: Lac Promoter Characterisation
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==[[Team:Imperial College London/Wetlab/Protocols/Autoinduction| Autoinduction]]==
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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/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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==[[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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<!--
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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
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===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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[[Team:Imperial_College_London/Wetlab/Protocols/IPTGgrowth| See the protocol for more details]]
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==Lac characterisation and IPTG effect on protein production==
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===Aims===
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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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===Assay===
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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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The experiment will generate OD and fluoresence data for RFP <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]]
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=Encapsulation assays=
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==Colanic Acid==
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===Aims===
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Colanic acid biosynthesis is both time consuming and metabolically expensive. If the E.ncapsulator is to be used in an industrial setting, then colanic acid mediated protection must be highly efficient. Protective efficiency can be defined as the percentage increase in fluorescence per µl of colanic acid produced (when compared to control cells). The following assay can be used to elucidate this parameter.
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===Assay===
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*Growth of cells (Assay preparation).
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*Measurement of cell density.
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*Measurement of packed cell volume (PCV).
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* Measurement of the % change in GFP fluorescence following acid incubation.
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[[Team:Imperial_College_London/Wetlab/Protocols/ColanicAcid| Click here for Colanic Acid assay details]]<br>
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-->
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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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