Team:Imperial College London/Wetlab/Protocols

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= Protocols =
= Protocols =
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# Calibrations
 
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#* Top 10 Growth Calibration
 
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# Promoter Characterisation
 
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#* Lac Promoter
 
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# Auto Induction
 
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#*
 
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#* [[Team:Imperial_College_London/Wetlab/Protocols/SecondaryCarbon| Secondary Carbon Source Experiment]]
 
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# Protein Production
 
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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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# Colanic Acid Encapsulation
 
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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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==[[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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<!--template:---------------------------------------------------------------------------->
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==[[Team:Imperial College London/Wetlab/Protocols/Calibration| Calibrations]]==
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{| style="color:#CCC; background-color:#325d97;" cellpadding="6" cellspacing="0" border="3"
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* [[Team:Imperial_College_London/Wetlab/Protocols/Abs |<b>CA1</b>]]: Optical Density Calibration
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! Section
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* GFP fluorescence calibration
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! Assay
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** [[Imperial_College_London/Wetlab/Protocols/FluorEx| <b>CA2</b>]]: External GFP fluorescence
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! Overview and Aims
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** [[Imperial_College_London/Wetlab/Protocols/FluorIn| <b>CA3</b>]]: Intracellular GFP fluorescence
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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==[[Team:Imperial College London/Wetlab/Protocols/PromoterCharacterisation| Promoter Characterisation]]==
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<!----------------------------------------biobricks---------------------------------------->
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* [[Team:Imperial_College_London/Wetlab/Protocols/IPTG-RFP |<b>PP1</b>]]: Lac Promoter Characterisation
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| 1.Calibration
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==[[Team:Imperial College London/Wetlab/Protocols/Autoinduction| Autoinduction]]==
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| [[Team:Imperial_College_London/Wetlab/Protocols/Abs| TOP10 Growth]]
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* [[Team:Imperial_College_London/Wetlab/Protocols/SecondaryCarbon |<b>AI1</b>]]: Secondary Carbon Source Experiment
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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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* [[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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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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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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| 1.Promoter Characterisation
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<!--
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| Blah
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[[Team:Imperial_College_London/Wetlab/Protocols/ColanicAcid|Colanic Acid ]]
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| To find
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# Trehalose Production
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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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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| 2.Auto-Induction
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<!--=Autoinduction assays=
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| [[Team:Imperial_College_London/Wetlab/Protocols/SecondaryCarbon|Secondary Carbon Source Diauxie Growth]]
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|
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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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* To determine the best secondary carbon source for optimal growth
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
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| 2.Auto-Induction
 
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| [[Team:Imperial_College_London/Wetlab/Protocols/Glucose delay|Glucose Time Delay]]
 
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| * Characterise the tunable time duration it takes before GFP expression (M2 activation)
 
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
 
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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/See Glucose time delay for details]]
 
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| <b>3.Protein Production</b>
 
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| [[Team:Imperial_College_London/Wetlab/Protocols/IPTGgrowth |IPTG Toxicity]]
 
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| To investigate the effect of our IPTG inducer on growth of our cultures.
 
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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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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
 
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| <b>3.Protein Production</b>
 
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| Cellulase
 
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| Aims
 
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
 
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| <b>3.Protein Production</b>
 
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| PAH
 
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| Aims
 
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
 
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| <b>4.Colanic Acid Encapsulation</b>
 
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| Colanic Acid
 
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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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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
 
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| <b>8.Genome Restriction</b>
 
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| [[Team:Imperial_College_London/Wetlab/Protocols/Restriction |In Vitro Restriction]]
 
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| * By running restriction digests on the genome of E.coli strains, we can investigate the efficiency of our restriction enzyme, taqI and dpnII, on genome deletion.
 
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* Run parallel digests with our chassis of choice (TOP10) and a Dam negative control (of which the DNA should not be methylated) to investigate the effects of methylation on cleavage.
 
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|- style="color:#333; background-color:#CCCCFF;" cellpadding="6" cellspacing="0" border="1"
 
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|}
 
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=Promoter characterisation assays=
 
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==Absorbance calibration==
 
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===Aim===
 
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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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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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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/Abs| See the protocol for more details]]
 
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=Autoinduction assays=
 
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<!--
 
==IPTG effect on growth==
==IPTG effect on growth==
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[[Team:Imperial_College_London/Wetlab/Protocols/IPTGgrowth| See the protocol for more details]]
[[Team:Imperial_College_London/Wetlab/Protocols/IPTGgrowth| See the protocol for more details]]
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-->
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==Lac characterisation and IPTG effect on protein production==
==Lac characterisation and IPTG effect on protein production==
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[[Team:Imperial_College_London/Wetlab/Protocols/IPTG-RFP| See the protocol for more details]]
[[Team:Imperial_College_London/Wetlab/Protocols/IPTG-RFP| See the protocol for more details]]
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<!--
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=Encapsulation assays=
=Encapsulation assays=
==Colanic Acid==
==Colanic Acid==

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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