Team:NYMU-Taipei/Project/Removal/Modeling

From 2009.igem.org

(Difference between revisions)
(New page: ===7/14 taq virus protein=== {{:Team:NYMU-Taipei/GEL|NYMU 2009 07 14 taq virus proteins.png|{{:Team:NYMU-Taipei/PCR|60|15|20|'''150'''|300|rp='''-'''|fp='''-'''}}|c= 2% agarose, 90V, 30min...)
 
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===7/14 taq virus protein===
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=== Oscillator Modelling 1 ===
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{{:Team:NYMU-Taipei/GEL|NYMU 2009 07 14 taq virus proteins.png|{{:Team:NYMU-Taipei/PCR|60|15|20|'''150'''|300|rp='''-'''|fp='''-'''}}|c=
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[[Image:NYMU oscillator modelling.png|800px]]
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2% agarose, 90V, 30min
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* <font color="red">'''TetR'''</font> concentration
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{{:Team:NYMU-Taipei/GELC|=
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* <font color="darkblue">'''CII'''</font> concentration.
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|0: marker 1kb+100bp|||n|=
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* 1/3min intervals.
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|1: gp120 full length|2621bp||w|=
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|2: gp120 a-b|2539bp||w|=
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|3: H1N1-HA full length|1742bp||f|=
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|4: H5N1-HA full length|1742bp||f|=
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|5: H1N1-NA full length|1460bp||w|=
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|6: H5N1-NA full length|1391bp||w|=
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|7: H5N1-NA a-b|1283bp||f|=
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|8: Positive Control BBa_E0240|1114bp (876+238)|238bp|w|=
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|9: H5N1-HA b-c|911||w|=
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|10:H1N1-NA c-d|606bp||w|=
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|11:H1N1-NA d-e|495bp||w|=
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|12:Positive Control pLac|437bp||w|=
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|13:H5N1-HA d-e|378bp||f|=
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|14:H1N1-NA b-c|315bp||f|=
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|15:H5N1-HA a-b|285bp||f|=
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|16:marker 100bp|||n}}
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}}
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{{:Team:NYMU-Taipei/GEL|NYMU 2009 07 14 taq virus proteins2.png|{{:Team:NYMU-Taipei/PCR|60|15|20|'''30'''|300|rp='''-'''|fp='''-'''}}|c=
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2% agarose, 90V, 30min
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{{:Team:NYMU-Taipei/GELC|=
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|0:marker 100bp|||n|=
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|1:H5N1-HA c-d|236bp||w|=
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|2:gp120 b-c|114bp||f|=
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|3:H5N1-NA b-c|126bp||f|=
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|4:H1N1-NA a-b|103bp||w|=
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|5:negative control|||n}}
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}}
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===7/20  pfu virus proteins===
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==== Variables Used ====
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{{:Team:NYMU-Taipei/GEL|NYMU 2009 07 20 pfu virus proteins1.png|{{:Team:NYMU-Taipei/PCR|60|15|20|'''120'''|300|rp='''-'''|fp='''-'''|polName=''pfu''|pol=0.5|ddH20=39.5|}}|c=
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{| border="1"
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2% agarose, 90V, 30min
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!    !!  Protein Name  !!  Half-life (min)  !!  Promoter  !!  Promoter Strength (relative)
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{{:Team:NYMU-Taipei/GELC|=
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|-
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|0: marker 1kb|||n|=
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! Inducer || CII  || 8 [1]  || pRE  || 0.025 (est)
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|1-3: gp120 b-c|114bp||w|=
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|-
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|4-6: H5N1-HA c-d|236bp||w|=
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! Repressor || TetR  || 40 (est due to LVA tag) || pTet  || 1
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|7:marker 1kb|||n}}
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|-
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}}
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! Reporter || GFP  || 2160 [2]  ||   ||  
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{{:Team:NYMU-Taipei/GEL|NYMU 2009 07 20 pfu virus proteins2.png|{{:Team:NYMU-Taipei/PCR|60|15|20|'''120'''|300|rp='''-'''|fp='''-'''|polName=''pfu''|pol=0.5|ddH20=39.5|}}|c=
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|}
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2% agarose, 90V, 30min
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Time segment used for the calculation: 1/3mins
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{{:Team:NYMU-Taipei/GELC|=
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Maximum amount of Repressors that can stop promoter activity: 10 (an arbitrary number)
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|0: marker 1kb|||n|=
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|1-2: H5N1_HA a-b|285bp||w|=
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|3-4: H5N1-HA d-e|378bp||w|=
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|5: marker 1kb|||n|=
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|6: Positive Control BBa_E0240|1114bp (876+238)|238bp|w|=
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|7: negative control|||n}}
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}}
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{{:Team:NYMU-Taipei/GEL|NYMU 2009 07 20 pfu virus proteins3.png|{{:Team:NYMU-Taipei/PCR|60|15|20|'''150'''|300|rp='''-'''|fp='''-'''|polName=''pfu''|pol=0.5|ddH20=39.5|}}|c=
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2% agarose, 90V, 30min
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{{:Team:NYMU-Taipei/GELC|=
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|0: marker 1kb|||n|=
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|1-2: H5N1-HA b-c|911bp||w|=
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|3-4: gp120 a-b|2539bp||f|=
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|5: marker 1kb|||n}}
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}}
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===7/20 taq virus proteins===
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==== Formulas used ====
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{{:Team:NYMU-Taipei/GEL|NYMU 2009 07 20 taq virus proteins.png|{{:Team:NYMU-Taipei/PCR|60|15|20|'''120'''|300|rp='''-'''|fp='''-'''}}|c=
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<html><style>span.bigger{font-size:141%;}</style></html>
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2% agarose, 90V, 30min
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Calculating the strength of the pTet promoter when there are repressors around:
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{{:Team:NYMU-Taipei/GELC|=
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|0: marker 1kb|||n|=
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|1: marker 100bp|||n|=
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|2: H1N1-HA full length|1742bp||f|=
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|3: H1N1-NA full length|1460bp||w|=
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|4: H5N1-NA full length|1391bp||f|=
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|5: H5N1-NA a-b|1283bp||f|=
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|6: H1N1-NA c-d|606bp||w|=
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|7: H1N1-NA d-e|495bp||w|=
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|8: H1N1-NA b-c|315bp||w|=
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|9: H1N1-NA d-e|495bp||f|=
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|10: H1N1-NA a-b|103bp||w|=
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|11: Positive Control BBa_E0240|1114bp (876+238)|238bp|w|=
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|12: negative control|||n|=
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|13: marker 100bp|||n|=
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|14: marker 1kb|||n}}
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}}
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===7/21 taq H1N1-HA H5N1-NA gp120b-c===
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<span class="bigger">pTet<sub>current</sub> = pTet<sub>max</sub>*1-1/Tet<sub>max</sub>*[TetR]</span>    ----- (1)
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{{:Team:NYMU-Taipei/GEL|NYMU 2009 07 21 H1N1-HA H5N1-NA gp120b-c.png|{{:Team:NYMU-Taipei/PCR|60|15|20|'''120'''|300|rp='''-'''|fp='''-'''}}|c=
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2% agarose, 90V, 30min
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Calculate the amount of each protein produced:
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{{:Team:NYMU-Taipei/GELC|=
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<span class="bigger">CII = pTet<sub>current</sub></span>    ----- (2)
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|0: marker 1kb|||n|=
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|1:-|||n|=
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<span class="bigger">TetR = [CII] * pRE<sub>max</sub></span>    ----- (3)
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|2: H5N1-NA a-b|1283bp||w|=
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|3: H5N1-NA full length|1391bp||w|=
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<span class="bigger">GFP = [CII] pTet<sub>current</sub></span>    ----- (4)
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|4: H1N1-HA full length|1742bp||w|=
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|5: Positive Control BBa_E0240|1114bp (876+238)|238bp|w|=
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|6: -|||n|=
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|7: marker 100bp|||n}}
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{{:Team:NYMU-Taipei/GELC|=
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|0: marker 1kb|||n|=
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|1: -|||n|=
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|2: -|||n|=
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|3: H5N1-NA b-c|126bp||w|=
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|4: gp120 b-c|114bp||w|=
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|5: negative control|||n|=
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|6: -|||n|=
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|7: marker 100bp|||n}}
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}}
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===7/22 pfu virus protein===
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{{:Team:NYMU-Taipei/GEL|NYMU 2009 07 22 pfu virus proteins1.png|{{:Team:NYMU-Taipei/PCR|60|15|20|'''120'''|300|rp='''-'''|fp='''-'''|polName=''pfu''|pol=0.5|ddH20=39.5|}}|c=
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2% agarose, 90V, 30min
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{{:Team:NYMU-Taipei/GELC|=
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|0: marker 1kb|||n|=
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|1-2: H1N1_NA a-b|103bp||w|=
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|3-4: gp120 b-c|114bp||w|=
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|5-6: H1N1-NA b-c|315bp||w|=
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|5-6: marker 100bp|||n}}
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}}
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{{:Team:NYMU-Taipei/GEL|NYMU 2009 07 22 pfu virus proteins2.png|{{:Team:NYMU-Taipei/PCR|60|15|20|'''120'''|300|rp='''-'''|fp='''-'''|polName=''pfu''|pol=0.5|ddH20=39.5|}}|c=
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2% agarose, 90V, 30min
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{{:Team:NYMU-Taipei/GELC|=
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|0: marker 1kb|||n|=
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|1-2:H1N1-NA d-e|495bp||w|=
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|3-4:H5N1-HA c-e|591bp||w|=
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|5-6:H1N1-NA c-d|606bp||w|=
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|7: marker 100bp|||n}}
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{{:Team:NYMU-Taipei/GELC|=
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|0: marker 1kb|||n|=
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|1-2:H5N1-NA a-c|1175bp||w|=
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|3: Positive Control BBa_E0240|1114bp (876+238)|238bp|w|=
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|4: negative control|||n|=
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|5: marker 100bp|||n}}
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}}
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===7/23 pfu virus protein===
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{{:Team:NYMU-Taipei/GEL|NYMU 2009 07 23 pfu virus proteins.png|{{:Team:NYMU-Taipei/PCR|60|15|20|'''120'''|300|rp='''-'''|fp='''-'''|polName=''pfu''|pol=0.5|ddH20=39.5|}}|c=
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The calculation of amount of substrates remaining w.r.t the half lives:
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2% agarose, 90V, 30min
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<span class="bigger">[CII] = [CII] x 0.5<sup>(&Delta;t / CII-t<sub>1/2</sub>)</sup></span>    ----- (5)
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{{:Team:NYMU-Taipei/GELC|=
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|0: marker 1kb|||n|=
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<span class="bigger">[TetR] = [TetR] x 0.5<sup>(&Delta;t / TetR-t<sub>1/2</sub>)</sup></span>    ----- (6)
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|1-2:H1N1-HA |1742bp||w|=
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|3-4:H5N1-HA |1742bp||w|=
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<span class="bigger">[GFP] = [GFP] x 0.5<sup>(&Delta;t / GFP-t<sub>1/2</sub>)</sup></span>    ----- (7)
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|5-6:H5N1-NA a-b|1283bp||w|=
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|7: Positive Control BBa_E0240|1114bp (876+238)|238bp|w}}
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==== Conclusion ====
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{{:Team:NYMU-Taipei/GELC|=
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|0: marker 1kb|||n|=
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The only variables are:
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|1-2:H1N1-NA c-e|1081bp||w|=
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* The relative strength of pRE relative to pTet (since it has not been measured yet)
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|3-4:H1N1_NA a-c|398bp||w|=
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** the weaker pRE is relative to pTet, the slower the oscillator dampens.
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|5-6:H5N1_NA b-c|126bp||w|=
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* Maximum amount of repressor such that adding more repressors will not affect the strength of pTet anymore.
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|7: negative control|||n}}
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** Amplitude of TetR oscillation is directly proportional.
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}}
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 +
==== References ====
 +
[1] Shotland, ''et al'': [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=94496 Proteolysis of Bacteriophage λ CII by Escherichia coli FtsH (HflB)]. J Bacteriol. 2000, 182(11): 3111–3116

Latest revision as of 21:03, 21 October 2009

Contents

Oscillator Modelling 1

NYMU oscillator modelling.png

  • TetR concentration
  • CII concentration.
  • 1/3min intervals.

Variables Used

Protein Name Half-life (min) Promoter Promoter Strength (relative)
Inducer CII 8 [1] pRE 0.025 (est)
Repressor TetR 40 (est due to LVA tag) pTet 1
Reporter GFP 2160 [2]

Time segment used for the calculation: 1/3mins Maximum amount of Repressors that can stop promoter activity: 10 (an arbitrary number)

Formulas used

Calculating the strength of the pTet promoter when there are repressors around: 

pTetcurrent = pTetmax*1-1/Tetmax*[TetR]     ----- (1)

Calculate the amount of each protein produced: 

CII = pTetcurrent     ----- (2)

TetR = [CII] * pREmax     ----- (3)

GFP = [CII] pTetcurrent     ----- (4)


The calculation of amount of substrates remaining w.r.t the half lives: 

[CII] = [CII] x 0.5(Δt / CII-t1/2)     ----- (5)

[TetR] = [TetR] x 0.5(Δt / TetR-t1/2)     ----- (6)

[GFP] = [GFP] x 0.5(Δt / GFP-t1/2)     ----- (7)

Conclusion

The only variables are:

  • The relative strength of pRE relative to pTet (since it has not been measured yet)
    • the weaker pRE is relative to pTet, the slower the oscillator dampens.
  • Maximum amount of repressor such that adding more repressors will not affect the strength of pTet anymore.
    • Amplitude of TetR oscillation is directly proportional.

References

[1] Shotland, et al: [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=94496 Proteolysis of Bacteriophage λ CII by Escherichia coli FtsH (HflB)]. J Bacteriol. 2000, 182(11): 3111–3116

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