Team:EPF-Lausanne/Our Project/TetR mutants/MITOMI data

From 2011.igem.org

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<html> <a href= >Row data</a></html>
<html> <a href= >Row data</a></html>
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===In vitro characterization===
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Using the <html> <a href="https://2011.igem.org/Team:EPF-Lausanne/Our_Project/TetR_mutants/MITOMI_data">MITOMI</a></html> technique we determined the DNA binding landscape of the TetR V36F mutant. To do so, first we designed and generated the library of double stranded DNA sequences that cover all possible single base substitution within the tetO operator sequence. Based on that library we measured the dissociation constants of the mutant to variable tetO-like sequences and determined the specificity of the mutant to the tet operator sequence (expressed as a PWM).
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WebLogo we obtained:
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[[File:EPFL2011_WTTetR_WebLogo.png|700px]]
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'''Reference:'''<p>
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Workman CT, Yin Y, Corcoran DL, Ideker T, Stormo GD, Benos PV.
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enoLOGOS: a versatile web tool for energy normalized sequence logos.
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Nucleic Acids Res. 2005 Jul 1;33:W389-92.</p>
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===Position Weight Matrix===
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''PO'''
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| align="center" style="background:#f0f0f0;"|'''A'''
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| align="center" style="background:#f0f0f0;"|'''T'''
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| align="center" style="background:#f0f0f0;"|'''C'''
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| align="center" style="background:#f0f0f0;"|'''G'''
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|-
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| 1||0.164072||0.519334||-0.0468541||0.22966
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|-
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| 2||0.642569||0.164072||0.188965||0.943314
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|-
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| 3||1.04481||0.558693||0.164072||0.848264
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|-
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| 4||0.959646||0.164072||2.17536||0.718959
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|-
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| 5||0.164072||1.19894||1.79469||1.56685
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|-
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| 6||1.46463||0.164072||1.74384||1.54845
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|-
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| 7||0.966397||1.07557||0.164072||1.72883
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|-
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| 8||0.164072||0.504312||0.755195||0.148902
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|-
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| 9||0.0876645||0.164072||0||0.0156484
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|-
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| 10||0.289722||0.164072||0.00707563||0.843474
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|-
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| 11||1.77568||1.3447||2.34804||0.164072
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|-
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| 12||0.164072||1.72354||1.62782||1.49176
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|-
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| 13||0.877518||0.164072||1.30879||1.9052
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|-
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| 14||0.164072||0.544642||0.3387||1.7537
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|-
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| 15||0.540091||0.8821||1.0861||0.164072
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|-
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| 16||-0.224147||0.280358||0.442769||0.164072
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|-
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| 17||0.31238||-0.0651059||0.164072||-0.303844
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|}
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Each row represents the changes in binding energy,  ΔΔG (kcal/mol), compared to the reference sequence upon the substitution to the indicated nucleotide at certain position within the target DNA element. Values are indicated in kcal/mol.
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We compared the measured DNA binding affinities of the V36F mutant to the affinities obtained for the  wt-TetR and found that V36F mutant interacts with the tetO as strong as the wild-type variant.
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[[File:EPFL2011_Scatter_plot_kd_wt_VF36.png|600px]]
=Mutant TetRs=
=Mutant TetRs=

Revision as of 02:18, 22 September 2011

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