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

From 2011.igem.org

(Difference between revisions)
(→Mutant TetRs)
(→Mutant TetRs)
Line 215: Line 215:
<html> <a href=http://partsregistry.org/Part:BBa_K613017>BBa_K613017</a></html>
<html> <a href=http://partsregistry.org/Part:BBa_K613017>BBa_K613017</a></html>
 +
 +
===In vitro characterization===
 +
 +
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 Y42F 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). For the Y42F mutant we observed the decrease of the specificity compared to the wild-type tetR sequence.
 +
 +
WebLogo we obtained for the Y42F mutant:
 +
 +
[[File:EPFL2011_WebLogo_Y42F.png|700px]]
 +
 +
 +
'''Reference:'''<p>
 +
Workman CT, Yin Y, Corcoran DL, Ideker T, Stormo GD, Benos PV.
 +
enoLOGOS: a versatile web tool for energy normalized sequence logos.
 +
Nucleic Acids Res. 2005 Jul 1;33:W389-92.</p>
 +
 +
 +
====Position Weight Matrix====
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''PO'''
 +
| align="center" style="background:#f0f0f0;"|'''A'''
 +
| align="center" style="background:#f0f0f0;"|'''T'''
 +
| align="center" style="background:#f0f0f0;"|'''C'''
 +
| align="center" style="background:#f0f0f0;"|'''G'''
 +
|-
 +
| 1||0.385241||0.85619||0||0.693793
 +
|-
 +
| 2||1.30965||0.385241||0.629213||1.2542
 +
|-
 +
| 3||1.18851||1.22909||0.385241||1.09825
 +
|-
 +
| 4||1.1084||0.385241||1.57045||1.20206
 +
|-
 +
| 5||0.385241||1.3955||1.10171||1.2069
 +
|-
 +
| 6||1.80251||0.385241||1.37974||1.23728
 +
|-
 +
| 7||1.25285||1.54821||0.385241||1.43937
 +
|-
 +
| 8||0.385241||0.475161||0.93954||0.488479
 +
|-
 +
| 9||0.341937||0.385241||0||0.053757
 +
|-
 +
| 10||0.973293||0.385241||0.426141||1.06563
 +
|-
 +
| 11||1.1587||1.50073||1.5542||0.385241
 +
|-
 +
| 12||0.385241||1.30762||1.11351||1.39762
 +
|-
 +
| 13||1.23638||0.385241||1.11282||1.24361
 +
|-
 +
| 14||0.385241||1.09074||0.75983||0.933921
 +
|-
 +
| 15||0.679315||1.33124||1.09937||0.385241
 +
|-
 +
| 16||0||0.467396||1.08277||0.385241
 +
|-
 +
| 17||0.664957||0.130021||0.385241||0
 +
 +
|}
 +
 +
Each row represents the changes in binding energy,  ΔΔG, 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.
 +
==Y42F K108E==
==Y42F K108E==

Revision as of 02:10, 22 September 2011

Retrieved from "http://2011.igem.org/Team:EPF-Lausanne/Our_Project/TetR_mutants/MITOMI_data"