Difference between revisions of "Part:BBa K613017"
(→‎In vitro characterization)
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===In vitro characterization===
 
===In vitro characterization===
  
−
Using the <html> <a href="http://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).
+
Using the <html> <a href="http://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:
 
WebLogo we obtained for the Y42F mutant:
Line 18: Line 18:
 
enoLOGOS: a versatile web tool for energy normalized sequence logos.
 
enoLOGOS: a versatile web tool for energy normalized sequence logos.
 
Nucleic Acids Res. 2005 Jul 1;33:W389-92.</p>
 
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
 +
|-
 +
|
 +
|}
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 21:44, 21 September 2011

TetR Y42F mutant

This is a TetR mutant that carry the Y42F mutation. Part of the EPFL2011 muTetR collection.

In vitro characterization

Using the MITOMI 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:

EPFL WebLogo Y42F.png


Reference:

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.


Position Weight Matrix

PO A T C 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

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]