Difference between revisions of "Part:BBa K613017"

Latest revision as of 03:51, 22 September 2011

TetR Y42F mutant

This is a TetR mutant that carries 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:

The measured binding affinities show no symmetry conservation, these experiment will have to be repeated in order to get a clearer idea of the binding affinities.

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

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.

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]

Designed sequence (1 mutation in yellow):

MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLFWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSH
RDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPA

FLFGLELIICGLEKQLKCESGS*

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K613017 short</partinfo>
-This is a TetR mutant that carry the Y42F mutation.
+This is a TetR mutant that carries the Y42F mutation.
 Part of the <html> <a href="http://2011.igem.org/Team:EPF-Lausanne/Our_Project/TetR_mutants/muTetRs">EPFL2011 muTetR collection. </a> </html>
@@ Line 12: / Line 12: @@
 [[Image:EPFL_WebLogo_Y42F.png|700px]]
+The measured binding affinities show no symmetry conservation, these experiment will have to be repeated in order to get a clearer idea of the binding affinities.
@@ Line 61: / Line 64: @@
 |-
 | 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.
 <!-- Add more about the biology of this part here
@@ Line 71: / Line 75: @@
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K613017 SequenceAndFeatures</partinfo>
+Designed sequence (1 mutation in yellow):
+'''MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTL'''<span style='background:yellow'>'''F'''</span>'''WHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSH<p>RDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPA</p><p>FLFGLELIICGLEKQLKCESGS*'''</p>