• Introduction
  • Home
  • The Team
  • Photo Gallery
• Our Project
  • Results Summary
  • Selection System
  • In-Vitro Characterization
  • In-Vivo Characterization
  • Microfluidics
  • Data
  • Attributions
• Tools
  • Gibson Assembly
  • MITOMI
• Notebook
  • Protocols
  • May
  • June
  • July
  • August
  • September
  • October
• Considerations
  • Human Practices
  • Safety
• Acknowledgements
  • Sponsors
  • Partners

MITOMI Data

In vitro Main | Why TetR? | Mutant TetRs | MITOMI Data | In-vivo & In-vitro outline

To find out more about the MITOMI method of characterization, please click here.

The raw data from the successful experiments can be found here.

Contents 1 Wildtype TetR 1.1 In Vitro Characterization 1.2 Position Weight Matrix 2 Mutant TetRs 2.1 V36F 2.1.1 In Vitro Characterization 2.1.2 Position Weight Matrix 2.2 E37A W43S T141A 2.2.1 In Vitro Characterization 2.2.2 Position Weight Matrix 2.3 P39K 2.3.1 In Vitro Characterization 2.3.2 Position Weight Matrix 2.4 Y42F 2.4.1 In Vitro Characterization 2.4.2 Position Weight Matrix 2.5 P39Q Y42M 2.5.1 In Vitro Characterization 2.5.2 Position Weight Matrix 3 Uncharacterized Mutant TetRs 3.1 Y42F K108E 3.2 V36F W43S 3.3 P39Q Y42M L197S 3.4 P39Q Y42M L52P 3.5 E37A P39K 3.6 E37A P39Q Y42F

Wildtype TetR

In Vitro Characterization

Using the MITOMI technique we determined the DNA binding landscape of the wild type TetR. To do so, we first designed and generated the library of double-stranded DNA sequences that cover all possible single base substitutions within the TetO operator sequence. Based on that library we measured the dissociation constants of the mutant to all the tetO-like sequences of the library. Then, we determined the specificity of the mutant to the Tet operator sequence, expressed as a position-weight matrix (PWM).

The enoLOGO we obtained for the wild-type is:

enoLOGO 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	0.164072	0.519334	-0.0468541	0.22966
2	0.642569	0.164072	0.188965	0.943314
3	1.04481	0.558693	0.164072	0.848264
4	0.959646	0.164072	2.17536	0.718959
5	0.164072	1.19894	1.79469	1.56685
6	1.46463	0.164072	1.74384	1.54845
7	0.966397	1.07557	0.164072	1.72883
8	0.164072	0.504312	0.755195	0.148902
9	0.0876645	0.164072	0	0.0156484
10	0.289722	0.164072	0.00707563	0.843474
11	1.77568	1.3447	2.34804	0.164072
12	0.164072	1.72354	1.62782	1.49176
13	0.877518	0.164072	1.30879	1.9052
14	0.164072	0.544642	0.3387	1.7537
15	0.540091	0.8821	1.0861	0.164072
16	-0.224147	0.280358	0.442769	0.164072
17	0.31238	-0.0651059	0.164072	-0.303844

Each row represents the changes in binding energy, ΔΔG (kcal/mol), compared to the reference sequence upon the substitution of the indicated nucleotide at a certain position within the target DNA element. Values are indicated in kcal/mol.

Mutant TetRs

V36F

BBa_K613013

In Vitro Characterization

Using the MITOMI 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).

The WebLogo we obtained for the V36F mutant:

WebLogo 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	0.164072	0.519334	0	0.22966
2	0.642569	0.164072	0.188965	0.943314
3	1.04481	0.558693	0.164072	0.848264
4	0.959646	0.164072	2.17536	0.718959
5	0.164072	1.19894	1.79469	1.56685
6	1.46463	0.164072	1.74384	1.54845
7	0.966397	1.07557	0.164072	1.72883
8	0.164072	0.504312	0.755195	0.148902
9	0.0876645	0.164072	0	0.0156484
10	0.289722	0.164072	0.00707563	0.843474
11	1.77568	1.3447	2.34804	0.164072
12	0.164072	1.72354	1.62782	1.49176
13	0.877518	0.164072	1.30879	1.9052
14	0.164072	0.544642	0.3387	1.7537
15	0.540091	0.8821	1.0861	0.164072
16	0	0.280358	0.442769	0.164072
17	0.31238	0	0.164072	0

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.

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 strongly as the wild-type variant.

E37A W43S T141A

BBa_K613015

In Vitro Characterization

Using the MITOMI technique we determined the DNA binding landscape of the TetR E37A W43S T141A 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).

The measured DNA binding affinities of the E37A W43S T141A mutant show that these mutations alter the sequence recognition, while the symmetry and the spacer affinities are conserved.

The WebLogo we obtained for the E37A W43S T141A mutant:

WebLogo 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	0.298921	0.228558	0.136478	0.0565634
2	0.217725	0.298921	0.0365532	0.101569
3	0.486164	0.884721	0.298921	0.689301
4	0.381337	0.298921	1.2275	0.443129
5	0.298921	0.829256	1.48516	0.861923
6	1.52008	0.298921	1.20145	1.04388
7	1.08347	1.57658	0.298921	2.1751
8	0.298921	0.506289	0.723699	0.34977
9	0.0627581	0.298921	0	0.0173955
10	0.49991	0.298921	0.250601	0.869584
11	1.58199	1.5844	2.35356	0.298921
12	0.298921	1.88623	1.34104	1.46941
13	1.00088	0.298921	1.01931	1.57583
14	0.298921	0.56873	0.306484	1.91995
15	0.65822	1.06676	1.10609	0.298921
16	0.290169	0.067613	0.660542	0.298921
17	0.557743	0.160129	0.298921	0.134017

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.

P39K

BBa_K613016

In Vitro Characterization

Using the MITOMI technique we determined the DNA binding landscape of the TetR P39K 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).

The WebLogo we obtained for the P39K mutant:

The strong difference of the binding affinities between the P39K mutant and the wtTetR, might be due to the altered recognition of the P39K. This mutant was shown to have a new recognition specificity for the tetO-4C operator in Helbl et al, 1998.

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.

Helbl, V., and Hillen, W. (1998). Stepwise selection of TetR variants recognizing tet operator 4C with high affinity and specificity. J Mol Biol 276, 313-318.

Position Weight Matrix

PO	A	T	C	G
1	0.259286	0.242355	0.296487	0
2	0.13749	0.259286	0	0.444867
3	0.149356	0.0391642	0.259286	0
4	0.000432676	0.259286	0.250803	0.09369
5	0.259286	0	0.183728	0.411598
6	0.104299	0.259286	0	0.157345
7	0.287302	0.0195324	0.259286	0.0244544
8	0.259286	0.286228	0.256377	0.0991247
9	0	0.259286	0.317281	0.536557
10	0	0.259286	0	0.31848
11	0.582859	0	0	0.259286
12	0.259286	0.184012	0.330234	0
13	0.0462643	0.259286	0.242912	0.357439
14	0.259286	0.011556	0.0598842	0.120511
15	0.228422	0.0270438	0.00125937	0.259286
16	0.102476	0.214565	0.0872813	0.259286
17	0.127319	0.112242	0.259286	0.299159

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

BBa_K613017

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.

The WebLogo we obtained for the Y42F mutant:

The measured binding affinities show no symmetry conservation and the contribution of the outer bases equals that of the inner. These experiment will have to be repeated in order to get a clearer idea of the binding affinities.

WebLogo 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 at the indicated nucleotide at a certain position within the target DNA element. Values are indicated in kcal/mol.

P39Q Y42M

BBa_K613019

In Vitro Characterization

Using the MITOMI technique we determined the DNA binding landscape of the TetR P39Q Y42M 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 P39Q Y42M mutant we observed the decrease of the specificity compared to the wild-type tetR sequence.

The WebLogo we obtained for the P39Q Y42M mutant:

WebLogo 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	0.455977	0.0513378	0.0469535	0.0942038
2	0	0.455977	0.0808971	0.0550434
3	0.024486	0	0.455977	0
4	0	0.455977	0	0.175581
5	0.455977	0	0.0549319	0
6	0.162363	0.455977	0.0897636	0.0689181
7	0	0.02975	0.455977	0.0170034
8	0.455977	0	0	0.0975597
9	0.134079	0.455977	0	0
10	0.486152	0.455977	0.0220095	0
11	0	0.0246322	0.0847586	0.455977
12	0.455977	0	0	0.0321177
13	0.20916	0.455977	0	0.0482279
14	0.455977	0	0.0837475	0.141681
15	0	0.107215	0	0.455977
16	0	0	0.269667	0.455977
17	0.0634094	0	0.455977	0.0582705

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.

Uncharacterized Mutant TetRs

Unfortunately we did not have time to finish the characterization of the following parts:

Y42F K108E

BBa_K613018

V36F W43S

BBa_K613014

P39Q Y42M L197S

BBa_K613020

P39Q Y42M L52P

BBa_K613021

E37A P39K

BBa_K61302

E37A P39Q Y42F

BBa_K613023