(Difference between revisions)

• 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

About MITOMI technique.

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

Contents 1 Wildtype TetR 2 Mutant TetRs 2.1 V36F 2.2 In vitro characterization 2.2.1 Position Weight Matrix 2.3 V36FW43S 2.4 V36FW43ST141A 2.5 P39K 2.6 Y42F 2.7 Y42F K108E 2.8 P39QY42M 2.9 P39Q Y42M L197S 2.10 P39Q Y42M L52P 2.11 E37A P39K 2.12 EA37PQ39YF42

Wildtype TetR

Row data

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).

WebLogo we obtained for the V36F mutant:

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 strong as the wild-type variant.

600px

V36FW43S

BBa_K613014

V36FW43ST141A

BBa_K613015

P39K

BBa_K613016

Y42F

BBa_K613017

Y42F K108E

BBa_K613018

P39QY42M

BBa_K613019

P39Q Y42M L197S

BBa_K613020

P39Q Y42M L52P

BBa_K613021

E37A P39K

BBa_K61302

EA37PQ39YF42

BBa_K613023