Part:BBa_K613015

TetR E37A W43S T141A mutant

This is a TetR with the triple mutation E37A W43S T141A. Part of the EPFL2011 muTetR collection.

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.

WebLogo we obtained for the E37A W43S T141A 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	0.298921	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.

In vivo characterization

This TetR mutant was characterized in vivo by putting it into pSB3K1 under a constitutive promoter (J23116). This plasmid was cotransformed with J61002 harbouring RFP under pTet promoter (B0040) in DH5alpha cells. Cells were grown in a medium containing Kanamycin & Amplicillin plus different concentrations of ATC, ranging from 0 to 2000 ng/mL. OD600 absorbence and RFP fluorescence were measured every 10 minutes during 12 hours on a platereader machine.

Induction curves

Fluorescence measurements (RFUs) were normalized by OD600 values.

In the absence of ATC, RFP expression in presence of mutant goes up to 2000 normalized RFUs, which is the same level of expression as for the wild-type TetR. This shows that the mutant is able to bind and inactivate pTet with the same strength as the wild-type. With 2000 ng/mL of ATC in the cell culture, RFP expression rises up to 16000 normalized RFUs. Even if ATC does inhibit TetR function, this inhibiton is less striking compared to the wild-type at the same ATC concentration. The E37A W43S T141A mutant may have an altered ATC binding property.

Dose-response curve

Fluorescence measurements (RFUs) were normalized by OD600 values. For each ATC concentration, we estimated the steady-state fluorescence expression by averaging the measurements over the last hour.

ATC action on the TetR mutant is clearly visible on this graph. From 200 ng/mL, RFP expression is reaching a plateau, indicating that the maximal TetR inhibition has been attained. The mutant is less repressed in presence of ATC than the wild-type protein.

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 (3 mutations in yellow):

MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVAQPTLYSHVKNKRALLDALAIEMLDRHHTHFCPLEGESW

QDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFALGCVLEDQ

EHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS*