Supporting Information
Analysis of Amino Acid Substitutions in AraC Variants that Respond to Triacetic Acid Lactone
Christopher S. Frei1, Zhiqing Wang1, Shuai Qian1, Samuel Deutsch2, Markus Sutter2, Patrick C.
Cirino1
1. Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX
77204, USA
2. Joint Genome Institute, 2800 Mitchell Drive Walnut Creek, CA 94598
Materials and Methods
General
Restriction enzymes, Phusion High-Fidelity DNA polymerase (Cat. No. M0530L), Gibson
Assembly Master Mix (Cat. No. E2611) and T4 DNA ligase (Cat. No. M0202L) were purchased
from New England Biolabs (Ipswich, MA). Oligonucleotides were synthesized by Integrated
DNA Technologies (Coralville, IA) and are reported in Table SI. Sanger DNA sequencing was
performed by SeqWright (Houston, TX). Molecular biology techniques for DNA manipulation
were performed according to standard protocols 1. The general cloning protocol for the
construction of vectors using the Gibson Assembly method is outlined in Figure S1.
Table S1 List of primers used in this study. The underlined sequence highlights the terminator
sequence incorporated into pFG29.
Primer name
pFG29-gib-for
pFG29-gib-rev
pET45-araC-LBD-for
Primer sequence (5'-3')
CGCCATTCAGGCTGCAACGACGGCCAGT
GAGCG
CCTTCCCAACAGTTGCAAAAAAAAAAGC
CCGCACTGTCAGGTGCGGGCTTTTTTCT
GTGTTTGCGCAATTAACCCTCACTAAAG
G
ATGACGACGACAAGAGTCCCATGGCTGA
AGCGCAAAATGAT
Plasmid/clone
pFG29
pFG29
pPCC1212
pET45-araC-LBD-rev
AGCTCCCAATTGGGATACCCTCACGACT
CGTTAATCGCTTCCATG
pPCC1212
pFG29_araC_GS_fwd_1
ATAAGAGACACCGGCATACT
All MutXXXXX mutants, recloned araC-TAL mutants
pCC1305_araCTAL_rvs
ATGCGTTGGTCCTCGCGC
All MutXXXXX mutants, recloned araC-TAL mutants
00001_1_WT_rvs
00001_2_TAL_fwd
10000_1_WT_rvs
10000_2_WT_fwd
00010_1_WT_rvs
00010_2_WT_fwd
00001
00001
10000
10000
00010
00010
00111_1_WT_rvs
ATACCATTCGCGAGCCTC
GAGGCTCGCGAATGGTAT
TACTCGTTTAACGCCCAT
GCCACCAGATGGGCGTTA
CAAGTGATGAATCTCTCCTGGC
GCCAGGAGAGATTCATCACTTGGGTCGT
CATCC
GCCAATCTCTCCTGGCGGGAACAGCA
TGCTGTTCCCGCCAGGAGAGATTGGCCA
CTTGG
ATCTCTCCTGGCGGGAACAG
00111_2_TAL_fwd
01000_1_WT_rvs
01000_2_TAL_fwd
01000_3_WT_fwd
01000_2_TAL_rvs
01011_1_WT_rvs
CTGTTCCCGCCAGGAGAGAT
TTAAACCCGCCACCAGATG
CCATCTGGTGGCGGGTTTAA
GCTGTTCCCGCCAGGAGAGATT
AATCTCTCCTGGCGGGAACAGC
CCACCAGATGGGCGTTAAAC
00111
01000
01000
01000
01000
01011, 01100, 01101, 10001,
01110, 10001, 10010, 10011,
10100, 10101, 01001, 01010,
10110
01011_2_TAL_fwd
GTTTAACGCCCATCTGGTGG
01011, 01100, 01101, 10001,
01110, 10001, 10010, 10011,
10100, 10101, 01001, 01010,
10110
01011_3_TAL_fwd
CTGTTCCCGCCAGGAGAGATTCATCACT
TGGG
CTGTTCCCGCCAGGAGAGATTGGCCACT
ACGGT
GCCAATCTCTCCTGGCGGGAACAG
GCCAGGAGAGATTGGCCACTACGGTCGT
CAT
GTAGTGGCCAATCTCTCCTGGC
CGGAGGCTCGCGAATGGTAT
ATACCATTCGCGAGCCTCCG
TAAACCCGCCACCAGATGGGC
GCCCATCTGGTGGCGGGTTTA
CGGAGGCTCGCGAATGGTAT
01011
00100_1_WT_rvs
00100_2_WT_fwd
01100_3_WT_fwd
01100_2_TAL_rvs
01101_3_TAL_fwd
01101_2_TAL_rvs
10001_3_TAL_fwd
10001_2_WT_rvs
01111_1_WT_rvs
01111_2_TAL_fwd
10001_3_TAL_fwd
00100, 11100
00100, 11100
00111, 01011, 10100, 01001,
01010, 10110, 11001, 11010
01100
01100
01101
01101
10001, 01110
10001, 01110
01111
01111
10001
10001_2_WT_rvs
10010_3_WT_fwd
10010_2_WT_rvs
10011_3_TAL_fwd
10011_2_WT_rvs
10100_3_WT_fwd
10101_3_TAL_fwd
10101_2_WT_rvs
10111_1_TAL_rvs
10111_2_WT_fwd
10111_3_TAL_fwd
10111_2_WT_rvs
11011_1_TAL_rvs
11011_2_TAL_fwd
11101_1_TAL_rvs
11101_2_TAL_fwd
11110_1_TAL_rvs
11110_2_WT_fwd
00101_1_WT_rvs
00101_2_TAL_fwd
00110_1_WT_rvs
00110_2_WT_fwd
01001_3_TAL_fwd
01010_3_WT_fwd
11010_2_WT_fwd
Adding_ScaI_Fragment_
1_fwd
araCTAL_5_AvrII_rvs
AraCTAL_5
pCC1305_araCTAL_rvs
pCC1321_PciI_PBAD
pCC1321_AgeI_PBAD
ATACCATTCGCGAGCCTCCG
CCGCCAGGAGAGATTCATCACTTGGGTC
GTCA
TGATGAATCTCTCCTGGCGG
GCCAGGAGAGATTCATCACTTGGGTCGT
CA
AGTGATGAATCTCTCCTGGC
CTGTTCCCGCCAGGAGAGATTGGCCACT
ACGGT
TCCCGCCAGGAGAGATTGGCCACTACGG
TCGTCAT
GCCAATCTCTCCTGGCGGGAACA
TTAAACCCGCCACCAGATG
CCATCTGGTGGCGGGTTTAA
GCTGTTCCCGCCAGGAGAGATT
AATCTCTCCTGGCGGGAACAGC
ATGAATCTCTCCTGGCGGGAACAGCA
TGCTGTTCCCGCCAGGAGAGATTCATCA
CTTGGGTC
GTAGTGGCCAATCTCTCCTGGC
GCCAGGAGAGATTGGCCACTACGGTCGT
CATCC
ATACCATTCGCGAGCCTC
GAGGCTCGCGAATGGTAT
GCCAATCTCTCCTGGCGGGAAC
CCCGCCAGGAGAGATTGGCCACTACGGT
CGTCA
CAAGTGGCCAATCTCTCCTGGCGGGAAC
A
TGTTCCCGCCAGGAGAGATTGGCCACTT
GGGTCGTCA
CTGTTCCCGCCAGGAGAGATTCATCACT
ACGGTCGTCA
CTGTTCCCGCCAGGAGAGATTCATCACT
TGGGTCGTCA
CTGTTCCCGCCAGGAGAGATTCATCACT
TGGGTCGTCA
CCCCAGCAGGCGAAAATCCTGTTTG
10001
10010
ATTTTGCGCTTCAGCCATCCTAGGTATC
TCCTGTG
ATGGCTGAAGCGCAAAATGATCC
ATGCGTTGGTCCTCGCGC
TTTTGCTGGCCTTTTGCTCAACTTTTCA
TACTCCCGCC
GCTTTTAATAAGCGGGGTTA
pPCC1321
10010
10011, 00011
10011, 00011
10100
10101
10101
10111
10111
10111
10111
11011
11011
11101
11101
11110
11110
00101
00101
00110
00110, 10110
01001, 11001
01010
11010
pPCC1321
pPCC1321
pPCC1321
pPCC1322
pPCC1322
Figure S1 General protocol for Gibson Assembly
Integration of a PBAD-bla reporter construct into HF19 for ampicillin selection. Strain SQ12
was created by integrating a fragment of DNA containing PBAD-bla (conferring ampicillin
resistance regulated by the AraC cognate promoter PBAD) into the genome of strain HF19 using
the “CRIM” method 2. The PBAD-gfpuv was amplified from plasmid pFG29 using primers
pPCC1215-gib-for and pPCC1215-gib-rev, and then inserted into NcoI-digested CRIM plasmid
pPCC20 3 by Gibson Assembly, resulting in plasmid pPCC1215. The bla gene was amplified
from pET45b using primers pPCC1217-gib-for and pPCC1217-fib-rev and cloned into
pPCC1215 digested with NdeI and SpeI using Gibson Assembly, resulting in plasmid
pPCC1217. Plasmid pPCC1217 was subsequently integrated into the chromosome of HF19 at
the HK022 site resulting in SQ11. Apramycin resistant colonies were selected and the integration
was verified by PCR. Removal of FRT-flanked apramycin resistance cassette was achieved as
described 4, resulting in strain SQ12.
Cloning of plasmids for screening and AraC library. Construction and expression of the mutant
library was carried out as previously described 5. Initial work was based on the dual plasmid
reporter system for AraC-controlled GFPuv expression as described previously 5, in which AraC
and the AraC combinatorial library (SLib4) are expressed from plasmid pPCC423 (maintained
by apramycin antibiotic resistance) under control of IPTG-inducible LacI. In this system, PBADcontrolled GFPuv expression occurs on a second plasmid, pPCC442 (maintained by
chloramphenicol resistance). Plasmid pPCC423 was subsequently modified to pPFG1
(described below) and the dual plasmid system was converted to a single plasmid pFG29 as
described below.
The PBAD-gfpuv reporter was cloned into plasmid pFG1 (yielding pFG29) using the Gibson
Assembly method. Primers pFG29-gib-for and pFG29-gib-rev were designed to amplify PBADgfpuv from pPCC442. Primer pFG29-gib-rev incorporated a terminator sequence
(AAAAAAAAAAGCCCGCACTGTCAGGTGCGGGCTTTTTTCTGTGTTT). PBAD-gfpuv was
amplified using Phusion polymerase. The resulting PCR product was gel purified. The pFG1
vector was cleaved with the FspI restriction enzyme. These fragments were mixed and
assembled according to the Gibson Assembly protocol. The resulting plasmid was named
pFG29, containing Ptac-araC and PBAD-gfpuv.
All AraC-TAL mutants of interest were cloned into plasmid pFG29 (from their pPCC423
source vector) for analysis following isolation from the SLib4 library. This was done by PCR
amplification of araC variants using primers pFG29-araC-GS and pPCC1305_araCTAL-rvs.
The products and pFG29 vector were subjected to sequential digest by AflII and BstapI. The
purified products were ligated using T4 DNA ligase and transformed into electroporation
competent MC1061 cells. Sequencing of the final clones confirmed the correct sequences.
AraC-TAL Variants Characterization and Analyses
Dose-dependent responses of AraC-TAL variants. To better characterize the newly isolated
AraC-TAL variants, we determined the dose-dependent response of each variant to TAL (1-25
mM) using the protocol outlined in the Materials and Methods section. Each variant shows a
dose-dependent response to TAL (Figure S2). However due to the toxicity of high
concentrations of TAL (>25 mM), the full dynamic range of response could not be determined
for most variants.
Figure S2 TAL-dependent dose response of AraC-TAL variants. Data is reported as the average
and standard deviation of three independent experiments in relative fluorescence units (A) and
fold-response (B). The fold-response was calculated by dividing the bulk fluorescence in the
presence of TAL by the bulk background fluorescence in the absence of TAL.
AraC-TAL fold-response depends on residue hydrophobicity and charge. The hydrophobicity
of each amino acid substitution was determined from residue sidechain hydrophobicity values
provided Kyte and coworkers 7. Plotting the total change in hydrophobicity for each variant
versus the respective response of the variant shows a positive correlation between an increase in
amino acid substitution hydrophobicity and response (Figure S2). The change in hydropathy
(ΔHydropathy) of the ligand binding domain (LBD) was calculated by summing the
corresponding hydropathy values for the substituted residues of each variant and subtracting the
wt-AraC value (Table S2). In addition to the hydropathy, the net charge was also calculated. In
all variants, the net charge was positive (at a neutral pH), but the variants with the greatest
charge were the least responsive.
Figure S3 Scatter plot representing the trend of hydrophobicity from amino acid
substitutions and fold-response to 5 mM TAL. The hydrophobicity data was calculated
from the hydrophobicity indices calculated by Kyte and coworkers. The red line in the
scatter plot represents the simple linear regression model fitted to the respective data. There
is a positive correlation showing that the fold-response to 5 mM TAL increases as the
hydrophobicity increases.
Table S2 Charge and hydrophobicity of amino acid substitutions in the AraC-TAL clones. (A)
Net change in charge (Δz) of the LBD shows there is a net positive charge for all AraC-TAL
variants. The net change in hydrophobicity was calculated according to (B) Kyte and coworkers.
Each AraC-TAL variant showed positive net charge and a positive net hydropathy (more
hydrophobic) in the LBD.
A
Clone
WT-AraC
AraC-TAL1
AraC-TAL2
AraC-TAL3
AraC-TAL4
AraC-TAL5
AraC-TAL6
AraC-TAL7
AraC-TAL8
AraC-TAL9
AraC-TAL10
8
P
V
G
S
S
I
G
V
G
T
G
Residue
24 80 82 93
T H Y H
I G L R
H H K L
I G I R
L G L R
L G I R
L H K V
L G L R
L H K F
I G L R
L G I R
8
0
0
0
0
0
0
0
0
0
0
0
Residue
B
24
0
0
0.09
0
0
0
0
0
0
0
0
80
0.09
0
0.09
0
0
0
0.09
0
0.09
0
0
z (pH 7)
82
93
0 0.09
0
1
1
0
0
1
0
1
0
1
1
0
0
1
1
0
0
1
0
1
zsum
0.18
1
1.18
1
1
1
1.09
1
1.09
1
1
Δz
0.00
0.82
1.00
0.82
0.82
0.82
0.91
0.82
0.91
0.82
0.82
Hydropathy (Kyte et al. 1982)
Clone
WT-AraC
8
P
24 80 82 93
T H Y H
8
-1.6
24
-0.7
80
82
-3.2 -1.3
93
-3.2
HI
-10.0
ΔHI
-
AraC-TAL1
AraC-TAL2
AraC-TAL3
AraC-TAL4
AraC-TAL5
AraC-TAL6
AraC-TAL7
AraC-TAL8
AraC-TAL9
V
G
S
S
I
G
V
G
T
I
H
I
L
L
L
L
L
I
G
H
G
G
G
H
G
H
G
L
K
I
L
I
K
L
K
L
R
L
R
R
R
V
R
F
R
4.2
-0.4
-0.8
-0.8
4.5
-0.4
4.2
-0.4
-0.7
4.5
-3.2
4.5
3.8
3.8
3.8
3.8
3.8
4.5
-0.4 3.8
-3.2 -3.9
-0.4 4.5
-0.4 3.8
-0.4 4.5
-3.2 -3.9
-0.4 3.8
-3.2 -3.9
-0.4 3.8
-4.5
3.8
-4.5
-4.5
-4.5
4.2
-4.5
2.8
-4.5
7.6
-6.9
3.3
1.9
7.9
0.5
6.9
-0.9
2.7
17.6
3.1
13.3
11.9
17.9
10.5
16.9
9.1
12.7
AraC-TAL10
G
L
G
I
R
-0.4
3.8
-0.4
-4.5
3.0
13.0
4.5
AraC-TAL variants show specificity towards TAL. The specificity of the AraC-TAL variants
was tested in the presence of two compounds similar to TAL, phloroglucinol and 2,6-dimethyl-γpyrone. The dose response was setup as described in the Materials and Methods. Phloroglucinol
and 2,6-dimethyl-γ-pyrone were prepared fresh and dissolved directly in the media to 50 mM.
Error bars were incorporated and represent the standard deviation of four replicate cultures.
Dilutions were made from this stock solution. None of the variants show a high response to
either compound. AraC-TAL6 shows a slight response to phloroglucinol at low concentrations.
Figure S4 Dose dependent responses of AraC-TAL variants to (A) phloroglucinol and (B) 2,6dimethyl-γ-pyrone.
L-Arabinose is Not an Inhibitor of TAL Response
The response of AraC-TAL1 in the presence of L-ara was investigated to determine the extent of
L-ara binding. The competition assay was setup following the protocol for deep-well plate dose
responses described in the Materials and Methods section. HF19 cells harboring either pFG29 or
pFG29-TAL1 were screened for response to TAL (0.5 – 25 mM) in the presence and absence of
1 mM L-ara in the media. Also, the response of AraC-TAL1 to 5 mM TAL in the presence of
varying concentrations of L-ara (0.001-10 mM L-ara) was determined. As can be seen in Figure
S5, the response of AraC-TAL1 was not affected by the presence of L-ara.
Figure S5 Competition assay to determine the effect of the presence of L-ara on AraC-TAL1
response to TAL.
AraC-TAL LBD Purification
Protein gel analysis of soluble LBD of AraC-TAL clones. Due to the low solubility of the
AraC-TAL1 LBD, we examined the remaining AraC-TAL variants to test whether any were
more soluble than AraC-TAL1. AraC-TAL variants were cloned into the pET45b vector and
expressed as described in the Materials and Methods section of this manuscript. The induced
cells were lysed by boiling in lysis buffer for 10 min. The soluble fraction of the lysed cells was
loaded onto a SDS-PAGE gel. Based on analysis of SDS-PAGE gel band intensities, no variants
appeared more soluble than AraC-TAL1.
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