Supplementary material
Cold shock induction of recombinantly-expressed Arctic environmental genes
Gro Elin Kjæreng Bjerga a, 1 , Adele Kim Williamson a a Norstruct, Department of Chemistry, Faculty of Science and Technology, University of
Tromsø, N-9037 Tromsø, Norway
1 Present address: Uni Research AS, Centre for Applied Biotechnology, Thormøhlensgt. 55, N-
5008 Bergen, Norway
Corresponding author:
Gro Elin Kjæreng Bjerga
Uni Research AS, Centre for Applied Biotechnology
Thormøhlensgt. 55
N-5008 Bergen, Norway

Table S1. Constructs cloned under control of the T7 promoter
Target Construct Plasmid Truncation Leader sequence
Native
Fusion tag
MZ0003 1
2
3
4
5
MZ0009 1
2
3
4
5
MZ0012 1
2
MZ0013 1
2
MZ0047 1
2
3
4
5
6
7 pET-
DEST42
No pET26b(+) N-terminal (1-25) pET32 N-terminal (1-25) pET30 pET30
N-terminal (1-25)
N-terminal (1-25) pET-
DEST42 pDEST17
None pDEST17 pDEST17
N-terminal (1-26)
N-terminal (1-26)
C-terminal (359-
449)
N-terminal (1-26)
C-terminal (340-
449)
No pDEST14 pET-
DEST42 pDEST17
None pET-
DEST42 pDEST17
No
None pDEST15 pET-
DEST42
N-terminal (1-38)
C-terminal (229-352)
N-terminal (1-33)
No pET-
DEST42
N-terminal (1-33) pET26b(+) N-terminal (1-33) pET26b(+) a No pET26b(+) a N-terminal (1-33) pDEST17 N-terminal (1-33)
PelB
No
No
No
Native
No
No
No
Native
No
No
Native
No
No
Native
No
PelB
Native
No
No
His (C)
His (C)
TRX-His (N),
TEV
His (N), TEV
His (C)
His (C)
His (N), TEV
His (N), TEV
His (N), TEV
None
His (C)
His (N), TEV
His (C)
His (N), TEV
GST (N), TEV
His (C)
His (C)
His (C)
His (C)
His (C)
His (N), TEV a) using sites that remove the E. coli PelB leader sequences encoded in the vector

Table S2. Expression conditions tested from T7 constructs. ++ indicates strong expression, + indicates a visible band, - indicates no expression T indicates toxicity (no cell growth).
Target
MZ0003
Construct 10-12 (ON) 15 (ON) 20 (ON) 37 (ON) Comment
1 a, + a
2 + a ++ a No over expression in the periplasmic fraction
3
4
+ a, d
+ b, c T d
+ , c
+ a
++ a
5 + d
MZ0009 1
2
b
++ c
b
++ c
3 ++ b, c, d ++ c
4
5
++ c
+ c
++ c
++ c
+ c
MZ0012 1
2
MZ0013 1
2
MZ0047 1
2
3
4
+
+
c
c
++ b, c, + d
a,b
++ c + b T d
a, + b
++ a
T a
T a
+ a + a
a,b
a, + b
++ a
T a
T a
++ a
Batch variation in expression
No over expression in the periplasmic fraction. Mixture of processed and unprocessed leader peptide
5
6
7 a) BL21Star pLys pRare b) BL21CodonPlus(DE3)RIL c) Rosetta2(DE3)pLysS d) ArcticExpress(DE3)RIL
+ c T b, d
T a
T a

Table S3. Solubility of proteins expressed under T7. S = soluble, (S)= small fraction soluble but most in the pellet, I = all insoluble, - is no expression, T is toxicity.
Target Construct 10-
12(ON)
MZ0003 1
2
3
4
5
MZ0009 1
2
3
4
MZ0012 1
2
MZ0013 1
2
MZ0047 1
2
3
4
5
6
7 a) BL21Star pLysS pRARE b) BL21CodonPlus(DE3)RIL c) Rosetta2(DE3)pLysS d) ArcticExpress(DE3)RIL
I c
I c
I d
15 (ON) 20 (ON) 37 (ON) Comment
a
(S) a
(S) a, d
(S) b (S) a
(S) b,d I c
b
(S) c
I c , (S) b, d
a,b
I c , (S) b ,T d
(S) a
a
T a
T a
(s) a (s) a
T b
(S) c
T a
T a
I a
I a
I a
I c
a,b
a
I a
T a
T a
I a
b
I c ,(S) c
I c
Soluble intracellular protein only indicating the leader peptide had not been processed
Batch variation in solubility
Mixture of processed and unprocessed leader peptide

Table S4. Primers used for generation of vectors and amplification and sequencing of cloned constructs.
Primer name
(restriction site)
TEV5' (BamHI)
TEV3' (BamHI) pCold-SUMO-F pCold-SUMO-R pCold-MBP-F pCold-MBP-R pCold-Trx-F pCold-Trx-R pCold-II-MZ0064-
TFEc_F pCold-II-MZ0064-
Sequence a (5'-3')
GATCCGAGAACCTTTACTTCCAGGGGG
Purpose
Primers for TEV insertion to pCold-II by oligonucleotide cloning
GATCCCCCCTGGAAGTAAAGGTTCTCG
CCATGAATCACAAAGTGCATCATCATCATCATCATAGCG
ACTCGGAAGTGAAC
Primers for TEV insertion to pCold-II by oligonucleotide cloning
Primers for gene-specific amplification (1 st PCR) designed for RF cloning of pCold-II-SUMO
GGTCGACAAGCTTGAATTCGGATCCACCGCCGATTTGTT
CACG
CCATGAATCACAAAGTGCATCATCATCATCATCATAAAA
CTGAAGAAGGTAAACTGGTAA
Primers for gene-specific amplification (1 st PCR) designed for RF cloning of pCold-II-SUMO
Primers for gene-specific amplification (1 of pCold-II-MBP-TEV st PCR) designed for RF cloning
CCCCTGGAAGTAAAGGTTCTCGGAAGATCCGGTACCAGT
CTGC
Primers for gene-specific amplification (1 st PCR) designed for RF cloning of pCold-II-MBP-TEV
CCATGAATCACAAAGTGCATCATCATCATCATCATAGCG
ATAAAATTATTCACCTGACTG
Primers for gene-specific amplification (1 st PCR) designed for RF cloning of pCold-II-TRX-TEV
CCCCTGGAAGTAAAGGTTCTCGGTATGGCCAGAACCAG
AACC
Primers for gene-specific amplification (1 st PCR) designed for RF cloning of pCold-II-TRX-TEV
CCATGAATCACAAAGTGCATCATCATCATCATCATCAAGT
TTCAGTTGAAACCACTCA
Primers for gene-specific amplification (1 st PCR) designed for RF cloning of pCold-II-TF-TEV
CGGATCCCTCGAGGGTACCGAGCGCCTGCTGGTTCATCA Primers for gene-specific amplification (1 st PCR) designed for RF cloning

TFEc_R
TF-F
MBP-F
MZ0009-RF-F2
MZ0009-RF-R5
MZ0012-RF-F3
MZ0012-RF-R2
MZ0013-RF-F3
MZ0013-RF-R2
MZ0003-RF-F
MZ0003-RF-R
MZ0047-RF-F
G
TGGAAGAGCAAGCTGTTGA of pCold-II-TF-TEV
Tag-specific primer for sequencing of inserts in pCold-II-TF-TEV constructs
GGTCGTCAGACTGTCGATGAAGCC
CCGAGAACCTTTACTTCCAGGGGCCGGCAGTGGACCTC
Tag-specific primer for sequencing of inserts in pCold-II-MBP-TEV constructs
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-TEV-based vectors
TCTTAGATTCTGTGCTTTTAAGCAGAGATTACCTAACTGG
CTCGCTTATTCTCG
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-TEV-based vectors
CCGAGAACCTTTACTTCCAGGGGCCCAGCTTCCGACCGA
TC
TCTTAGATTCTGTGCTTTTAAGCAGAGATTACCTAAGTG
GCCATAACGGACGC
CCGAGAACCTTTACTTCCAGGGGTATCACCGCTTTGGCG
AG
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-TEV-based vectors
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-TEV-based vectors
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-TEV-based vectors
TCTTAGATTCTGTGCTTTTAAGCAGAGATTACCTAGACTG
CCCCTGAATGCTG
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-TEV-based vectors
CCGAGAACCTTTACTTCCAGGGGCAGCCGCGCGGATTCA
AC
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-TEV-based vectors
TCTTAGATTCTGTGCTTTTAAGCAGAGATTACCTATTCTG
CACCGCCCGTCAT
CCGAGAACCTTTACTTCCAGGGGACCGCCGTTCCCCAAC
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-TEV-based vectors
Primers for gene-specific amplification (1 st PCR) designed for RF cloning

MZ0047-RF-R pCold-F pCold-R
MZ0003-RF-F2
MZ0047-RF-F2
MZ0013-RF-F4
MZ0012-RF-F5
MZ0009-RF-F4
TC
TCTTAGATTCTGTGCTTTTAAGCAGAGATTACCTAGAAAT
CGTCAGGCCTAACGC to pCold-II-TEV-based vectors
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-TEV-based vectors
ACGCCATATCGCCGAAAGG Vector-specific primers for sequencing of pCold-II-based plasmids
TGGCAGGGATCTTAGATTCTG Vector-specific primers for sequencing of pCold-II-based plasmids
CTCATCGTGAACAAATCGGCGGTCAGCCGCGCGGATTCA
AC
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-SUMO
CTCATCGTGAACAAATCGGCGGTACCGCCGTTCCCCAAC
TC
CTCATCGTGAACAAATCGGCGGTTATCACCGCTTTGGCG
AG
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-SUMO
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-SUMO
CTCATCGTGAACAAATCGGCGGTTTCCGACCGATCATAC
GC
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-SUMO
CTCATCGTGAACAAATCGGCGGTGCAGTGGACCTCGTC G
G
Primers for gene-specific amplification (1 st PCR) designed for RF cloning to pCold-II-SUMO

Supplementary figure labels
Figure S1. Alignment of MZ0009 with characterized GH18 chitinases.
The relevant region containing the substrate binding motif (SXGG) and the catalytic motif (DXDXE, wherein catalytic residues are highlighted with black circles) of MZ0009 and aligned sequences of characterized GH18 chitinases is shown. The label on the left side of each sequence gives its name,
UniProt ID [1] and organism: SERMA, Serratia marcescens; SACD2, Saccharophagus degradans (strain
2-40); VIBHA, Vibrio harveyi; STRCO, Streptomyces coelicolor. Amino acid numbering is indicated on the left of each sequence. Secondary structure information is retrieved from S. marcescens ChiB
(UniProt ID: Q54276, PDB ID: 1E15). Dark backgrounds indicate alignment of identical residues, and lined boxes indicate alignment of similar residues. The alignment was constructed using the multiple
alignment program ClustalX2.0 [2]. Sequence similarities and secondary structure information
(stands and helices are shown as black arrows and spirals above the alignment) from aligned
sequences was rendered by ESPript3.0 [3].
References
1. Consortium, T.U., Activities at the Universal Protein Resource (UniProt). Nucleic Acids
Research, 2014. 42(D1): p. D191-D198.
2.
3.
Larkin, M.A., et al., Clustal W and Clustal X version 2.0. Bioinformatics, 2007. 23(21): p.
2947-8.
Robert, X. and P. Gouet, Deciphering key features in protein structures with the new
ENDscript server. Nucleic Acids Research, 2014. 42(W1): p. W320-W324.