An improved tetracycline-inducible expression system for fission yeast
Xiao-Hui Lyu

Introduction
In Schizosaccharomyces pombe, the thiamine-repressible nmt1 promoter and its weakened
variants have been the most commonly used inducible expression system for the last 30 years1.
However, this system has drawbacks including long induction time, incompatibility with richmedia, and requirement for medium switch. Tetracycline-inducible promoters do not have such
limitations but have not been widely used due to low expression levels at the induced state.
Recently, we have constructed a new tetracycline-inducible system based on a promoter
named enotetS, which is constructed by replacing a 19-bp sequence in the promoter of theeno101 gene with a single 19-bp tet operator sequence2. The enotetS promoter is fully induced
within a few hours upon the addition of the inducer anhydrotetracycline, and reaches anexpression level higher than that of the strong adh1 promoter.
Furthermore, for applications that need lower expression levels, we construct four weakened
variants of the enotetS promoter by inserting GC-rich sequences of different lengths, whichinhibit expression to different degrees3. The resulting promoter series spans a broad range of
expression levels, matching the range of the nmt promoter series but providing moreintermediate levels. For ease of use, we construct plasmid vectors that contain both atetracycline-inducible promoter and a cassette expressing the TetR repressor. This systemenhances the ability to manipulate gene expression in fission yeast.

Plasmids information(Expression level: PenotetS> PenotetSW1> PenotetSW2> PenotetSW3> PenotetSW4)
plasmid

GCK File NamepDB5318

pAde6-pCMV-tetR-PenotetS-mECtrine-ADH1 terminator-hphMX

pDB5319

pAde6-pCMV-tetR-PenotetSW1-mECtrine-ADH1 terminator-hphMX

pDB5320

pAde6-pCMV-tetR-PenotetSW2-mECtrine-ADH1 terminator-hphMX

pDB5321

pAde6-pCMV-tetR-PenotetSW3-mECtrine-ADH1 terminator-hphMX

pDB5322

pAde6-pCMV-tetR-PenotetSW4-mECtrine-ADH1 terminator-hphMX

How these plasmids were constructed:
1. Replace a 19-bp sequence between the TATA box and TSS of Peno2764 (refined fromPeno101) with a 19-bp tet operator sequence to get the enotetS promoter.
2. Integrate the enotetS promoter into the SIV plasmid.
3. A cassette expressing the TetR repressor is inserted in reverse upstream of the enotetSpromoter.
4. Insert different stem-loop sequences at the 5’ UTR (before the NheI restriction site).
Notes for using these plasmids:
1. Restriction site for changing different tag: NheI & BglII
2. Linearization Site: NotI

Results
To characterize the expression level and other related features of the enotetS series promoters,
we integrated the above plasmids (pDB5318-pDB5322) into LD1 (h− leu1-32 ura4-D18) andquantified the mECtrine fluorescence detected by dragonfly confocal microscopy.
(The following data may be update.)
1. The effect of ahTet concentration on PenotetS expression
EMM-PenotetS

YES-PenotetS
4000

mECtrine fluorescence, A.U.

3000

2000

1000

0

3000

2000

1000

concentration of ahTet (ug/ml)

1

1

5

5

5

5

0.

0.

0.

2.

2.

02

0.

0.

402
0.

4

52.

00

52.

0.

50.

0

50.

00

10.

0.

1
0.

02
0.

4020.

400
0.

000
0.

000mECtrine fluorescence, A.U.

4000

concentration of ahTet (ug/ml)

2.5 μg/ml of ahTet is a suitable concentration in both YES and EMM medium.
2. Fluorescence induction kinetics
Cells were induced with 2.5 μg/ml ahTet and sampled at different time. The expression levels
at different time points were normalized to the last time point.
YES-PenotetSW2

YES-PenotetS
100%

100%
64% 72%

88% 92%103% 108% 99% 94% 100%
51%

29%

70%

88% 91% 101% 100% 98%

102%100%

8242585% 87% 99% 84% 90% 91%

100%82529%

10%10%9%1%9%2%

1%

2%0%0%0%0%00.5

124681012222324

0250.5124610122223Time (h)Time (h)EMM-PenotetS

EMM-PenotetSW2
100%

100%
53%10%66%

85% 89%103%

88% 96% 93% 100%
52%

18%

10%

65%

18%

4%

4%

1%

1%
1%

1%
0%

0%

0%
00.5

1246810Time (h)12222324

250%
00.5

124610Time (h)12222324

3. Comparison of expression levels
YES
Low scale:High scale:

YES-comparison
104
2.99E3
2.10E3mECtrine fluorescence, A.U.

1.71E3103
4.22E2

1.01E2102

5.56E1

1.44E1

101

7.56

2.00
1.24

1.164tS
W

Pe

note

te
noPe

rep.

d.
in
4-tS
W

3-

rep.

d.
Pe
notet

SW

SW

2-tet

Pe
nonoPe

3in

rep.

d.
tetS

W

SW

1Pe
notet

W
tetS
noPe

2in

rep.

d.
1inp.

SW

tet
Pe
nonotetS-reintS
Pe

Pe

note

Padh

1d.

100

Expression level (induced):
PenotetS > Padh1 > PenotetSW1 > PenotetSW2 > PenotetSW3 > PenotetSW4

EMM

EMM-comparison
104
3.41E3

3.18E3

2.04E3

1.76E3mECtrine fluorescence, A.U.

103

5.35E2
3.76E2102

1.23E2

1.21E2
6.62E1

7.33E1

1.61E1

101

6.45

4.98

1.34

100
3.27E-1
1.59E-1

10-1
2.99E-2

Pa
Pn dh
1mt1
indPn.
m
t1-rPnepm.
t4
Pn 1-ind.
mt4
1Pn
rep.
mt8
1-in
Pnd.
mt8
1Pe
re
nop.
tetS
Peinnd.
Pe otetS
noretep.
tS
PeW
no1in
te
Pe tSW d.
no1retep.
tS
PeW
no2intePe tSW d.
no2retep.
tS
PeW
no3intePe tSW d.
no3retep.
tS
PeW
no4inted.
tS
W
4rep.

10-2Expression level (induced):
PenotetS > Pnmt1 > PenotetSW1 > Padh1 > PenotetSW2 > Pnmt41 > PenotetSW3 >PenotetSW4 > Pnmt81

Reference
1. Basi, G., Schmid, E. & Maundrell, K. TATA box mutations in the Schizosaccharomyces pombe nmt1
promoter affect transcription efficiency but not the transcription start point or thiamine repressibility.
Gene 123, 131–136 (1993).
2. Murphy, K. F., Balázsi, G. & Collins, J. J. Combinatorial promoter design for engineering noisy geneexpression. Proc. Natl. Acad. Sci. U. S. A. 104, 12726–12731 (2007).
3. Lamping, E., Niimi, M. & Cannon, R. D. Small, synthetic, GC-rich mRNA stem-loop modules 5’ proximal
to the AΜG start-codon predictably tune gene expression in yeast. Microb. Cell Factories 12, 74 (2013).
4. Wang, H. et al. Identification and refinement of two strong constitutive promoters for gene expression
system of Schizosaccharomyces pombe. World J. Microbiol. Biotechnol. 30, 1809–1817 (2014).