Synthetic Control of Transcription: From Hybrid Promoters to Promoter Engineering to Synthetic ...

42 Pages·2016·3.24 MB·English

by  Hal Alper

Engineering Conferences International
ECI Digital Archives
Metabolic Engineering IX Proceedings
Summer 6-6-2012
Synthetic Control of Transcription: From Hybrid
Promoters to Promoter Engineering to Synthetic
Operon Design
Hal Alper
The University of Texas at Austin
Follow this and additional works at:http://dc.engconfintl.org/metabolic_ix
Part of theBiomedical Engineering and Bioengineering Commons
Recommended Citation
Hal Alper, "Synthetic Control of Transcription: From Hybrid Promoters to Promoter Engineering to Synthetic Operon Design" in
"Metabolic Engineering IX", E. Heinzle, Saarland Univ.; P. Soucaille, INSA; G. Whited, Danisco Eds, ECI Symposium Series, (2013).
http://dc.engconfintl.org/metabolic_ix/15
This Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in
Metabolic Engineering IX by an authorized administrator of ECI Digital Archives. For more information, please [email protected].
Synthetic control of transcription
:
From hybrid promoters to promoter engineering
to synthetic operon design
Hal Alper
Department of Chemical Engineering
The University of Texas at Austin
h1 [email protected]
September 7, 2005
http://www.che.utexas.edu/alper_group/
Metabolic Engineering has opened up
possibilities…
• Metabolic
engineering
advances have
expanded the breath
of chemicals
pprroodduucceedd bbyy cceellllss
– Pharmaceuticals and
Nutraceuticals
– Fuels
– Commodity/Specialty
Chemicals
– Polymers and
Precursors Curran & Alper, Metabolic
Engineering, 2012
Laboratory for Cellular June 6, 2012
Hal Alper
and Metabolic Engineering Slide # 2
…but, requires synthetic control elements...
Pathway Control Circuit Design
A
B
C D
Heterologous Expression Probing via Graded Expression
HAT
A promoter
HAT*
promoter
B D
promoter
C
promoter
Laboratory for Cellular June 6, 2012
Hal Alper
and Metabolic Engineering Slide # 3
…with a range of expression capacities.
Native range of mRNA levels promoter
Require a promoter series(s)
that span above the highest
promoter
native expression down to
essentially zero expression:
promoter
Affords both amplification
and knockdowns.
promoter
Holstege, et al. (1998), Cell 95:717-728.
Laboratory for Cellular June 6, 2012
Hal Alper
and Metabolic Engineering Slide # 4
Synthetic control of transcription occurs at
many levels
Synthetic
5’UTR and MCSs
Transcriptional Genetic Context
Amplifiers
EElleemmeennttss tthhaatt
Influence
Terminators and
Expression
3’UTR
Mutant Promoters
Selection Markers
and Vectors
Laboratory for Cellular June 6, 2012
Hal Alper
and Metabolic Engineering Slide # 5
Designing novel genetic control elements
• Promoter Engineering is an effective method for
generating a collection of genetic control elements
Prior Promoter engineering example
+
GFP
Range of promoter
Promoter Promoter strength to control gene
Library expression
Alper et al. PNAS, 2005.
Laboratory for Cellular June 6, 2012
Hal Alper
and Metabolic Engineering Slide # 6
Development of diversified promoters
Mutant
T
CYC1
Promoter yECitrine
Nevoigt et al. AEM, 2006.
Laboratory for Cellular June 6, 2012
Hal Alper
and Metabolic Engineering Slide # 7
Development of novel low-strength promoters
Mutant
T
CYC1
Promoter yECitrine
4
3.5
11
CC
FF
TT 3

oo
tt

ee 22..55
vv
ii
tt
aa
elel 2
RR

ee
cc 1.5
nn
ee
cc
ss 1
ee
rr
oo
uu
0.5
ll
FF
0
Promoter
Laboratory for Cellular June 6, 2012
Hal Alper
and Metabolic Engineering Slide # 8
An expanding range of yeast promoters
Native range of mRNA levels
Holstege, et al. (1998), Cell 95:717-728.
Laboratory for Cellular June 6, 2012
Hal Alper
and Metabolic Engineering Slide # 9