This document describes a study that characterized different combinations of promoters and terminators in yeast to better understand how they regulate gene expression. Various promoter-terminator plasmid combinations were constructed and their impact on yellow fluorescent protein (YFP) expression was measured. The same promoter combined with different terminators resulted in up to 180-fold differences in protein expression. Characterizing these regulatory elements will allow optimizing gene expression and minimizing unwanted recombination when integrating long pathways for applications like biofuel production.

1. Characterization of S. cerevisiae promoter-terminator combinations to better regulate gene expression.
Nestor Orozco, Luke Latimer, Ilyssa Evans, and John Dueber
Department of Bioengineering and Chemistry, Energy Biosciences Institute, University of California Berkeley
Abstract
Introduction
Methods
Results
Discussion
Conclusion
YFP expression for different plasmids in yeast.
I would like to express my gratitude towards Professor John Dueber, and the NIH
Bridges to Baccalaureate Program for the opportunity to work in the Dueber Lab.
Also, I would like to express my gratitude towards my mentor Luke Latimer for his
co-operation, guidance and advise which helped me the most in the development of
this project and to my labmates Ilyssa Evans and Angel Asante for their support and
collaboration.
This project was supported by the Energy Biosciences Institute and Award Number
R25GM095401 from the National Institute of General Medical Sciences. The
content is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institute of General Medical Sciences or
the National Institutes of Health.
Promoters and terminators are regulatory DNA elements that
control gene expression. A promoter is a region of DNA
upstream of a gene that associates with transcription factors
while terminators are at the end of a gene and cause the
termination of transcription. To vary protein expression,
different promoters can be used to drive a gene. The study of
terminators, and their effect on protein levels, is largely
ignored and in most labs only one or two different terminators
are used for heterologous gene expression. This is
problematic during the integration of multiple genes as
repeated sequences in terminators may cause undesirable
recombination events. To begin addressing this problem for
multi-enzyme pathway integrations, we cloned different
combinations of promoters and terminators and observed their
impact on expression in yeast using a fluorescence protein as
a read out for gene expression. The total combinations of the
plasmids were expected to be 90. The same promoter
combined with different terminators can result in over 180 fold
differences in protein expression. Characterization and
cloning of these terminators will allow future work in our lab
and other labs to effectively assemble and integrate longer
gene pathways and tailor recombinant protein expression.
References
• The S. cerevisiae yeast is widely used as a eukaryotic
expression system for fermentations
• Gene expression is the process by which information from
a gene is used in the synthesis of a gene product.
• Gene expression is dependent on:
• A promoter: a region of DNA upstream of a gene that associates
with transcription factors
• A terminator: a region of DNA at the end of a gene that ends
transcription
• Most labs use only a few terminators for gene expression
• These may not be the best for high expression
• Characterization of more terminators could help reduce
undesired recombination events during integrations when
creating long pathways.
• Yamanishi, M. (2012) characterized 5300 different
terminators for gene expression with promoter TDH3
driving green fluorescence protein (GFP) in S. cerevisiae.
• This project consisted of further characterizing some of
these terminators with different promoters and a different
gene.
• The promoter, gene, and terminator sequences are in
charge of gene expression. The other parts of the DNA
sequences form the backbone of the DNA which has
encoded resistance to antibiotics and other biological
information.
Golden Gate
• The golden gate protocol uses restriction enzymes to
assemble in one step, separate DNA fragments
together into an acceptor vector.
Digestion and ligation
• Digestion is cutting DNA into and or making different
fragments of DNA. Special restriction enzymes
recognize specific sequences in the DNA molecules
hydrolyzing the phosphodiester backbone and cleaving
the DNA.
1. Yamanishi, M., Ito, Y., Kintaka, R,. Imamura, C,. Katahira, S., Ikeuchi, A., Moriya, H., and Matsuyama,
T. (2012). A Genome-Wide Activity Assessment of Terminator Regions in Saccharomyces
cerevisiae Provides a ″Terminatome″ Toolbox. ACS Syn. Biol. A-J.
2. Hillson, N.J., Rosengarten, R.D., and Keasling J.D. (2012) j5 DNA Assembly Design Automation
Software. ACS Syn. Biol. 1 (1), 14-21.
3. Mumberg, D., Müller, R., and Funk, M., (1994 December); Regulatable promoters of Saccharomyces
cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic
Acids Res, 5767, 5768.
• The promoter/terminator combinations influences gene
expression.
• The characterization of promoter/terminator combinations
can help to tailor gene expression.
• This can help optimize and regulate gene expression and
also minimize undesired recombination by creating longer
pathways with no repeat sequences.
• The creation of longer and optimized pathways integrated
into the genome can help in the production of biofuels
and the consumption of xylose for the creation of ethanol.
• Pathway optimization can save time and money in the
long run for producers and consumers.
Ratio of YFP expression for experimental
terminators compared to reference ADH1 with
various promoters
• Most gene expression with Terminators VMA2t, and JJJ2t
• Least gene expression with Terminators KSP1t, and ALY2t.
• Resulting in a difference of up to 180 fold in expression
• Most consistent terminators with varying promoters are CBR1t and
RPL15At
• Terminators effect on expression are most likely to be independent
of the promoter
• The results are inconsistent with Yamanishi, M. (2012) for some
terminators.
• More promoter-terminator combinations could be tested with more
promoters as well as with a different gene.
• There terminator combinations can be applied toward long
metabolic pathway engineering such as the consumption of Xylose
for the production of ethanol.
• It is performed by inputting a mix of up to 10 undigested
plasmids in a restriction-ligation and then transforming
the resulting product into competent cells. For this
project 7 different plasmids were used.
• After digestion the resulting DNA
fragments are separated by agarose
gel electrophoresis and then the desire
band can be cut out and purified.
• Ligation uses the help of an enzyme to join DNA
fragments which are joined together and the fragments
are inserted into a plasmid. These are joined together
by the formation of phosphodiester bonds. Ligation is
normally performed using T4 DNA ligase.
Fluorescence per optical density helps us find the relative
fluorescence per cell mass.
Commonly used terminator ADH1 is used as reference.
It is used to compare with other terminators.
​ 𝐹 𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝑝𝑒𝑟 𝑐𝑒𝑙𝑙 𝑚𝑎𝑠𝑠/𝐴𝐷𝐻1 𝐹𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝑝𝑒𝑟 𝑐𝑒𝑙𝑙
𝑚𝑎𝑠𝑠
Logarithmic interpretation of fluorescence per mass cell
of various promoter-terminator combination for an easier
interpretation of data.
Acknowledgements
Terminator
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