poster nih summer 2013

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 TerminatomeToolbox. 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. /1 Logarithmic interpretation of fluorescence per mass cell of various promoter-terminator combination for an easier interpretation of data. Acknowledgements Terminator Promoter Promoter Terminator Terminator Promoter Promoter Terminator

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Page 1: Poster NIH summer 2013

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.

𝐹𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒  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

Promoter

Promoter

Terminator

Terminator

Promoter

Promoter

Terminator