EscheRatio coli A Ratio Sensor for Synthetic Biology

Christopher Brunson, Francisco Cai, Laura O’Brien, Gregory Owen, Karina Padilla, Alex Virrueta Drew Endy, Christina Smolke

Department of Bioengineering, Stanford University, Stanford, CA 94305

Motivation

• Lee SK, Keasling JD. A propionate-inducible expression system for enteric bacteria. Appl Environ Microbiol. 2005 Nov;71(11):6856-62.

• Khlebnikov A, Risa O, Skaug T, Carrier TA, Keasling JD. Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture. J Bacteriol. 2000 Dec;182(24):7029-34.

• Khlebnikov A and Keasling JD. Effect of lacY Expression on Homogeneity of Induction from the Ptac and Ptrc Promoters by Natural and Synthetic Inducers. Biotechnol Prog 2002 April; 18 672-674.

• Khlebnikov A, Skaug T, Keasling JD. Modulation of gene expression from the arabinose-inducible araBAD promoter. J Ind Microbiol Biotechnol. 2002 Jul;29(1):34-7.

Faculty Sponsors: Drew Endy, Christina Smolke, Demir Akin Post-Graduate/Graduate Student Mentors: Graham Anderson, Ryan Bloom, Jerome Bonnet, Andy Chang, Ying Lei, Francois St. Pierre, Isis Trenchard, Rayka Yokoo, Chris VanLang Director: Anusuya Ramasubramanian Funding: Draper Fisher Jurvetson Venture Group, Stanford VPUE, Bio-X

Motivation In order to be a part of a real-world application, every biological device must have a means of interfacing with its environment. A vast range of potential target processes in biology are dependent upon ratios, yet synthetic biologists have had no device capable of using those ratios as an input source. This year our team engineered two systems to unlock the potential of ratios for synthetic biology. Our sensors allow E. coli to sense the ratio of two different chemicals in its environment and produce a protein output based on that ratio without requiring human action.

sRNA Inhibition System

Twitter: A Platform for Communication Our team created an iGEM 2010 list on Twitter to allow teams to help each other solve lab problems and share ideas. We chose Twitter because of its ease of use, speed and breadth of distribution, brevity of messages, and pre-existing iGEM community. Twitter's list function allowed us to easily gather teams into one conversation, while its 140-character limit on tweets ensured that that conversation never became bogged down with lengthy posts. Our list contains 57 of this year’s teams, several of whom used Twitter to help each other troubleshoot their projects.

References & Acknowledgements

Device Overviews After researching several candidate biological mechanisms and reviewing scenarios in which our sensors would be used, we decided to pursue two different systems for ratio sensing: one based on sRNA interference and one based on a kinase-phosphatase pair. While both systems receive two input signals, the output they give is different, allowing them be applied in different situations. Both of our devices are orthogonal to the host cell and modular with respect to input and output chemicals.

Kinase-Phosphatase System

Registry Error: Barcodes

Our second sensor design responds to a range of ratios, varying the intensity of its output in response to the ratio of input chemicals.

Production of the transcription factor is under the control of a constitutive promoter, which maintains a basal concentration. The kinase (PknH) that acts on the transcription factor is under the control of a promoter positively regulated by one input (here arabinose). The phosphatase is similarly controlled by a promoter regulated by the other input (here AHL).

One of the input chemicals leads to the phosphorylation of a transcription factor (EmbR), while the other leads to its de-phosphorylation. Only the phosphorylated version of the transcription factor can activate the promoter upstream of the output protein (here GFP).

During sequencing, we found a 25 bp sequence in RFP (part E1010) not mentioned in the Registry description. Further research revealed that this sequence, which appeared between the terminator and the BioBrick suffix, was one of the genetic barcodes inserted into the original Registry parts. None of the parts that have these barcodes mention them in the DNA sequences listed in the Registry, which should be noted by teams planning on using these parts in the future.

Fig 3 - Teams collaborating via our Twitter list

Fig 4 – Some of the teams on our Twitter list

Fig 1 – An example of an application in which ratios are important. Last year our team developed a device to treat Inflammatory Bowel Disease, an autoimmune condition caused by an imbalance in the ratio of two types of T cells.

Our first sensor design is tuned to a single ratio of input chemicals and tells the user if the input chemicals are present in relative concentrations below, at, or above that ratio. The two input chemicals each bind to a receiver upstream of an sRNA and a reporter protein. Each sRNA is complementary to a specific "RSID" sequence that includes the RBS of the opposite reporter protein.

When the sRNA's bind to their target RSID sequences, they cover the RBS, preventing the translation of the reporter protein. The sRNA/reporter protein pair that receives the greater stimulus from the environment wins out over the other sRNA/reporter protein pair and determines the cell's output. We have designed the sRNA and RSID's so that we can use different receivers and reporters with this system.

Fig 5 – a parts-level diagram of our sRNA system showing the interaction between each sRNA and its respective RSID

The RSID: Improving sRNA InhibitionG The RSID (Ribonucleotide Sequence ID) is a synthetic DNA target sequence that is inserted upstream of the output proteins. Each sRNA recognizes and binds to a specific RSID, covering the adjacent RBS which prevents the translation of the downstream output protein. Because the RSID's are independent from the output protein, new output proteins may be inserted without affecting the performance or design of the internal mechanism.

We set out to add new tools to the synthetic biology toolbox. We: • Designed and built two complementary ratiometric sensors

• sRNA/mRNA providing an ultrasensitive response • kinase/phosphatase providing a graded response

• Tested and characterized the sRNA/mRNA system • Established a Twitter community to encourage collaboration

Summary Fig 6 - sRNA Inhibits L-arabinose Induction of GFP

(mean fluorescence plus one standard deviation)

We grew E. coli overnight with AHL. The cells contained either our L-arabinose-inducible GFP construct, or the inducible GFP construct plus the AHL-inducible sRNA construct. For cells containing the sRNA construct, after incubation with AHL, we backdiluted in media containing AHL and L-arabinose. Cells with the GFP construct were induced and began to express GFP, but cells containing the same construct along with the sRNA did not express GFP. We conclude that our GFP construct can be induced by L-arabinose, and that our sRNA construct inhibits GFP expression.

Characterization

Fig 2 – Our systems sense two inputs and calculate a ratio

Fig 7 – device level (left) and parts level (right) diagram for our kinase-phosphatase system showing the competitive actions of the kinase and phosphatase on the transcription factor