Team:ETH Zurich/Biology/MolecularMechanism

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Can you feel the smoke tonight?

Circuit design

Plasmid design

Cloning strategy

We combined a Smoke-sensitive band-pass filter with GFP output with a quorum-sensing diffusive mechanism that alarms the user of the system to high Xylene levels by expressing RFP.

Circuit design

Circuit operations for SmoColi exposed to medium, low and zero xylene concentration resulting in a GFP band. Triangles indicating the gradient of AHL (blue) and sensor molecule (violett), receptively. Non active interactions and non expressed Proteins are indicated light colors.

SmoColi is a bacterio quantifier which can be activated with different signal, we implemented two of them acetaldehyde and xylene. The concentration gradient which is needed for SmoColi is either naturally or achieved by synthetic cellular degradation. For example in case of Xylene we included the upper Tol pathway of Pseudomonas putida in SmoColi [1]. In contrast acetaldeydhde is naturally degraded.

In our system we can use different small molceules as an activating input for the bandpass-filter [2]. The repressor which is activated by the small molecule is constantly expressed and enhances the expression of the LacI_M1 repressor (codon-modified LacI) and the lambda repressor CI, which are under control of the Xyl-Promotor, respectively. High xylene concentration results in high cytoplasmic levels of CI and of LacI_M1 and repression of the green fluorescent protein (GFP). Cells that are far from the input have low xylene concentrations, because of xylene degradation. Accordingly, LacI_M1 and CI are only expressed at basal level. Without repression of the lamda promoter wild-type LacI is produced and represses the production of GFP. At the same point the N-Acyl homoserine lactone (AHL) Synthase LuxI is produced, AHL binds to LuxR which is constantly expressed and represses the red fluorescent protein (RFP). AHL is a quorum-sensing molecule with a high diffusion rate, it diffuses through the whole tube and represses RFP production in the whole tube, even in cells where no AHL is produced.

Circuit operations for SmoColi exposed to very high xylene concentration resulting in an alarmsystem which turns the whole channel red. The triangle is indicating the sensor molecule concentration. Non active interactions and non expressed Proteins are indicated light colors.

If the xylene concentration is too high and it can not be degraded within the tube, no AHL is produced. Without AHL LuxR does not repress RFP and the whole tube turns red. To obtain better dynamics we tagged GFP and TetR with LVA tags.

Finally we can also use a negative input for our system, in this case we have to introduce an additional inverter to invert the negative input signal into a positive one. Therefore the tetracycline repressor protein (TetR) was used in case of AlcR. If acetalydeyhde is present AlcR binds to the promotor of TetR and inhibit its represssion, resulting in no TetR.

Plasmid design

For the biological implementation of the SmoColi system, we constructed a three-plasmid system with different antibiotic resistances. This setup was chosen in order to clone the whole SmoColi system into the same bacterial cells.

The vectors used in our setup are the parts pSB6A5, pSB3C5, pSB3K3 and pSB4K5. These plasmids contain different origins of replication, thus resulting in different copy numbers of the correlating plasmid in e.coli. pBR322, p15A and pSC101 origins are used as a stable three-plasmid system [3]. The copy number influences the amount of protein produced by the cell. Depending on the function of a particular protein in our system we chose on which plasmid the corresponding gene should be cloned.

http://www.springerlink.com/content/3fj1xxl9p42kx8l5/

Cloning strategy

References

[1] [http://aem.asm.org/cgi/content/abstract/64/2/748 Sven Panke, Juan M. Sánchez-Romero, and Víctor de Lorenzo: Engineering of Quasi-Natural Pseudomonas putida Strains for Toluene Metabolism through an ortho-Cleavage Degradation Pathway, Appl Environ Microbiol, February 1998, 64: 748-751]

[2] [http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html Subhayu Basu, Yoram Gerchman1, Cynthia H. Collins, Frances H. Arnold & Ron Weiss: A synthetic multicellular system for programmed pattern formation, Nature 2005, 434: 1130-11342]

[3] [http://www.springerlink.com/content/3fj1xxl9p42kx8l5/ Karl Friehs Plasmid Copy Number and Plasmid Stability, Advances in Biochemical Engineering/Biotechnology, 2004, 86: 22-192]

[4] [http://openwetware.org/wiki/Arking:JCAOligoTutorial8 OpenWetWare Arking:JCAOligoTutorial8, 0 Jul 2008, 04:32 UTC. 21 Sep 2011, 01:32]

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	For the biological implementation of the SmoColi system, we constructed a three-plasmid system with different antibiotic resistances. This setup was chosen in order to clone the whole SmoColi system into the same bacterial cells.		For the biological implementation of the SmoColi system, we constructed a three-plasmid system with different antibiotic resistances. This setup was chosen in order to clone the whole SmoColi system into the same bacterial cells.

-	The vectors used in our setup are the parts pSB6A5, pSB3C5, pSB3K3 and pSB4K5. These plasmids contain different origins of replication, thus resulting in different copy numbers of the correlating plasmid in ''e.coli''. pBR322, p15A and pSC101 origins are used as a stable three-plasmid system. The copy number influences the amount of protein produced by the cell. Depending on the function of a particular protein in our system we chose on which plasmid the corresponding gene should be cloned.	+	The vectors used in our setup are the parts pSB6A5, pSB3C5, pSB3K3 and pSB4K5. These plasmids contain different origins of replication, thus resulting in different copy numbers of the correlating plasmid in ''e.coli''. pBR322, p15A and pSC101 origins are used as a stable three-plasmid system [[#Ref3\|[3]]]. The copy number influences the amount of protein produced by the cell. Depending on the function of a particular protein in our system we chose on which plasmid the corresponding gene should be cloned.