Team:ETH Zurich/Biology/MolecularMechanism

Bandpass

Where it comes from...

In 2005 Ron Weiss et al. constructed the first band-pass filter device in synthetic biology. Therfore he combined a high pass and a low pass filter. We used his pioneering network design as a basis in the planing process for our band-pass filter implementation [9].

Quorum sensing of the alarmsystem

Where it comes from...

The luminescence operon in Vibrio fischeri is regulated by the transcriptional activator (LuxR). The lux box is a 20 bp inverted repeat sequence at a position centered 42.5 bases upstream of transcriptional start. LuxR binds to the lux box in presents of AHL. LuxR is needed to activate the σ⁷⁰ RNA Polymerase. The Vibrio fischeri lux operon consists of seven genes, the first one is luxI. The luxI gene encodes for the enzyme for AHL synthesis.

How we used it

For our system we used a Promoter which is repressed by luxR!(BBa_R0061) It was designed by placing the lux box between and partially overlapping the consensus -35 and -10 site of an artificial lacZ promoter. In presents of AHL LuxR binds to the lux box and inhibits binding of the RNA Polymerase. [7]

Plasmid design

Plasmid design for the AlcR system Transcriptional terminators included for each gene. Plasmid copy numbers adapted from [4].

Plasmid design for the XylR system Transcriptional terminators included for each gene. Plasmid copy numbers adapted from [4]. For a detailed description of the pCK04AxylR plasmid see [1].

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 and plays a crucial role in our design, especially for the bandpass filter. Depending on the function of a particular protein in our system we had to evaluate on which plasmid the corresponding gene should be cloned.

In the xylene-responsive system we included an artificial degradation pathway (xylWCMABN) so that we could establish a xylene gradient. For this purpose the plasmid pCK04AxylR was added [1]. To avoid incompatibilities of plasmids we adapted the rest of our system and changed the composition of the genes on each vector compared to the AlcR system.

Cloning strategy

By cloning our parts we tried to stick as much as possible to the strategies proposed in the registry of standard biological parts [5].

• If available, genes and promoters in SmoColi were obtained from the iGEM spring 2011 distribution kit. A codon-optimized version of alcR and P_tet-lacI_M1 were synthesized. P_alc- and P_U-promoters were obtained by PCR.

• After isolation of the DNA, ribosome binding sites and prefix (5' gaattcgcggccgcttctagag 3') respectively suffix (5' tactagtagcggccgctgcag 3') were added to the gene by polymerase chain reaction. The PCR products were purified and afterwards digested with XbaI-PstI.

• Plasmid vectors containing the corresponding promoter were linearized by SpeI-PstI and the XbaI-PstI fragment containing the gene of interest was ligated into the backbone.

• In a next step we isolated a EcoRI-SpeI fragment with promoter, ribosome binding site and gene and inserted it into a EcoRI-XbaI cut vector containing a transcriptional terminator.

• In the end all the constructed parts were assembled on the corresponding vectors for the final SmoColi system. Some parts (especially lacI in an actively expressed form) appeared to be negatively selected by e.coli. So we had to assemble the parts in a way that lacI was always repressed.

Definition of ribosome binding sites

Another important role in the synthesis of proteins plays the ribosome binding site (RBS), where the ribosomes are bound to the mRNA at the initiation of the translation process. The ribosome binding sites which we included in our system were designed in several different ways:

• For the bandpass filter we implemented the ones which are also used for the plasmids in [2].

• For the other parts we either tried to use naturally ocurring ribosome binding sites or took specific ones from the Anderson library of ribosome binding sites [6].

The strength of all the ribosome binding sites used was estimated with a RBS calculator [7]. Although the efficiency of protein synthesis is not directly proportional to the estimated values, they can still indicate whether a certain RBS is more or less strong. Below you can find a list of the different ribosome binding sites used, their estimated binding strength and in front of which genes they were put.

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]

[5] [http://partsregistry.org/Help:Contents Registry of Standard Biological Parts Help:contents 21 Sep 2011, 02:59]

[6] [http://partsregistry.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Anderson J. Christopher Anderson Ribosome Binding Sites/Prokaryotic/Constitutive/Community Collection Registry of Standard Biological Parts, 21 Sep 2011, 08:49]

[7] []

[8] [http://jb.asm.org/cgi/content/abstract/182/3/805 Kristi A. Egland and E. P. Greenberg: Conversion of the Vibrio fischeri Transcriptional Activator, LuxR, to a Repressor , Journal of Bacteriology, 2000, 182: 805-811]

[9] [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]