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From 2011.igem.org

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Module 1: Phyto-Route

Chemotaxis is the movement of bacteria based on attraction or repulsion of chemicals. Roots secrete a variety of compounds that E. coli are not attracted to naturally. Accordingly, we engineered a chemoreceptor into our chassis that can sense malate, a common root exudate, so that it can swim towards the root. Additionally, E. coli are actively taken up by plant roots, which will allow targeted IAA delivery into roots by our system.

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Design

We aim to ensure that the specifications that were drawn up are considered in the design.

1. The bacteria should actively move towards roots.

• While malate-responsive sensors do not naturally occur in E. coli, they have been identified in several other bacteria species, including the soil microbe Pseudomonas putida. We therefore chose to engineer the chemoreceptor mcpS from the P. putida KT2440 strain. McpS is a receptor that responds to a number of TCA cycle intermediates such as malate, fumarate, oxaloacetate, succinate, citrate, isocitrate and butyrate [1]. P. putida and E. coli differ in their chemotaxis systems, but have structurally similar chemotaxis proteins. Since these proteins are structurally similar, it is reasonable to assume that the mcpS domain will interact with the Che proteins in E. coli and the bacteria will be able to perform chemotactic response upon malate binding to an introduced receptor mcpS. In addition to the mcpS chemoreceptor, we are also introducing another malate responsive chemoreceptor PA2652 from the Pseudomonas aeruginosa PA01 strain [2]. With PA2652 we are applying the same logic as with mcpS, in that structural similarity is enough for these proteins to be compatible with native chemotaxis system of E. coli. The introduction of two different malate chemoreceptors to different cells, allows us to compare different responsiveness of rewired chemotaxis based on structural similarity of introduced receptors to the common E. coli receptors.

  

• Genetic constructs for expression of malate chemoreceptor are not complicated, however they are modular. Expression of the constructs is under constitutive expression of promoter J23100. It is provided from the registry in the biobrick K398500, in the backbone vector pSB1C3. The coding sequence has been removed from this construct using PCR, providing us with backbone vector with the constitutive promoter, to be assembled using Gibson or CPEC assembly. Ribosome binding sites (RBS) have been generated using Salis RBS calculator with relative translation initiation rates (TIR): 44050 & 42010 for mcpS and PA2652 respectively. The coding sequences for mcpS and for PA2652 have been codon optimised using our codon optimisation software, so that sequence is codon optimised for E. coli. We exploit terminator embedded in the backbone vector pSB1C3 for termination of transcription. In addition, modularity of our system is ensured by putting 15 bp insulator sequence between the RBS and the promoter. This sequence was specifically designed to make the promoter interchangeable without affecting the RBS strength and therefore translation initation rate is not affected by changing a different promoter in front of the insulator sequence. This allows us to change the promoter strength simply by changing promoter without modifying the whole genetic construct, thus it improves modularity of the construct. The insulator sequence acts as a starting point for PCR and it can be used to amplify the receptor sequence without the promoter.

2. Uptake of bacteria into roots.

• Root uptake of both E. coli and the bakers’s yeast S. cerevisiae can be observed in the model organism Arabidopsis thaliana and in tomato plants. It is a process that occurs naturally (althought it yet remains to be observed in soil) and we do not need to incorporate additional genes into our design.

References

[1] Lacal, J., Alfonso, C., Liu, X. & et al. (2010) Identification of a chemoreceptor for tricarboxylic acid cycle intermediates: differential chemotactic response towards receptor ligands. Journal of Biological Chemistry, 285 (30), 23126 – 23136.

[2] Alvarez-Ortega, C. & Harwood, C. S. (2007) Identification of Malate Chemoreceptor in Pseudomonas aeruginosa by Screening for Chemotaxis Defects in an Energy Taxis-Deficient Mutant. Applied and Environmental Microbiology, 73 (23, 7793 - 7795.
Paungfoo-Lonhienne et al. (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS ONE 5(7): e11915. http://www.nih.gov/science/models/arabidopsis/index.html