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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.

Design

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

1. The bacteria should sense malate and actively move towards roots.

• While malate-responsive sensors do not naturally occur in E. coli, they have been identified in several other bacterial species, including the soil microbes Pseudomonas aeruginosa PA01 strain & Pseudomonas putida KT2440 strain. PA2652 is a malate-responsive chemoreceptor found in P. aeruginosa and mcpS is a receptor found in P. putida that responds to a number of TCA cycle intermediates including malate^[1][2]. The molecular mechanisms of chemotaxis in Pseudomonas aeruginosa and P. putida differ from that of E. coli. However, there is a high degree of structural similarity between the proteins that make up the chemotaxis pathways in these organisms. It is therefore reasonable to assume that the PA2652 or mcpS domain will interact with the native chemotaxis pathway in E. coli and that the bacteria will be able to perform a chemotactic response upon malate binding to an introduced receptor.

2. Efficient expression in our chassis.

• We have used our codon optimisation software to optimise the PA2652 construct ( BBa_K515102) for expression in E. coli.

3. The construct must be as modular as possible.

• Genetic constructs for expression of the malate chemoreceptor are not complicated. However, they are modular. At present, expression of the constructs is regulated by the constitutive promoter J23100. This is one of the strongest constitutive promoters in the registry. We chose this promoter as it is easier to tune down expression of a construct than to tune it up. To tune the expression of the genetic construct we have introduced a 15 bp insulator sequence, which allows easy interchangeability of the promoter using PCR and thus fine-tuning of expression levels. It ensures that the ribosome binding site's TIR (translation initiation rate) does not change when the promoter is replaced. The TIRs for PA2652 and mcpS are 42010 and 44050, respectively.

4. Uptake of bacteria into roots.

• Root uptake of both E. coli and the bakers’s yeast Saccharomyces cerevisiae can be observed in the model organism Arabidopsis thaliana and also in tomato plants. This 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 for uptake to take place.

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 and Harwood CS (2007) Identification of malate chemoreceptor in Pseudomonas aeruginosa by screening for chemotaxis defects in an energy taxis-deficient mutant. Applied and Environmental Microbiology 73 7793-7795.