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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 bacteria species, including the soil microbes Pseudomonas aeruginosa PA01 strain & Pseudomonas putida KT2440 strain. PA2652 is a malate responsive chemoreceptor found in P. aeruginosa & mcpS is a receptor found in P. putida responding to a number of TCA cycle intermediates including malate^[1][2]. The molecular mechanism of chemotaxis in Pseudomonas aeruginosa & P. putida are different to that of E. coli, however there is high degree of structural similarity between the proteins that make up the chemotaxis pathway. Since these proteins are structurally similar, it is reasonable to assume that the PA2652 or mcpS domain will interact with the native chemotaxis pathway in E. coli and the bacteria will be able to perform chemotactic response upon malate binding to an introduced receptor.

2. The construct must allow tunable expression.

• 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. This is one of the strongest constitutive receptors provided from the registry 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 15 bp insulator sequence, which allows easy interchangeability of the promoter using PCR. us and 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.

3. Construct should be modular.

In addition to be able to modify promoter strength, insulator sequence also ensures that Ribosome binding site´s TIR (translation initiation rate) does not change, when the promoter is replaced. This then leads to modular construct with replaceable promoters, however with the same rate of translation. TIRs are 42010 & 44050 for PA2652 and mcpS respectively. Ribosome binding sites have been generated using Salis RBS calculator.

3. 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 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.