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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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Assembly

We have chosen to test two chemoreceptors. McpS from P. putida and PA2652 from P. aeruginosa. In order to test the receptor´s ability to rewire chemotaxis towards malate, we had to assemble the construct first. We have codon optimised the sequences for E. coli. Both McpS and PA2652 are long constructs (1923bp & 1689bp respectively, just for the coding sequence), therefore we decided to get them synthesised in two fragments. The assembly into the vector pSB1C3 was done by one of the multifragment assembly strategies. When we ordered the sequences we added MlyI sites (a blunt end endonuclease) at both ends of the sequences. It allowed us to excise our sequences through a simple digestion. This new standard allows the user to skip the first PCR step thereby decreasing the probability of a mutation occurring.

In parallel to ordering the sequences for synthesis, we asked Dr Juan Luis Ramos, who has been working with mcpS gene and who is a leading expert in the study of McpS, to provide us with this gene. He agreed to help us and we obtained from him a non-codon optimised mcpS gene under the control of an unknown promoter and RBS in pRK415 vector.

Due to our limited timeframe, Eurofins did not manage to synthesise the mcpS fragments in time, and therefore we were not able to assemble this construct. However, the PA2652 fragments were delivered on time and they were assembled into a pSB1C3 vector using CPEC assembly.

Assembly of PA2652 using CPEC

We assembled our two PA2652 fragments along with the promoter-containing backbone using CPEC. CPEC (Circular Polymerase Extension Cloning) is a primer-independent PCR assembly technique which relies on overlaping sequences between each part to be assembled. With a denaturing step, the double stranded DNA is melted, allowing compatible single stranded ends of each part to joined. For this reason it is essential that the parts are designed with homologous ends (the fragments we used were designed with 60 bp overlaps). The annealed overlapping ends then serve as primers for polymerase extension to join the parts into a seamless construct.

We quickly verified the assembly by doing a PCR on the CPEC product with our standard sequencing primers which anneal to the pSB1C3 backbone. We expected a PCR product of around 2kbp in size. We also transformed cells with the assembled construct and performed a colony PCR. The PCR products were run on an analytical gel (shown below) and all of the bands corresponded to the expected sizes.

DNA was mini-prepped from colonies and sequenced by Eurofins. The sequences came back positive so we could move on and start characterizing the PA2652 construct.

Gel 12: Colony PCR of 19 colonies picked from cells transformed with CPEC assembled PA2652 construct, about half have the correct size insert, these will be inoculated and miniprepped. Gel 13: Two more colony PCRs which were unsuccesfull, followed by five colony PCRs from negative control colonies (assembly of backbone vector without insert)showing backbone vector (6a) in one. The next well is a positive control colony PCR with plasmid 6a. The final two wells are analytical PCRs of the CPEC assembly and negative control with sequencing primers showing the correct size band for the assembled insert.