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Team:Imperial College London/Project Chemotaxis Assembly
From 2011.igem.org
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.
Assembly
Fig. 1: Assembly strategy for our Phyto-Route construct.
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 (see Fig. 2 and 3) 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.
Fragments for codon optimised mcpS receptor have not arrived in time and therefore could not have been assembled. However Juan L. Ramos of Consejo Superior de Investigaciones Científicas in Spain was able to provide us with non-codon optimised mcpS gene with unknown promoter and ribosome binding site in the pRK415 vector. We have pereformed tests on this construct as well.
Fig 2. Schematic of the final construct. Click on the parts to be directed to the registry.
