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Revision as of 00:54, 21 September 2011

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

Product testing at Syngenta.

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.

Fig. 1: 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. Fig. 2: Two more colony PCRs which were unsuccessful followed by five colony PCRs from negative control colonies (assembly of backbone vector without insert). The next well is a positive control colony PCR with the same vector plasmid. The final two wells are analytical PCRs of the CPEC assembly and negative control with sequencing primers showing the correctly sized band for the assembled insert.