Team:NortheasternU-Boston/Design

Project Design

Expression without Cloning!

A key advantage of cell-free systems is that they allow expression directly from linear amplified DNA rather than from a bacterial plasmid. This enabled our overall part design scheme of IDT synthesized G-blocks containing each of our AMPs between our desired 5’UTR and Terminator, allowing us to express these AMPs after a single round of PCR with overhanging primers containing promoter regions. This schema allowed us to test numerous AMPs as well as GFP and methionine aminopeptidase under the control of two promoters without having to go through the time consuming process of cloning a bacterial plasmid for each expression construct.

This allows for an extremely iterative design process where a part can be conceived of, a G-block synthesized, a PCR amplification performed, and a part tested in a cell-free expression system within a matter of two weeks.

Experimental Plans

We set out to answer key questions concerning what level of complexity of AMP could be produced in our cell-free system. We also wanted to discover whether or not we could rescue AMP function through treatment with methionine aminopeptidase in the case where a non-canonical N-terminal methionine was added to the peptide as a start codon for expression. AMP effectivity is gauged via bacterial killing effectivity measured by OD600 growth curves or various other assays. Our experimental design would then be to test our panel of AMPs across promoters and in the presence or absence of methionine aminopeptidase also produced via cell-free reactions. Pardee 2016 reported that the AMPs that they produced in cell-free reactions were equally viable in purified and unpurified form, therefore purification was a step that we avoided in order to create a streamlined workflow directly from synthesis to amplification to expression to characterization.

Citations

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