Revision as of 13:40, 17 October 2018

Production

In the Design section, we designed StarCore sequences as compound BioBricks fusing an AMP sequence to a multimeric core. In this section, we use cell-free expression to produce StarCore proteins.

Cell-free expression allows us to bypass the toxic effect that our living cells would have if they produced our StarCore for hours but also allows us to have high throughput expression and to quickly screen many compounds.

Results

Expression of the StarCore Fusion Proteins

StarCore fusion proteins were expressed using the myTXTL Sigma 70 Master Mix Kit, generously provided by our team sponsor, Arbor Biosciences. As an expression vector, we used pACYCDuet-1 from Novagen. This vector is widely used for protein production in strains of E. coli that express T7 polymerase such as BL21 (DE3). It contains a T7 promoter upstream of a strong RBS and a lac operator, allowing IPTG-controllable protein expression.

To express from the T7 promoter, it was necessary to first produce T7 polymerase in the cell-free extract. For this purpose, we used the plasmid P70a-T7rnap, supplied by the manufacturer. We also included 100 uM IPTG in the master mix, to relieve lac repression.

Figure 1:Binary regulation used for cell-free expression.

Cell-free extracts were assayed for the presence of StarCore proteins by a variety of methods, described in the Characterization page. Unfortunately, none of these assayes produced evidence of successful protein expression and this despite our tests with the same constructs encoding eGFP instead of our StarCore.

Box 1: Why are StarCore Proteins Difficult to Express?

Although our cell-free mix performed well for expression of GFP controls, we were not able to achieve sufficient yields of StarCore proteins. It is clear that AMPs present special challenges to cell-free expression. Here are some reasons we came up with for why StarCores might be difficult to express with high yield.

Unbalanced amino acid usage, with a high density of lysine and arginine residues.

High density of positive charges, leading to misfolding and aggregation.

Direct toxicity to cell components caused by AMP activity.

Commercial Sourcing of StarCore Fusion Proteins

Fortunately, we had arranged an alternate source for StarCore proteins. The Bioneer company generously offered to sponsor us by giving us access to their ExiProgen™automated protein synthesis platform. This is a fully automated system that takes synthetic DNA as input and performs cell-free expression and protein purification.

Like us, Bioneer found most of the StarCore constructs to be difficult to clone, express and purify. However, thanks to their efforts we were able to obtain 11 StarCore proteins at high yield whose expected masses were compared to those observed on gels to verify plasmid designs and transformations (see the “Design” part).

Figure 2:Gel of colony-PCR allowing to verify the starCore expression (the stars reveal the bands of interest; L for ladder).

StarCore descriptions and controls are summarized in the table below, according to their constitution, mass and concentration: