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 was indispensable for this project, because the proteins we are making are inherently toxic to bacteria. Our cell-free expression platform also allowed us to produce and screen many constructs quickly, without the intermediate step of bacterial transformation.

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. 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.

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.

Box 1: Why are StarCore Proteins Difficult to Express?

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 1 StarCore proteins can be expressed and purified.Team sponsor Bioneer provided us this compound image of an SDS-PAGE gel with his-tag purified StarCore proteins. The red stars indicate specific bands corresponding to expected StarCore size.

Table 1 Yields of successfully produced StarCore Proteins.11 proteins were obtained by team sponsor Bioneer using their automated ExiProgen system.

Discussion

We successfully obtained 11 StarCore proteins. Next, these proteins were passed to the Testing group to determine if they were effective against bacteria.

Methods

Cell-Free Mix Preparation

Cell free expression was performed using the myTXTL Sigma 70 Master Mix Kit (Arbor Biosciences, #507024).

Following Sun et al., (2013), we also prepared our own cell-free lysate to test the relative efficacy of the commercial extract. The protocol was modified by using sonication instead of use of a bead beater.

50 mL of E. coli DH5a were cultured overnight in LB, then washed multiple times in cold water. Cells were gently pelleted at 4 C then resuspended in 1 mL of S30A buffer (14 mM Mg-glutamate, 60 mM K-glutamate, 50 mM Tris, 2 mM DTT, pH 7.7)

Cells were sonicated using a Vibra-Cell™ Ultrasonic Liquid Processors VCX 130 with the following settings: 40 s ON—1 min OFF—40 s On—1 min OFF—40 s ON. Output frequency 20 kHz, amplitude 50%.

Amino acid and energy mixes were added to the cell lysate to support in-vitro translation. The final cell lysate contained 3 mM Magnesium glutamate, 8 mM Potassium glutamate, 1.5 mM of each amino acid (except leucine), 1.25 mM leucine, 50 mM HEPES, 1.5 mM ATP and GTP, 0.9 mM CTP and UTP, 0.2 mg/mL tRNA, 0.26 mM CoA, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid, 1 mM spermidine, 30 mM 3-PGA, 2% PEG-8000.

Cell-Free Mix Calibration

Protein expression was induced by adding 8 ul of commercial or lab-made cell-free mix to 2 ul of plasmids carrying the genes of interest.

We compared the protein production between our “home made” master mix and the commercial product of our sponsor.

Figure 2: A) Commercial cell-free mix outperforms standard lysates;B) Two-plasmid system drives cell-free expression with the T7 polymerase. Control plasmid sigma70-eGFP expresses GFP from a standard promoter at high yield. To express from plasmid T7pr-eGFP, it is necessary to first express the T7 polymerase from a separate plasmid. A plasmid ratio of 1:20 was empirically determined to produce the best yield.

In addition, we also performed tests to better characterized cell-free expression. These tests revealed that our plasmids expressed litely more eGFP as those already optimized for the cell free mix that come from our sponsor when a binary regulation with a ratio of 1:20 is occuring.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France

paris-bettencourt-2018@cri-paris.org

Team:Paris Bettencourt/Production