Team:Paris Bettencourt/Production
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.
| 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. |
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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).
Discussion
Using the cell-free system in collaboration, we successfully obtained 11 StarCore proteins. The expression system produced StarCores and these proteins were run on protein gels and the protein concentration was determined using a nanodrop. Successively, these proteins were then transferred to the Testing group to determine their killing efficacy on the test bacterium. In future, one needs to improve the efficiency of protein expression permitting multi-utility.
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.
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.
paris-bettencourt-2018@cri-paris.org 
