Design and Results

RNA Thermoswitches

Cell-free, synthetic biology systems open new horizons in engineering biomolecular systems which feature complex, cell-like behaviors in the absence of living entities. Having no superior genetic control, user-controllable mechanisms to regulate gene expression are necessary to successfully operate these systems. We have created a small collection of synthetic RNA thermometers that enable temperature-dependent translation of membrane proteins, work well in cells and display great potential to be transferred to any in vitro protein synthesis system.

Description

Fig. 1 A simplified mechanism of action of RNA thermometers. At lower temperatures the secondary messenger RNA (mRNA) stem-loop masks the ribosome binding site (RBS). Higher temperature induces melting of the hairpin which reveals the RBS to allow ribosome binding and initiation of translation.

Background

RNA thermometers are RNA-based genetic control tools that react to temperature changes¹. Low temperatures keep the mRNA at a conformation that masks the ribosome binding site within the 5’ end untranslated region (UTR). Masking of the Shine-Dalgarno (SD) sequence restricts ribosome binding and subsequent protein-translation. Higher temperatures melt the hairpins of RNA secondary structure allowing the ribosomes to access SD sequence to initiate translation ¹. In terms of applicability of RNA thermometers in in vitro systems, they display certain advantages over ribo- or toehold switches: they do not require binding of a ligand, metabolite or trigger RNA to induce the conformational change^2,3, therefore are especially compatible with our liposome IVTT system. Keeping that in mind we have explored literature ^1,4 and found five different RNA thermoswitches that we decided to test and build into our system in order to delay the translation of fusion construct bearing beta-barrel membrane protein. Furthermore, understanding the importance of expanding the library of well characterized and widely-applicable biobricks, we have de novo designed ( check RNA Thermoswitches model) six completely unique heat-inducible RNA thermometers.

Results

With custom IDT primers with overhangs bearing thermoswitch sequences we performed a PCR from a GFP gene containing plasmid and inserted RNA thermometers GJ_x (link: BBa_K2622010, BBa_K2622011, BBa_K2622012, BBa_K2622013, BBa_K2622014) upstream the GFP gene. Another set of primers was used to produce RNA thermometers Sw_x (link: BBa_K2622016, BBa_K2622017, BBa_K2622018, BBa_K2622019, BBa_K2622020, BBa_K2622021). PCR was successful and all products were the same size as expected for Sw_x constructs ~76 bp (Fig. 2). DNA gel electrophoresis was not performed for GJx constructs, because whole plasmid was multiplied and only 40-60 bp were inserted.

Fig. 2 Electrophoresis gel of PCR products: 6 - Sw2, 7 - Sw3, 8 - Sw6, 9 - Sw7, 10 - Sw9, 11 - Sw11.

pRSET plasmid and Sw_x PCR products were digested with restriction enzymes and ligated, while GJ_x PCR products were phosphorylated and ligated to produce plasmids from linear products. DH5α competent cells were transformed and plated on lysogeny broth (LB) media with ampicillin (Amp) and grown for 16 hours. Positive colonies were selected by colony PCR or restriction analysis (Fig. 3 and Fig. 4) and grown in 5 mL LB media. Plasmids were purified and BL21 competent cells were transformed. Three tubes of every construct plus plasmid with GFP without RNA thermometer were grown till OD₆₀₀ reached 0.4. Control samples were taken and protein expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG). One tube of every construct was grown in 24 ˚C, 30 ˚C, and 37 ˚C. Samples were taken after 1 and 2 hours. SDS-PAGE was run (for elaborate protocol see Notebook/Protocols). Fig. 5, Fig 6 and Fig. 7 depicts GFP expression at different temperatures. Although our RNA thermometers were designed to melt at 37 ˚C, some displayed leakiness to different extent. GJ3 (link:BBa_K2622011) RNA thermometer was the leakeast and allowed for GFP translation at lower temperatures. On the other hand, when grown at 37 ˚C, it unlocked the translation of GFP to highest yields. GJ2 (link:BBa_K2622010) was less leaky, but inhibited protein translation more strictly when grown at 37 ˚C. GJ6 (link: BBa_K2622012), GJ9 (link:BBa_K2622013), and GJ10 (link: BBa_K2622014) suppressed GFP production at 24 ˚C and 30 ˚C at similar level. They also inhibited translation to some extent at higher temperatures, meaning their melting temperature was not reached. Altogether these results prove, that our synthetic thermoswitches are temperature-responsive and act in physiological temperature range needed for IVTT reaction and also for BamA folding and membrane insertion.

Fig. 3 Restriction analysis of GJ_x constructs

Fig. 4 Colony PCR of RNA thermometers in pSB1C3 plasmid.

Fig. 5 expression at 24 ˚C. On the right you can see GFP expression without RNA thermometer.

Fig. 6 GFP expression at 30 ˚C. On the right you can see GFP expression without RNA thermometer.

Fig. 7 GFP expression in 37 ˚C. On the right you can see GFP expression without RNA thermometer.

Discussion

As described in other sections of the Design and results page (check BAM Complex), beta-barrel bearing proteins are assembled into the membrane by the BAM protein complex machinery. The key protein BamA is itself a membrane protein, whose folding and insertion into membrane where it helps assemble target proteins, last up to two hours. In order to prevent the aggregation of our fusion proteins after encapsulating their gene-bearing plasmids and purified BamA mRNA into liposomes, we needed to develop a modulatory regulatory tool to lock the translation of our membrane proteins to allow enough time for the encapsulated BamA to fold and insert into the liposome membrane.

Fig. 8 Associational scheme of thermoswitches’ action in the SynDrop system. Not locking the concomitant translation of our target protein and BamA results in target protein aggregation due to insufficient membrane insertion and assembling potential of BamA.

Fig. 9 Associational scheme of thermoswitches’ action in the SynDrop system. Locking up translation gives time for proper folding and insertion of BamA and prevents undesirable aggregation of target membrane proteins.

Additionally, while creating SynDrop, we have considered various options on how to make our complex cell-free system more user-controllable and predictable. Cell-free systems are becoming an attractive platform for in vitro compartmentalization and protein research, and although usually compositionally sensitive, they also offer a platform for building synthetic genetic regulatory tools or logic gates. Both the need to control the translation time of target genes and desire to provide more modularity for our synthetic system, led us to exploring RNA thermometers as a viable option to perform these tasks. They have minimal molecular burden and are easy to modulate. These properties encouraged us to developed a library of synthetic RNA thermometers suitable to translationally regulate the expression of our fusion constructs in bacteria with a further possibility to transfer them to IVTT systems. All of the RNA thermometers including those we found in literature and our de novo modelled ones were optimized for best performance at 37^oC, bearing in mind their future transition to IVTT system, whose optimum performance temperature is also 37^oC. Consequently, our experiments showed that our synthetic RNA thermometers, despite their simplistic structures compared to naturally occurring ones, efficiently triggered the expression of target constructs at 37^oC, and successfully locked it at lower temperatures having made them an ideal complement to our liposome IVTT system. All of our thermoswitches unlocked the expression to similarly high levels at 37^oC, but differed in terms of leakiness and success at inhibiting translation at lower temperatures.

Conclusions

We proved that herein described synthetic RNA thermometers enable high-yield expression of our constructs in an inducible temperature range. What is more important, this spectrum of temperature is compatible with currently used in vitro transcription and translation systems. Synthetic Thermoswitches allow the user-controllable and responsive protein translation for custom experiments. Finally, we introduced six de novo designed RNA thermoswitches which will have been used by future iGEM teams having to work both with cell-free and in vivo synthetic biology systems.

References

1. Neupert J, Karcher D, Bock R. Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli. Nucleic Acids Res. Oxford University Press; (2008); 36:e124–e124.
2. Narberhaus F, Waldminghaus T, Chowdhury S. RNA thermometers. FEMS Microbiol. Rev. Wiley/Blackwell (10.1111); (2006); 30:3–16.
3. Storz G. An RNA thermometer. Genes Dev. Cold Spring Harbor Laboratory Press; (1999); 13:633–6.
4. Sen S, Apurva D, Satija R, Siegal D, Murray RM. Design of a Toolbox of RNA Thermometers. ACS Synth. Biol. (2017); 6:1461–70.

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