Lorem ipsum dolor, sit amet consectetur adipisicing elit. Esse beatae assumenda eaque ex recusandae pariatur sunt soluta modi facere laborum exercitationem odio iure magnam obcaecati quos voluptatibus placeat, ratione harum! Provident, maxime ipsum veniam, rerum facere ad vero fugit ipsa natus recusandae sit voluptatum architecto laudantium vitae necessitatibus! Nesciunt illum porro sint odio sequi reprehenderit. Sint eligendi ex impedit recusandae! Alias obcaecati impedit iure recusandae quas asperiores tempore sint, consectetur veniam provident iste nulla fugit velit aliquam expedita, assumenda repellat dolorem dolore! Sit quis dolorem ad pariatur repellat reiciendis officiis. Asperiores molestiae eos quo inventore recusandae quae placeat delectus, natus sint. Ullam quas culpa nobis exercitationem omnis animi velit, deleniti fugit! Sapiente aperiam sit minima nostrum, rerum quae laudantium vero? Qui blanditiis, excepturi veritatis eaque temporibus voluptate maxime facere laborum voluptatem rerum ex a ipsum voluptatibus tempore, saepe sunt omnis nostrum sint? Voluptatum facilis omnis ea accusantium explicabo magnam architecto! Repellendus incidunt doloremque, a cum voluptates esse officia quia veniam architecto. Quibusdam deserunt nulla, dolore perspiciatis accusantium ad aliquam voluptatem iste iure, quae minus ipsa voluptates, sit voluptatum consequatur tempora? Architecto impedit in repellendus, quo dolorum sequi voluptatum omnis maxime perspiciatis aspernatur obcaecati sint iusto tenetur praesentium labore. Natus eveniet quaerat recusandae dignissimos nam sed distinctio quos fugiat aliquid eligendi. Molestias inventore nobis minus aspernatur recusandae asperiores excepturi ipsam voluptas quos, quae voluptatem voluptatibus vero veniam dolores fuga aliquid neque ut dolorem beatae cum temporibus tempora? Iure expedita debitis corrupti. Quae quam earum impedit laborum, nobis aspernatur fugit enim consequuntur provident laboriosam obcaecati doloremque ipsam quis modi quos ratione ipsum beatae? Voluptas, debitis eum! Dolorem accusantium et rem. Veniam, dicta! Sequi aut, eos id nemo maiores iste! Dicta cum eos, incidunt aperiam voluptate facilis vero vel deleniti inventore accusantium cupiditate saepe dolore atque quisquam voluptates aliquam amet a! Hic, consectetur.

Background

Insertion of many membrane proteins in prokaryotes as well as the endoplasmic reticulum of eukaryotes is facilitated by various translocons. These complexes interact with the ribosome during protein synthesis and direct the newly forming peptide into the translocon pore, or directly into the membrane. However, for the translocon to function properly, it requires many auxiliary components (signalling sequence, chaperones, insertion mechanism), which would be far too many for our system to remain stable. Since we could not simply transfer the entire translocation system into SynDrop, we hypothesized that some of its functionality could be emulated in different ways.

Nickel-chelating lipids and polyhistidine tags as potential solution

Specifically to localize the ribosomes near the membrane and reduce the exposure of MP transmembrane domains to the aqueous environment, we eventually chose a method that has already been successfully used to attach other proteins to membranes - via a his-tag to nickel ions, chelated by specific lipids composing membrane^1,2. Our reasoning for this was threefold. Firstly, this would have allowed ribosomes to be correctly positioned. If the ribosomes were localized near the membrane, but the peptide exit tunnel hadn’t been consistently pointed towards the membrane, the entire benefit of reducing MP aggregation would be lost. By being able to create his-tagged fusion proteins specifically of the ribosomal subunits localized near the exit tunnel, we guaranteed that the exit tunnel could never point away from the membrane. Secondly, general protein synthesis should be the least impeded, compared to alternatives, such as introducing a lipid anchor via an enzyme, or creating a fusion protein with a self inserting MP. These latter methods would completely immobilize the ribosome onto the membrane, removing degrees of freedom. With the polyhistidine-nickel interaction the ribosome would simply exhibit a much greater affinity towards the membrane, while still being able to detach and reattach as needed. Lastly, the simplicity for this method seemed to be the greatest, as only modified ribosomes as well as the incorporation of nickel-chelating lipids during liposome synthesis was needed - this was far more important than it can appear, because it was vital for our overall system to be as robust as possible.

Results

Designing the modifications

The basic procedure we designed to obtain our modified ribosomes is as follows: first, using CRISPR-Cas9, we modify the genome of E. coli (native strain MG1655) to incorporate his-tags onto the ribosomal subunits, as well as a Strep-tag onto the L12 subunit for ease of purification³. In literature a his-tag was described to aid purification when fused to subunit L12, however we exchanged it for a Strep tag, as it is also short in sequence and while being biologically does not interfere with the functionality of other his-tags. The subunits localized near the exit tunnel we chose as potential targets, in order of priority, were L24, L23, and L29. These subunits were chosen as they had free, unfolded C ends exposed to the surface at nearest proximity to the exit tunnel. These subunits are also not integral to the overall function of the ribosome, meaning that our modifications would have little to no impact on peptide synthesis^4,5,6,7. After the E. coli genome is successfully modified, the ribosomes are purified with the help of the previously incorporated Strep tag, and the final product is used for IVTT reactions.

Modification procedure

CRISPR-Cas9 was utilized via pCas9 and pTargetF plasmids ⁸. pCas9 constitutively expresses Cas9, which induces a double-strand DNA break at a specific target sequence, that is complementary to the guide RNA. The guide RNA sequences for our targets were introduced via reverse PCR into the pTargetF plasmid series, which then supplies it to Cas9. After the double stranded break occurs, the HDR (homology directed repair) mechanism is activated, which repairs the genome according to a supplied donor sequence. This process is highly efficient with the assistance λ-red proteins, expressed from pCas9. The donor sequence has ~300 bp length homology arms and the insertion sequence that can include either the His or Strep tag to be fused with the chosen ribosomal subunit C’-end, as well as an in frame selection marker (select antibiotic resistance genes). pCas9 expresses a gRNA targeting the ori of pTargetF, therefore the cells are automatically cured from pTargetF after each modification. Additionally, pCas9 has a temperature-sensitive ori, it is cured by growing the cells at 37 ^oC.

For multi-gene editing, we chose to supply the donor sequence as a linear DNA strand (PCR product). Due to financial reasons, to construct the donor DNA sequence we performed separate PCRs of the homology arms (from the E. coli genome), selection marker (antibiotic resistance genes from available plasmids) (Fig. 4). The oligomers had the his and strep tag sequences incorporated into them alongside 2 different restriction sites. In case the distance between the ribosomes and the membrane wall was too small for our system to be efficient, we also designed alternative variants the would feature the his-tags connected via a highly flexible two-glycine-four-serine linker (GGSSSS), which is a highly popular linker for artificial fusion proteins.

When the homology arm and marker gene pcr products are restricted and ligated, the end result is a single sequence with an antibiotic resistance gene flanked on either side by ~300 bp homology arms, in addition to the according tag fused to the selected subunit’s gene. The entire linear DNA product was amplified via PCR using primers that annealed to the ends of the strand. Large amounts of nonspecific product was present even after performing PCR of the whole ligated sequence, therefore gel purification was necessary after each amplification (Fig. 5).

The genome modifications were then carried according to Protocols our protocol. Although cPCR gave us mixed results, we could not verify any colonies that afterwards grew on our selected marker antibiotics, and thus could not continue our experiments with them. It appears most likely that the genome modifications were not entirely successful, due to the somewhat unstable nature of the ligated linear DNA used for the donor sequence.

Conclusion and Discussion

Although well described and planned in theory, our ribosome attachment system did not yield desired results. We hypothesize, that the underlying cause may include a flawed implementation of the CRISPR-Cas9 system: in our case we were forced to incorporate selection markers due to the functionality of the specific expression plasmids we used, which lead to the construction of unstable donor sequences. While this is unfortunate, we are still confident that our idea is worth further investigation: as we could not reproduce genome modifications on the subunit that has already been reportedly modified in a similar manner, it does not suggest that our primary concept is unworkable. Moving forward, we will most likely move to an alternative CRISPR-Cas9 expression system that would allow for optimized modifications, such as removing the requirement for a selection marker, which in turn would allow us to construct more robust donor sequences. Nonetheless, herein we provide a concept and initial tools for ribosome engineering. This demonstrates great potential in synthetic biology, especially in cell-free biomolecular systems like SynDrop.

References

1. 1.Chikh, G., Li, W., Schutze-Redelmeier, M., Meunier, J. & Bally, M. Attaching histidine-tagged peptides and proteins to lipid-based carriers through use of metal-ion-chelating lipids. Biochimica et Biophysica Acta (BBA) - Biomembranes 1567, 204-212 (2002).
2. 2.Blanchette, C., Fischer, N., Corzett, M., Bench, G. & Hoeprich, P. Kinetic Analysis of His-Tagged Protein Binding to Nickel-Chelating Nanolipoprotein Particles. Bioconjugate Chemistry 21, 1321-1330 (2010).
3. 3.Ederth, J., Mandava, C., Dasgupta, S. & Sanyal, S. A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli. Nucleic Acids Research 37, e15-e15 (2008).
4. 4.Spillmann, S. & Nierhaus, K. The ribosomal protein L24 of Escherichia coli is an assembly protein. Journal of Biological Chemistry (1978). at http://www.jbc.org/content/253/19/7047.long
5. 5.GU, S. The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome. RNA 9, 566-573 (2003).
6. 6.STOFFLER-MEILICKE, M., DABBS, E., ALBRECHT-EHRLICH, R. & STOFFLER, G. A mutant from Escherichia coli which lacks ribosomal proteins S17 and L29 used to localize these two proteins on the ribosomal surface. European Journal of Biochemistry 150, 485-490 (1985).
7. 7.Noeske, J. et al. Synergy of Streptogramin Antibiotics Occurs Independently of Their Effects on Translation. Antimicrobial Agents and Chemotherapy 58, 5269-5279 (2014).
8. 8.Jiang, Y. et al. Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Applied and Environmental Microbiology 81, 2506-2514 (2015)

Lorem ipsum dolor, sit amet consectetur adipisicing elit. Esse beatae assumenda eaque ex recusandae pariatur sunt soluta modi facere laborum exercitationem odio iure magnam obcaecati quos voluptatibus placeat, ratione harum! Provident, maxime ipsum veniam, rerum facere ad vero fugit ipsa natus recusandae sit voluptatum architecto laudantium vitae necessitatibus! Nesciunt illum porro sint odio sequi reprehenderit. Sint eligendi ex impedit recusandae! Alias obcaecati impedit iure recusandae quas asperiores tempore sint, consectetur veniam provident iste nulla fugit velit aliquam expedita, assumenda repellat dolorem dolore! Sit quis dolorem ad pariatur repellat reiciendis officiis. Asperiores molestiae eos quo inventore recusandae quae placeat delectus, natus sint. Ullam quas culpa nobis exercitationem omnis animi velit, deleniti fugit! Sapiente aperiam sit minima nostrum, rerum quae laudantium vero? Qui blanditiis, excepturi veritatis eaque temporibus voluptate maxime facere laborum voluptatem rerum ex a ipsum voluptatibus tempore, saepe sunt omnis nostrum sint? Voluptatum facilis omnis ea accusantium explicabo magnam architecto! Repellendus incidunt doloremque, a cum voluptates esse officia quia veniam architecto. Quibusdam deserunt nulla, dolore perspiciatis accusantium ad aliquam voluptatem iste iure, quae minus ipsa voluptates, sit voluptatum consequatur tempora? Architecto impedit in repellendus, quo dolorum sequi voluptatum omnis maxime perspiciatis aspernatur obcaecati sint iusto tenetur praesentium labore. Natus eveniet quaerat recusandae dignissimos nam sed distinctio quos fugiat aliquid eligendi. Molestias inventore nobis minus aspernatur recusandae asperiores excepturi ipsam voluptas quos, quae voluptatem voluptatibus vero veniam dolores fuga aliquid neque ut dolorem beatae cum temporibus tempora? Iure expedita debitis corrupti. Quae quam earum impedit laborum, nobis aspernatur fugit enim consequuntur provident laboriosam obcaecati doloremque ipsam quis modi quos ratione ipsum beatae? Voluptas, debitis eum! Dolorem accusantium et rem. Veniam, dicta! Sequi aut, eos id nemo maiores iste! Dicta cum eos, incidunt aperiam voluptate facilis vero vel deleniti inventore accusantium cupiditate saepe dolore atque quisquam voluptates aliquam amet a! Hic, consectetur.

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 () 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 , 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.