iGEM 2024 — Team sydney-australia

{% extends "base.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

{% extends "base.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Transforming R bodies into a chemotherapeutic endosomal escape platform through iterative optimisation cycles.{% endblock %}
{% block page\_content %}

## Menu

[Introduction](#introduction)
[1. Expression & Purification](#express-purify)
[Cycle 1.1](#cycle-1.1)
[Cycle 1.2](#cycle-1.2)
[Cycle 1.3](#cycle-1.3)
[Cycle 1.4](#cycle-1.4)
[2. Genetic Assembly & Conjugation](#assembly-conjugation)
[Cycle 2.1](#cycle-2.1)
[Cycle 2.2](#cycle-2.2)
[Cycle 2.3](#cycle-2.3)
[Cycle 2.4](#cycle-2.4)
[3. Cleavable Linkers & In Vitro Application](#cleavable-invitro)
[Cycle 3.1](#cycle-3.1)
[Conclusion](#conclusion)
[Engineering Philosophy](#engineering-philosophy)

## Introduction

Endolysosomal sequestration is a major rate-limiting step in intracellular delivery of biological or chemical therapeutics. In order to elicit responses, therapeutic agents must access the cytoplasm to interact with their corresponding intracellular targets. Most small molecules or macromolecules do not readily diffuse across the cell membrane but are taken up into cells by endocytosis. An exceedingly minor proportion escapes membrane-bound endocytic vesicles prior to acidic and hydrolytic degradation. Many treatment paradigms are therefore marred by poor bioavailability, impaired pharmacokinetics and off-target toxicity.

The ideal drug delivery system would overcome endosomal entrapment, feature multi-drug compatibility, high bioavailability and stimulus responsiveness, and be cost-effective. To resolve this unmet need, our team designed a modular drug delivery platform to mechanically disrupt endosomal compartments as they acidify. We engineered Reb A and Reb B monomer peptides from polymeric *Caedobacter taeniospiralis* refractile bodies (R bodies) with bioconjugation handles. We demonstrate that modified R bodies retain their mechanical piston-like function, and conjugate them with fluorescent markers to demonstrate their unusually efficient endocytic uptake and cytotolerance. Herein our team also documents the initial iteration of refractile bodies conjugated with acid-labile cargo, aimed at verifying drug release from ruptured endosomes. Our findings highlight the promise of R bodies as efficient, cheap platforms for orthogonal drug and/or excipient conjugation and delivery.

Our project was divided into five phases: expressing and purifying wildtype R bodies, creating new R body variants engineered to display conjugation tags, verifying our bioconjugation strategies, and testing the in vitro application of our modified variants.
For more detail on [results](https://2024.igem.wiki/sydney-australia/results) and [protocols](https://2024.igem.wiki/sydney-australia/protocols), refer to our respective wiki pages.

Check out our [Engineering Philosophy](#engineering-philosophy) for the team philosophy that enabled our engineering success!

---

## 1. Expression & Purification

Our team found immediate success with R body expression, but purification proved substantially more challenging. R bodies are not amenable to purification by chromatographic techniques due to their poor solubility in aqueous media and unusual size (Cai, 2023; Polka et al. 2016). Polka et al. (2016) describe a simple but relatively harsh purification method that separates R bodies from cellular components through lysis, enzymatic cleanup, centrifugation and repeated washing. However, our team was unable to successfully replicate this protocol. It took us over 6 months of DBTL optimisation cycles to reach sufficient purity for downstream applications. Our team initially used an mNeonGreen-R body fluorescent fusion protein constructed by Polka et al. (2016) to visualise whether R bodies retained their pH-induced extension after our relatively harsh treatment. Once we gained access to more powerful imaging modalities we learnt that the fluorescent R bodies had been giving us repeated false negatives for extension, and finally used wildtype R bodies to verify successful extension post-purification. Our team quickly took to heart that treating every failure as a chance to refine our approach was the cornerstone of progress!

---

### Cycle 1.1

![](https://static.igem.wiki/teams/5121/engineering/design.webp)

##### Design and Build

A wildtype and fluorescent construct were expressed. Different expression conditions (enriched media, codon optimisation) were evaluated for improving yield.

![](https://static.igem.wiki/teams/5121/engineering/test.webp)

##### Test and Learn

Fluorescence assays were performed as a proxy for R body expression, as no method has been developed for their biochemical analysis. Enriched medium enhanced yield of fluorescent protein but extended expression time. Codon optimisation had no effect.

![](https://static.igem.wiki/teams/5121/engineering/next.webp)

##### Next Cycle

Fluorescence results were promising, however to be certain of successful expression and R body assembly we needed to purify the protein and identify it through its characteristic contraction-extension behaviour.

#### Design and Build

Cycle 1.1 optimised conditions for R body expression. Our team was fortunate to inherit two stocks of DH5α strains containing R body plasmids (Reb constructs) created by Polka et al. 2016, namely Reb1 and its fluorescent version Reb206 (Figure 1).

![](https://static.igem.wiki/teams/5121/engineering/eng-fig1.png)

**Figure 1: Schematic overview of constructs used for R body expression in Cycle 1.1.** A) Reb1 construct containing the wildtype R body operon containing RebA, RebB, RebC and RebD genes. RebA and RebB genes encode R body monomers. RebC is reported to incorporate into R bodies at very low frequencies. RebD’s function is unknown and SZU-China 2019 report successfully expressing R bodies without the RebD gene. B) Reb206 construct containing an mNeonGreen-RebB fusion monomer under a weak RBS for stochastic incorporation into the R body structure, and mCherry under control of a strong RBS to help confirm protein expression.

As DH5α E. coli are a standard strain for plasmid propagation (Trivedi et al., 2014), we relied on standard protocols for transferring each construct into BL21 E. coli for recombinant protein expression. See our [Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) page for more details. For protein expression, we faced the following challenge: as complete R body dissociation has not yet been achieved no quantitative methods exist for reliably measuring R body concentrations or yields. As we were limited in imaging modalities, we could only approximately quantify Reb206 expression via fluorescence assays. We therefore decided to optimise expression conditions using Reb206 as a proxy for wildtype expression (the latter was required for conjugation cycles). We decided to first attempt expression at high temperature (37°C) for each construct as it was reported to be optimal for R bodies (Polka et al. 2016; Schrallhammer et al. 2012). We also attempted to optimise Reb206 expression through culturing in LB, enriched TPM medium or culturing a codon-optimised version of Reb206 produced in-house by Team PI Yu Heng Lau in 2018 (YHReb206). We tested enriched media as R body assembly is an energy-intensive process: cells enter a vegetative state once induced (Pond et al., 1989).

#### Test and Learn

Standard protocols sufficed for propagating our plasmid constructs and transforming them into BL21. We wanted to verify whether any of our experimental conditions (enriched medium or codon optimisation) improved R body yield. This was an important consideration for our future work in [Entrepreneurship](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27entrepreneurship%27%29%20%7D%7D). We used mNeonGreen fluorescence per cell as a proxy measure of R body expression (Figure 2). Though we applied this data in the Learn part of our DBTL cycle, it should be noted that without data on relative rates of fusion versus wildtype RebB monomer expression, rates of monomer incorporation into assembled R bodies, or rates of successful R body assembly given monomer expression, drawing correlation between monomer expression (fluorescence data) and R body yield is only approximate.

![](https://static.igem.wiki/teams/5121/engineering/engineering-reb1-expression.webp)

**Figure 2. Fluorescence and growth measurements during R body expression.** A) mNeonGreen fluorescence per cell was used as a proxy for R body expression. Four constructs were expressed in either LB or TPM medium, following which an aliquot was taken and mNeonGreen fluorescence as a function of cell density was measured. Non-fluorescent wildtype Reb1 was used as a negative control. Statistical analysis by one-way ANOVA with Tukey multiple comparisons test, error bars are ±SD. B) OD600 measurements of bacterial cultures across different media and constructs. The dashed line indicates the cutoff value of OD600 = 0.4 where cultures were induced with IPTG.

We learnt that codon optimisation did not improve R body yield, but enriched media more than doubled R body yield. However, it also doubled the time taken for the expression culture to reach OD600 = 0.4. As our future work required small yields we continued expressing with LB media. Expression in TPM remained an important consideration for future entrepreneurship work, particularly with respect to scaling-up operations.

#### Next Cycle

Although we collected evidence that suggested R bodies were successfully expressed, we could not be certain of correct assembly at high temperatures. We could not know how robust R body assembly is at high temperatures, and were concerned that accelerated protein aggregation and misfolding at high temperatures might impede assembly (Rosa et al., 2017). In cycle 1.2, We needed to purify the protein and confirm their contraction-extension function to confirm our success.

---

### Cycle 1.2

![](https://static.igem.wiki/teams/5121/engineering/design.webp)

##### Design and Build

We tried to replicate Polka et al.’s protocol (see [Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D#purification)) to purify Reb206. We chose to test our optimisation cycles on fluorescent Reb206 R bodies as it would enable easy downstream analysis.

![](https://static.igem.wiki/teams/5121/engineering/test.webp)

##### Test and Learn

Observation by naked eye and microscopy revealed extensive DNA and bacterial contamination. Loss of R bodies was observed in supernatants when following the Polka et al. (2016) protocol.

![](https://static.igem.wiki/teams/5121/engineering/next.webp)

##### Next Cycle

We focused on improving lysis, removing DNA contamination, and preventing R body loss in supernatants.

#### Design and Build

We now needed to design an unconventional protein purification strategy. R bodies are not amenable to purification by chromatographic techniques such as FPLC due to their poor solubility in aqueous media and unusual size (Cai, 2023; Polka et al. 2016). However, R bodies are reported to be stable under extreme treatments such as 5% mercaptoethanol, 8M urea, boiling, 6M guanidine thiocyanate and even shearing into fragments does not abrogate their pH-sensitive mechanical transitions (Cai, 2023; Pond et al., 1989). This enables, indeed necessitates, the use of harsh purification conditions which all reported purification protocols exploit (Lalucat, 1988). Various cell lysis methods are typically followed by DNase treatment for clean up, and rounds of harsh detergent washes and centrifugation to pellet clean R bodies. We therefore first chose to replicate Polka et al.’s purification strategy with fluorescent Reb206, as we were limited in our imaging modalities. While wildtype Reb1 are not visible with standard brightfield microscopy, individual Reb206 proteins can be easily visualised with a fluorescence filter due to their size. Thus we could visually confirm whether R bodies were purified and whether they survive (i.e. retain pH-sensitive extension) post-purification.

#### Test and Learn

Replicating Polka et al.’s protocol yielded very poor results. We did not obtain a fine, white sediment as reported in relevant literature but rather a stringy, viscous mass indicative of DNA contamination (Figure 3). We tried to resuspend the pellet to measure A260:A280 to confirm the presence of DNA, but resuspension was incomplete and the opaque, viscous solution did not yield intelligible data. Microscopy at pH 7.5 (when the sample should feature homogeneous masses of small contracted coils) revealed amorphous heterogeneous clumps with both fluorescent and non-fluorescent components, and extensive bacterial contamination. When resuspended in pH 5 buffer to induce R body extension, only a transient inflation of these clumps was observed (Video 1). This implied some mechanical force was being generated (which was further evidence that expression in Cycle 1.1 was successful), but perhaps complete conformational change was impeded by impurity.

![](https://static.igem.wiki/teams/5121/engineering/r-body-purify-polka.webp)

**Figure 3. Results of R body purification achieved by replicating Polka et al.’s protocol.** A) R body solution in Cycle 1.2 after attempted purification visualised under brightfield microscopy at pH 7.5. B) R body solution in Cycle 1.2 after attempted purification visualised under a fluorescent filter at pH 7.5. C) Falcon tube containing R body pellet purified in Cycle 1.2. The inset is reproduced from Polka et al. (2016) and shows the features of an adequately purified R body sample. Our sample is stringy, viscous and difficult to resuspend. See our Gallery for a video of us reacting to this pellet!

As we were replicating Polka et al.’s protocol, our team also observed cloudy white supernatants matching descriptions of suspended R body solutions, and observed fluorescence in supernatant fractions. This suggested R bodies were not precipitating, either due to low centrifugation speeds or DNA contamination impeding settling (or a combination thereof).

#### Next Cycle

Our team confirmed that fluorescent structures, most likely Reb206 R bodies, were trapped within amorphous clumps. Observation and microscopy indicated the greatest degree of contamination originated from DNA and unlysed bacteria. In the next DBTL cycle we focused on eliminating this contamination, and avoiding R body loss in supernatants.

---

### Cycle 1.3

![](https://static.igem.wiki/teams/5121/engineering/design.webp)

##### Design and Build

We experimented with a variety of methods to improve purification, including varying enzymatic incubation conditions, ionic strength, sonication or detergent type.

![](https://static.igem.wiki/teams/5121/engineering/test.webp)

##### Test and Learn

We successfully purified the sample but still could not observe extension. This suggested contamination was not preventing extension.

![](https://static.igem.wiki/teams/5121/engineering/next.webp)

##### Next Cycle

We gained access to improved imaging equipment. This eliminated our reliance on fluorescence and enabled us to test our purification protocol on wildtype R bodies (Reb1) as a positive control.

#### Design and Build

After a literature review our team concluded that Polka et al.’s approach represented the archetypical R body purification method, and no protocol existed that departed from Polka et al.’s main principles. We were on our own. Over the next half-year our team trialed a variety of methods to improve lysis and eliminate DNA contamination. Table 1 compiles the approaches sampled by our team. We collate them here into a single DBTL cycle as our fundamental goal - visualising Reb206 extension by achieving sufficient purity - remained unchanged. The loss of R bodies into supernatant observed in Cycle 1.2 was easily resolved by increasing centrifugation speeds.

#### Test and Learn

In order to best understand the changes made we recommend referring to our Protocols page for comparison to the original purification method.

| Modification | Rationale |
| --- | --- |
| Lengthening enzymatic and detergent incubations | Lengthened incubation with lysozyme and DNase to maximise their effect yielded no improvement in purity, although it improved bacterial lysis. Lengthened incubation with 1% SDS solution before washing, inspired by Bedingfield et al. (1984), likely enabled more contaminants to be solubilised into the supernatant and improved purity appreciably. The SDS incubation also facilitated lysis. |
| Increasing lysozyme and DNase concentrations in the initial incubation step | Increasing concentrations only marginally improved purity. Stringiness and amorphous clumps remained. |
| Introducing a second lysozyme and DNase incubation | Given that longer incubations and higher concentrations in the first incubation step only marginally improved purity, we hypothesized the enzymes might be inactivated during the first incubation step and replaced them with fresh enzyme in a second incubation step. This improved purity significantly. |
| Altering buffer composition | Although Polka et al. utilised EDTA in buffers, likely to inactivate intracellular proteases, reports of R body resistance to proteolytic digestion (Pond et al., 1989) and concerns that EDTA might impair DNase and lysozyme activity led us to remove it. No appreciable difference was observed with or without EDTA. |
| Cell lysis by sonication | Sonication was trialed to improve lysis. Harsh sonication produced foaming and deteriorated purity. Gentle sonication yielded approximately equivalent results to high lysozyme concentrations, so for fear of mechanically damaging R bodies sonication was abandoned. |
| Increasing ionic strength | High ionic strength has been reported to separate DNA from histone proteins by screening electrostatic attraction (Zhong et al., 2022). We tested increasing salt concentrations but observed on improvement. |
| Washing with nonionic Triton X-100 detergent instead of SDS | There appears little consensus in the literature as to which detergent is preferable (Polka et al., 2016; Schralhammer et al., 2012). We found that switching from SDS to Triton had the greatest effect on improving purity. |

**Table 1. Modifications made to Polka et al.’s protocol to optimize R body purification.**

Combining these strategies ultimately yielded a pure sample of Reb206 (see Figure 4 in Cycle 1.4), but still no extension at low pH could be visualized with brightfield microscopy. We had ruled out contamination as the issue behind extension. It was nonetheless a worthwhile effort as speaking with experts such as [Professor John Rasko](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#prof-john-rasko) and [Dr Nicholas Hunt](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#dr-nicholas-hunt) made us acutely aware of the rigorous purity standards necessary for administration in patients, meaning significant progress had been achieved towards our goal of developing a human therapeutic.

#### Next Cycle

We sought access to improved imaging modalities so that fluorescence would be unnecessary to detect extension. This would allow us to test our purification protocol on wildtype R bodies (Reb1) as a positive control.

---

### Cycle 1.4

![](https://static.igem.wiki/teams/5121/engineering/design.webp)

##### Design and Build

We tested our purification protocol on Reb1 as a positive control.

![](https://static.igem.wiki/teams/5121/engineering/test.webp)

##### Test and Learn

Reb1 extension was clearly observed, although not all R bodies particles were functional.

![](https://static.igem.wiki/teams/5121/engineering/next.webp)

##### Next Cycle

Future experiments would look at improving the proportion of functional Reb1 R bodies and elucidating the cause behind Reb206’s failed extension.

#### Design and Build

In this cycle we gained access to phase contrast and differential interference contrast (DIC) functionality. We could now examine the effects of our purification strategy on Reb1 to determine whether issues with extension were Reb206-specific or a result of our purification protocol.

#### Test and Learn

We observed Reb1 extension at low pH, which confirmed that Reb206 was non-functional. Surprisingly, even in pH 3 a significant proportion of our R bodies remained contracted. Overall this results enabled us to move onto the next phase of our project: designing and expressing R body variants for bioconjugation.

![](https://static.igem.wiki/teams/5121/engineering/206vreb1.webp)

Figure 4. Comparison of our purified Reb206 samples to R bodies purified by Polka et al. (2016). A) Purified Reb206 sample visualised under 63x phase contrast microscopy in pH 3 acetic acid. Although the sample is clean, the R bodies remained contracted. B) Panels showing contracted R bodies visualised by phase contrast microscopy at pH 6.1, reproduced from Polka et al. (2016). Clumping and morphology identical to our samples is observed. C) Panels showing extended R bodies visualised by phase contrast microscopy at pH <5.7, reproduced from Polka et al. (2016). Extension is clear. D) Purified Reb1 sample visualised under 6x phase contrast microscopy in pH 3 acetic acid. The sample is mostly clean and extended R bodies are clearly visible. Red arrows show examples of extended R bodies. The blue arrow shows a coiled, contracted R body.

#### Next Cycle

Our results opened several avenues of investigation: why were our Reb206 constructs non-functional? Why did some Reb1 R bodies extend at low pH but not others? In future cycles team would likely trial different expression conditions to assess whether temperature-dependent misassembly may have contributed to these observations. We would also investigate whether gentler purification might improve functional R body yield.

---

## Genetic Assembly & Conjugation

We wanted to engineer R bodies with a modular cargo-carrying function using orthogonal conjugation strategies. Physical models by Cai (2023) resolve the ribbon-like structure of R bodies composed of RebA and RebB monomers aligned along their vertical axis (that is, horizontal dimensions aligned in the same plane). The N termini and C termini are spatially separated across the outer and inner ribbon surface. This structure allows us to engineer up to four different conjugation handles at once onto the N termini and C termini of RebA and RebB. Orthogonality is crucial as it increases the combinations of cargo or excipient molecules that may be conjugated at once without interference. Polka et al.’s Reb206 construct also demonstrates that multiple versions of a single Reb monomer integrate may integrate into one R body. In future investigations, characterizing monomer assembly mechanisms and ratios will further expand the variety of conjugation handles that can be simultaneously displayed.

![](https://static.igem.wiki/teams/5121/engineering/eng-fig5.png)

**Figure 5. Example modular R body design with conjugation-ready linkers available at both ends of each monomer.**

Our team considered a range of conjugation strategies. We ultimately selected:

* Sortase A enzymatic ligation due to its reliability and FDA-approval in antibody-drug conjugates (Chen et al. 2016; Taylor et al. 2022). See Cycle 2.1.
* Non-canonical amino acid incorporation by amber codon suppression due to easy testing (Lang & Chin, 2014; Nodling et al., 2019; Waals & Ovaa, 2014). See Cycle 2.2.
* Thiol-maleimide conjugation due to its in vitro stability and precedent in FDA-approved trastuzumab (Fontaine et al., 2014). See Cycle 2.3.
* 2-pyridinecarboxyaldehyde selective N terminus modification as it can be applied post hoc to any engineered R body variant (Macdonald et al., 2015). See Cycle 2.4.

We created new [Part](https://2024.igem.wiki/sydney-australia/parts) for each strategy. Our team also considered using tyrosine-based PTAD click chemistry (Dorta et al., 2020). We designed primers and began the cloning procedure but did not complete testing due to time constraints. Several other strategies were deemed compatible with R bodies but were not tested: native chemical ligation, serine-threonine peptide ligation, typical and atypical split intein conjugation, SpyTag-SpyCatcher, protein farnesyltransferase and N-myristoyltransferase. See our [Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D) page for more details. Future investigation of these strategies would enhance the R body conjugation toolbox.

---

### Cycle 2.1

![](https://static.igem.wiki/teams/5121/engineering/design.webp)

##### Design and Build

Sortase A enzymatic ligation required cloning an LPXTG motif onto Reb monomers and a GGG motif onto a fluorescent marker.

![](https://static.igem.wiki/teams/5121/engineering/test.webp)

##### Test and Learn

R body extension post-modification was tested and confirmed. Conjugation was immediately successful.

![](https://static.igem.wiki/teams/5121/engineering/next.webp)

##### Next Cycle

Quantifying conjugation yields will be important for characterisation and therapeutic application.

#### Design and Build

*S. aureus* sortase A (SrtA) catalyses a highly specific, directional transpeptidase reaction between a C-terminal LPXTG motif and N-terminal oligoglycine (Antos et al., 2009; Figure 6). The closest existing analogue to our drug delivery system, conventional ADCs, suffer from pharmacologic unpredictability due to heterogeneous modification and variable drug-to-antibody ratios (Lucas et al., 2018). SrtA conjugation has thus emerged as a promising option to improve ADC pharmacologic predictability and has been used to generate enhanced counterparts of several ADCs (Beerli et al., 2015). Applied to our delivery system, SrtA site-specific conjugation would generate homogeneously modified drug carriers with defined drug-to-carrier ratios.

As the SrtA reaction is directional, we were limited to conjugating on the C-terminus. We also wanted to conjugate a fluorescent cargo (we chose mNeonGreen due to its improved stability) for easy downstream validation. Research from Chen et al. (2016) had optimised an enhanced SrtA (eSrtA) enzyme that ligated best with an LPETG motif, and this enzyme was commercially available. Thus three constructs needed to be created and expressed: mNeonGreen with an N-terminus oligoglycine, and Reb1 where RebA or RebB had a C-terminus LPETG motif. Hereon we refer to these constructs as GGG-mNeonGreen, RebA-LPETG and RebB-LPETG respectively.

To clone our constructs, overlap extension PCR was used with Reb1 or mNeonGreen templates to insert the necessary sortase motifs and overlaps. The fragments were combined by Gibson assembly. They were transformed into DH5α E. coli for amplification and BL21 E. coli for expression. Meanwhile eSrtA was expressed from a commercial plasmid (Addgene #75144) and purified by the 6xHis/Ni-NTA system (see Results for details).

![](https://static.igem.wiki/teams/5121/engineering/liorimage2.png)

**Figure 6. Schematic overview of Sortase A cloning strategy.**
The final product is an R body with mNeonGreen conjugated via an LPETGGG linker to the C-terminus of RebA or RebB.

#### Test and Learn

We wanted to confirm that C-terminal (or N-terminal) modifications to R body monomers preserve their extension-contraction behaviour. RebA-LPETG and RebB-LPETG both exhibited this activity (Figure 7A). As relatively large peptide motifs did not obstruct R body mechanical transitions we concluded the very C-terminal of R bodies was unlikely to feature specific interactions needed for conformational change.

Our choice of a fluorescent cargo allowed us to easily and qualitatively verify that conjugation was successful (Figure 7B). These results are a scientific curiosity as they represent successful aqueous-phase conjugation on a resuspended insoluble protein, which is also one of the largest natural protein assemblies on which conjugation has been attempted to the extent of our knowledge.

![](https://static.igem.wiki/teams/5121/engineering/sortase-a-conj.webp)

**Figure 7. Sortase conjugation of RebA-LPETG and RebB-LPETG.** A) RebA-LPETG R bodies extended in pH 2 HCl-KCl buffer observed at 40x under DIC. A ROI (white square) is magnified to demonstrate the thin straight lines that are extended R bodies. B) Washed pellet of RebB-LPETG R bodies conjugated with GGG-mNeonGreen (left) compared to a washed control pellet of RebB-LPETG R bodies (right) that had been incubated with GGG-mNeonGreen without eSrtA. A distinct yellow hue is observed in the conjugated pellet. C) Washed pellet of RebA-LPETG R bodies conjugated with GGG-mNeonGreen (left) compared to a washed control pellet of RebA-LPETG R bodies (right) incubated without eSrtA.

#### Next Cycle

As no biochemical tests exist to quantify R body yields or concentrations, we could not quantify the efficiency of conjugation. This will be critical to characterise prior to any therapeutic application. In our protocol we also used an enormous excess of GGG-mNeonGreen and eSrtA to force reaction equilibrium towards the product. Future cycles could reduce and optimise reagent concentrations, which would assist in scaling up production for entrepreneurial purposes.

---

### Cycle 2.2

![](https://static.igem.wiki/teams/5121/engineering/design.webp)

##### Design and Build

Non-canonical amino acid (ncAA) incorporation by amber codon suppression requires cotransformation with an aminoacyl-tRNA synthetase/tRNACUA plasmid and a modified Reb1 plasmid with an amber (TAG) codon. An ncAA compatible with copper-catalysed azide-alkyne cycloaddition (CuAAC) was chosen for downstream dye conjugation.

![](https://static.igem.wiki/teams/5121/engineering/test.webp)

##### Test and Learn

Conjugation failed on the first attempt but was successful on the second after a rate-accelerating ligand was introduced.

![](https://static.igem.wiki/teams/5121/engineering/next.webp)

##### Next Cycle

Improving efficiency of conjugation and once again quantifying conjugation yields will be important for characterisation and therapeutic application.

#### Design and Build

Amber codon suppression is a genetic code expansion technique that introduces non-canonical amino acids (ncAA) with powerful reactive handles into proteins. An ncAA added to the cell growth medium is specifically recognized by an orthogonal aminoacyl-tRNA synthetase and attached to an orthogonal amber suppressor tRNA, which is decoded by the ribosome in response to an amber codon (TAG) introduced into the gene of interest, allowing the synthesis of a protein with a site-specifically introduced ncAA (Lang & Chin, 2014; Figure 8).

Amber codon suppression can be challenging to implement and its efficiency varies unpredictably with sequence context, cell type, specific ncAA or translation rates (Meineke et al., 2023; Bartoschek et al., 2021). Therefore we chose to engineer our TAG amber codon only at the N-terminus of RebA or RebB monomers. Thus, if the ncAA was not successfully incorporated we would obtain no R body product, and the mere presence of purified R bodies signifies successful ncAA incorporation. Validating ncAA incorporation at the C-terminus poses a far greater problem that we did not attempt due to time constraints. The same overlap extension PCR technique with Gibson assembly as in Cycle 2.1 was used to create our constructs, hereon named TAG-RebA and TAG-RebB.

Once the ncAA is incorporated into our protein, our team needed to apply an orthogonal chemical conjugation strategy to a fluorescent marker. We chanced upon a paper by Chin et al. (2021) which described incorporation of p-azido-L-phenylalanine (AzF) in E. coli. This ncAA is compatible with copper-catalysed azide-alkyne cycloaddition (CuAAC), a widespread click chemistry reaction that occurs under biologically-relevant conditions, produces a stable product and is orthogonal. The process was reported to be efficient in several contexts and the reagents were inexpensive.

We therefore cotransformed BL21 E. coli with TAG-RebA or TAG-RebB and pEVOL AzF (the plasmid encoding the AzF-tRNA synthetase/tRNACUA pair) and cultured in medium with AzF. Purified R bodies were conjugated with sulfo-Cy5 alkyne dye.

#### Test and Learn

R body purification yielded appreciable pellets which implied amber codon suppression had been successful. However, subsequent CuAAC conjugation with dye was unsuccessful. As we were confident in ncAA incorporation, we troubleshooted the CuAAC reaction by adding a water-soluble ligand, THPTA, which coordinates Cu(I) and accelerates the reaction by preventing Cu(I) oxidation into Cu(II). This afforded excellent results (Figure 9). Demonstrating that R bodies are amenable to ncAA incorporation adds powerful site-specific optionality to our conjugation chassis. We report observing TAG-RebA and TAG-RebB extension under powerful brightfield microscopy but due to time constraints are unable to present DIC images in this wiki.

![](https://static.igem.wiki/teams/5121/engineering/eng-fig-8.png)

**Figure 8. Schematic of AzF incorporation via amber codon suppression with subsequent coupling to sulfo-Cy5 dye through CuAAC reaction.** 5-ethynylpicolinaldehyde conjugation and the CuAAC click reaction of sulfo-Cy5 onto alkyne-functionalised R bodies is shown.

![](https://static.igem.wiki/teams/5121/engineering/tag-conj-photo.webp)

**Figure 9. TAG-RebA (left) and TAG-RebB (right) pellets conjugated with sulfo-Cy5 alkyne dye.** The pellets have been washed thrice with water. A control pellet where the CuAAC reaction was conducted without copper catalyst is inset for comparison.

#### Next Cycle

Once again, as no biochemical tests exist to quantify R body yields or concentrations we could not quantify the efficiency of conjugation. This will be an important consideration in future applications. Attempting AzF incorporation at the C-terminus of RebA or RebB is difficult to validate, but if R bodies can be at least partially dissociated into monomer peptides, MS/MS could confirm the presence of AzF at the correct position. However, now that we have confirmed that amber codon suppression is successful at the N-terminus, we could verify AzF incorporation at the C-terminus by simply reacting a fluorophore like sulfo-Cy5 alkyne to verify its presence. Lastly, experiments with different ncAAs would help with expanding the R body conjugation repertoire.

---

### Cycle 2.3

![](https://static.igem.wiki/teams/5121/engineering/design.webp)

##### Design and Build

Selective modification at the N-terminus with various 2-pyridinecarboxyaldehydes (2-PCA) would introduce orthogonal functional groups. As this reaction’s only requirement is exposed N termini, 2-PCA can in theory be utilised with any R body construct. We tested its application in wildtype R bodies with an alkyne 2-PCA derivative and a sulfo-Cy5 azide dye.

![](https://static.igem.wiki/teams/5121/engineering/test.webp)

##### Test and Learn

Conjugation was unsuccessful on the first attempt and optimised by changing reaction temperature in a second cycle.

![](https://static.igem.wiki/teams/5121/engineering/next.webp)

##### Next Cycle

In future cycles we would continue characterising this strategy. The most pressing question is verifying that 2-PCA conjugation indeed is compatible with all other R body constructs. We could also exchange the order of reaction steps to enable robust chemical analysis of intermediate products.

#### Design and Build

Modification of native proteins by 2-pyridinecarboxaldehyde (2-PCA) derivatives affords excellent site specificity, mild reaction conditions and no inherent requirement for genetic engineering (MacDonald et al., 2015). This imine condensation reaction occurs specifically at peptide N-termini, yields a stable junction (imidazolidinine) and shows no cross-reactivity with lysine ɛ-amino groups (Figure 10). Moreover, the reaction is largely independent of the N-terminus residue identity, meaning 2-PCA conjugation should be compatible with our N-terminus engineered constructs. In this cycle we therefore aimed to simply replicate the 2-PCA reaction on wildtype R bodies using an alkyne derivative, 5-ethynylpicolinadehyde, reported by Bridge et al. (2021) to enable CuAAC-mediated fluorescent tagging. We used sulfo-Cy5 azide dye because its deep blue colour creates a simple visual output to verify conjugation success.

Although this is a two-step process (5-ethynylpicolinadehyde conjugation, then CuAAC tagging) best monitored by tandem MS, R bodies are incompatible with mass spectrometer as they cannot be decomposed into monomer peptides (and the extreme conditions required would likely create undesirable chemical modifications). Therefore our iterative design cycle relied on empirically testing our product and careful observation. Our optimisation and success with CuAAC in Cycle 2.2 gave us confidence that poor results could be attributed to the 5-ethynylpicolinadehyde reaction step, and that is where we would focus our troubleshooting.

Alternatively, we could have performed the CuAAC-mediated 2-PCA reaction with sulfo-Cy5 azide first, identified the product via NMR or MS, then proceeded to conjugation with R bodies. However we opted against this approach as not only would the sulfo-Cy5 azide group reduce the water solubility of 5-ethynylpicolinaldehyde, but we were also concerned that the resulting product from such a direction would yield a 2-PCA derivative with decreased reactivity, owing to the different electronic properties of the CuAAC triazole product (as opposed to the alkyne) which could be undesirably less electron-withdrawing upon the carbonyl carbon. Future cycles would determine whether these concerns hold ground and help understand the effect of reaction order on conjugation efficiency.

![](https://static.igem.wiki/teams/5121/engineering/eng-fig10.png)

**Figure 10. Complete reaction scheme of the 2-PCA approach.**
5-ethynylpicolinaldehyde conjugation and the CuAAC click reaction of sulfo-Cy5 onto alkyne-functionalised R bodies is shown.

#### Test and Learn

As always, our testing phase aimed to verify extension post-conjugation and observe color change associated conjugation. Our first conjugation was unsuccessful, but after reviewing optimisations reported by Bridge et al. (2021) we concluded that the reaction was temperature sensitive and retried at higher temperatures. This time both our negative control and experimental pellets were blue (Figure 11A). We hypothesized that the sulfo-Cy5 azide dye might associate strongly and non-specifically with R bodies (e.g. due to π-π stacking) and washed thoroughly with 20% ethanol to better solubilise the dye. This yielded confirmation that we had conjugated wildtype R bodies with 2-PCA chemistry (Figure 11B). Once again we report observing 2-PCA conjugated R bodies to extend under powerful brightfield microscopy but due to time constraints are unable to present DIC images in this wiki.

![](https://static.igem.wiki/teams/5121/engineering/eng-fig11.png)

**Figure 11. Attempts at 2-PCA conjugation with sulfo-Cy5 azide dye.**
A) Control 2-PCA conjugation attempt where no Cu catalyst was added (left) compared to experimental 2-PCA conjugation attempt (right). Both pellets have been washed thrice with water but are identically blue, although sulfo-Cy5 azide should not be conjugated to the control pellet. B) Control pellet (left) compared to experimental pellet (right) after three 20% ethanol washes. The control pellet is white whereas the experimental pellet is blue.

#### Next Cycle

To better characterise this strategy changing the order of reactions would allow us to assess intermediate products by NMR or MS. As always, once a method for biochemical analysis of R body concentrations/yields is developed, characterising conjugation yields will be necessary. The purpose of the 2-PCA approach was to enable compatibility with all other N-terminal modifications (potentially enabling two orthogonal strategies to be applied at one site), so evaluating compatibility with, for instance, TAG-RebA or TAG-RebB, would be valuable in future cycles.

---

### Cycle 2.4

![](https://static.igem.wiki/teams/5121/engineering/design.webp)

##### Design and Build

Thiol-maleimide conjugation is a popular, robust conjugation strategy specific to exposed cysteine residues. We engineered N-terminus and C-terminus cysteines attached by a flexible linker to RebA or RebB.

![](https://static.igem.wiki/teams/5121/engineering/test.webp)

##### Test and Learn

Plasmids for N RebA and C RebA encountered issues with transformation. N RebB was successfully conjugated to sulfo-Cy5 maleimide dye but was not observed to extend. C RebB could not be conjugated to sulfo-Cy5 maleimide dye.

![](https://static.igem.wiki/teams/5121/engineering/next.webp)

##### Next Cycle

An unusual pellet appearance leads us to believe that Cys-N RebB does not due to a purification error. We need to replicate the purification protocol to be certain whether purification may have damaged the sample.

#### Design and Build

Maleimide–thiol conjugation is one of the most commonly used methods for bioconjugation, and offers a very fast reaction to quantitatively conjugate proteins under mild conditions (Merten et al. 2019). The reaction employs a thiolate from a surface-exposed cysteine residue to undergo addition to the double bond of a maleimide, forming a thiosuccinimide or succinimidyl thioether junction (Figure 12A). Current FDA-approved ADCs Brentuximab and Trastuzumab employ a thiol-maleimide adduct for its site-specificity and junction stability (Fontaine et al., 2014). Reb A and Reb B monomers contain no cysteine residues: this allows our team to define conjugation sites by engineering cysteine residues onto the N-terminus or C-terminus of Reb A or Reb B (hereon named N RebA, C RebA, N RebB and C RebB). Since surface-exposure of the thiol group is essential, we once again appended the cysteine residue to a flexible GGGGS linker. Our payload was fluorescent sulfo-Cy5 maleimide dye.

#### Test and Learn

Despite being the most established approach, thiol-maleimide was the most difficult conjugation strategy for our team to perfect. Due to difficulties with transformations, work with N RebA and C RebA was suspended. The thiol-maleimide reaction was successful on N RebB but not C RebB (Figure 12B). Presently, our best theory for why this may be suggests that the density of exposed cysteine residues in close proximity favours disulfide bridge formation, though the success of N-terminus conjugation and presence of TCEP in reaction buffers renders this explanation unlikely. Moreover, while C RebB was observed to readily extend in acid, N RebB did not (Figure 12C and 12D), although an unusual pellet appearance leads us to believe this may be a result of a purification error. This result does demonstrate that there are differences in chemical reactivity or accessibility to different parts of the R body monomer. Prior to us demonstrating this difference in chemical functionality, only Cai (2023) had provided some deuterium exchange data which vaguely suggested there may be accessibility differences between monomer termini. Our finding recapitulates the importance of our site-specific conjugation design. If the site of conjugation has significant influence on chemical functionality, one could envision opportunities such as modulating accessibility to the chemical environment of the endosome and altering cargo cleavage kinetics through rational choice of conjugation site.

![](https://static.igem.wiki/teams/5121/engineering/eng-fig12.png)

**Figure 12. Overview of the thiol-maleimide conjugation strategy.**
A) Mechanism of the thiol-maleimide reaction. A thiol, oxidised to thiolate by a base or nucleophile, undergoes Michael addition onto a maleimide moiety. Reproduced from Wu et al. (2019). B) C RebB (left) and N RebB (right) pellets after thiol-maleimide reaction with sulfo-Cy5 maleimide. Both pellets were washed thrice with water. C RebB is distinctly white and unconjugated, while N RebB displays a deep blue hue caused by sulfo-Cy5.

#### Next Cycle

The cause of Cys-N RebB's failure to extend needs to be determined. The first priority would be to replicate purification alongside a milder protocol and determine whether a purification error or harsh conditions are responsible for extension failure. If not, we would assume that the N-terminus linker impedes structural transition, which could be validated by testing whether other N-terminus constructs with identical linkers, like TAG-RebA or TAG-RebB, can extend.

---

## Cleavable Linkers & In Vitro Application

Incredibly, we had been able to troubleshoot and successfully apply every conjugation strategy to an unusually large, insoluble protein. Our team now wanted to perform in vitro testing to 1) characterise the tendency of cells to endocytose R bodies, and 2) characterise the efficiency of endosomal rupture and drug release by R bodies.

---

### Cycle 3.1

![](https://static.igem.wiki/teams/5121/engineering/design.webp)

##### Design and Build

Internalisation of R bodies was investigated using fluorescent cargo molecules mNeonGreen and aldoxorubicin.

![](https://static.igem.wiki/teams/5121/engineering/test.webp)

##### Test and Learn

We observed that R bodies are non-cytotoxic, favourably but heterogeneously endocytosed, and accumulate in dose-dependent fashion. The favourable endocytosis of such an enormous protein is a surprising discovery.

![](https://static.igem.wiki/teams/5121/engineering/next.webp)

##### Next Cycle

The ultimate test of our product is its ability to enhance endosomal escape for membrane-impermeant cargo. Unfortunately we ran out of time to complete this characterisation and it would be our highest priority in the next DBTL cycle.

#### Design and Build

To characterise endocytotic efficiency we chose to use RebA-LPETGGG-mNeonGreen and RebB-LPETGGG-mNeonGreen constructs. mNeonGreen retains its fluorescence in the acidic endosomal environment (Steiert et al., 2018) and is attached to the R body via a non-cleavable linker. This would allow us to track the number of R bodies endocytosed. For cell culture we used EXPI293 cells in suspension, which are derived from a neoplastic human embryonic kidney (HEK) cell line.
We made this choice after our [interview with Dr. Kristina Cook](https://2024.igem.wiki/sydney-australia/human-practices#dr-kristina-cook), who suggested that delivery of R bodies to leaky tumour vasculature/interstitial fluid via trans-arterial chemoembolisation (TACE) would be an optimal implementation strategy. Dr. Cook advised that it could limit premature drug release into systemic circulation, minimising patient side effects, and would work synergistically with our stable platform by allowing them to stay longer in the vicinity of the tumour before (presumably) eventual breakdown. A suspension EXPI293 culture allows us to most closely mimic the delivery of R bodies to tumour cells in interstitial fluid.

Drug release from the R body delivery platform requires cleavable linkers. The cargo must be released prior to R body extension in the endosomal milieu, since endosomal contents will be rapidly diluted after membrane rupture. We considered two approaches: acid-labile groups and protease-labile sequences (such as for cathepsin B). Acid-labile groups, such as hydrazones or oximes, must exhibit favorable kinetics for most cargo molecules to be released prior to endosome rupture, and might suffer from stability issues in vivo. Protease-labile sequences, however, might suffer from incomplete cleavage if membrane rupture proceeds prior to endolysosomal fusion, and cleavage will likely vary significantly between cell types in vivo.

We ultimately chose to characterise our delivery platform with aldoxorubicin as a proof-of-concept. Aldoxorubicin is a prodrug of doxorubicin with an acid-labile hydrazone group and maleimide-functionalised linker. In vivo it is bound to an exposed cysteine on circulating serum albumin and concentrated in neoplasms via the enhanced permeability and retention (EPR) effect. The acidic microenvironment of the tumour facilitates hydrazone hydrolysis and doxorubicin release into the tumour interstitial fluid (Cranmer, 2019; Mita et al., 2014). We selected aldoxorubicin due clinical precedent (it has undergone Phase I clinical trials) and immediate compatibility with our thiol-maleimide conjugation strategy. Another practical advantage of aldoxorubicin is that it fluoresces at around 600nm. We hoped to demonstrate that internalising aldoxorubicin-conjugated R bodies into the acidic endosomal environment achieves targeted intracellular cleavage, and that the cytotoxic effect is enhanced by endosomal rupture. Unfortunately, as Cycle 3.1 was undertaken in parallel with Cycle 2.4 we learned too late that Cys-N RebB did not extend at low pH, meaning our design did not examine the effect of endosomal rupture. We present our results here to only demonstrate that R bodies effectively enable aldoxorubicin entry into endosomes and presumably its hydrolysis therein. Subsequent tests with R bodies that have their extension function will characterise the contribution of endosomal escape to targeted cytotoxicity.

#### Test and Learn

In order to assess endocytic efficiency we calculated the average number of RebA-LPETGGG-mNeonGreen bodies per cell at different dilutions and incubation times. The mean number of R bodies per cell scaled with dilution factor and averaged 4 R bodies per cell (when a 2 mL pellet was diluted 1:8 times). However, the mean number of R bodies yields a misleading impression: as seen in Figure 13 the number of R bodies per cell follows a bimodal distribution. At high concentration (1:8 dilution), a majority of cells internalised 5-10 R bodies and the remaining proportion internalised 0-1 R bodies (see Figure 14D). The cause and therapeutic implications of this heterogeneity merit further investigation. Notably, RebA-LPETGGG-mNeonGreen constructs did not exert cytotoxic effects (Figure 14D) and up to 20 R bodies were observed accumulating inside cells in a vesicular pattern without notable deleterious effect (the cells continued doubling every 24h, they appear morphologically healthy, and absence of trypan blue staining indicates the cell membrane is intact). Thus we conclude that our drug delivery system is intrinsically non-cytotoxic, matching reports by Pond et al. (1989), and that its cytotoxic activity can be modulated by its cargo. Further, its average intracellular accumulation is ostensibly dose-dependent, which is highly promising for therapeutic application. We also believe the ability of R bodies to facilitate internalisation of membrane-impermeant molecules is highly compatible with the development of large macromolecules such as cyclic peptides into therapeutic options. Cyclic peptides, such as those developed using the RaPID platform, have picomolar binding affinities to target proteins but are limited by cellular impermeability.

![](https://static.igem.wiki/teams/5121/engineering/eng-fig13.png)

**Figure 13. Endocytic efficiency of RebA-LPETGGG-mNeonGreen constructs.**
A 2 mL pellet of RebA-LPETGGG-mNeonGreen was resuspended and plated at 1:8 and 1:16 dilution into suspension EXPI293 cell culture. The cultures were incubated for 24 h or 48 h. Aliquots of cell culture were collected, mixed with tryphan blue at 1:1 ratio and five images were taken of random areas using a Zeiss Axio Imager Z1. The number of R bodies per cell was counted and averaged across all images.

Aldoxorubicin conjugated onto Cys-N RebB (aldoxorubicin-Cys-N RebB) produced similar data. It is important to keep in mind that Cys-N RebB does not extend under pH 5, and we verified that aldoxorubicin-Cys-N RebB too did not extend. After 24 h of cell incubation we observed a vesicular distribution of brightly fluorescent particles which we assumed to be R bodies, and a diffuse cytoplasmic gradient (Figure 14A and 14B). It is likely that the R bodies localised to the endosome, and as the endosome matures and acidifies aldoxorubicin is cleaved off and diffuses through the vesicular membrane into the bulk cytoplasm. The relatively high permeability of cell membranes to aldoxorubicin is well known (Yousefpour et al., 2019). This might be why we observed a relatively lower number of R bodies in living cells and a high density of aldoxorubicin in apoptotic cells (Figure 14A and 14C). Such an interpretation already highlights the efficient ability of R bodies to deliver intracellular cargo. However, we cannot rule out that the source of aldoxorubicin observed within cells is slow cleavage from R bodies present in solution (i.e. diffusion through the plasma membrane from the extracellular environment).

Overall, this experiment yields little concrete data due to suboptimal design. It confirms the ability of R bodies to deliver aldoxorubicin into cells, and suggests that they assist intracellular accumulation. However the endosome-rupturing function has not been examined and no cytotoxicity measurements can be made given the starting concentration of R bodies cannot be quantified.

![](https://static.igem.wiki/teams/5121/engineering/eng-fig14.png)

**Figure 14. Endocytosis of aldoxorubicin-N’ RebB conjugates (A-C) and RebA-LPETGGG-mNeonGreen (D) in EXPI293 cells.**
A-C) DIC microscopy, fluorescence at 567 nm, and both modalities overlaid, respectively, of EXPI293 cells incubated with aldoxorubicin-N’ RebB. The R bodies were sterilised with 100% ethanol post-conjugation and then added to EXPI293 cell suspension culture. The cells were incubated at 37°C for 24h. Tryphan blue was added in 1:1 ratio to an aliquot of the cell culture to quench extracellular fluorescence. Images produced on Zeiss Axio Imager Z1. White squares in A and C indicate cells with particularly high intracellular concentrations of aldoxorubicin, likely those that have internalised the most R bodies (or internalised them earliest). A distinctly vesicular pattern (yellow square in B) overlaid with extensive cytoplasmic diffusion in most live cells is observed. The white arrow indicates a clump of contracted aldoxorubicin-Cys-N RebB bodies. D) EXPI293 cells were incubated with RebA-LPETGGG-mNeonGreen. The R bodies were sterilised with 100% ethanol post-conjugation and then added to EXPI293 cell suspension culture. The cells were incubated at 37°C for 24h.

Incidentally, as we were forced to sterilise the R bodies with 100% ethanol to prevent cell culture contamination, we confirmed that RebA-LPETGGG-mNeonGreen is stable in 100% ethanol and preserves its extension function (see Figure 8). Ethanol typically induces protein aggregation and unfolding by weakening non-local hydrophobic forces and enhancing local polar interactions, resulting in destabilisation of the native hydrophobic core and increased formation of local secondary structures (Singh et al., 2010). Our conjugation handles are engineered at exposed surface microenvironments that are most likely not involved in protein folding (indeed, most of our designs rely on exposed, flexible, terminal glycine-rich linkers averse to forming secondary structures that will expose functional groups like thiols or AzF to the chemical environment). The stability of RebA-LPETGGG-mNeonGreen is very likely a function of intrinsic R body stability, which has been reported elsewhere (Pond et al., 1989). We are confident that future investigation will reveal that all conjugated Reb constructs are stable in 100% ethanol. The ability to sterilise a protein with 100% ethanol without structural damage should enable more efficient purification, sterilisation for therapeutic application and a greater variety of compatible chemical reactions. It is a novel and promising discovery for a therapeutically targeted protein construct.

#### Next Cycle

This round of characterisation convincingly demonstrates that R bodies are non-cytotoxic and efficiently deliver cargo into endosomes. The next cycle of in vitro testing must characterise the endosomal escape function using a cleavable linker. For instance, engineering a cathepsin B cleavage sequence into RebA-LPETGGG-mNeonGreen would allow us to test for diffuse fluorescence in the bulk cytoplasm. As mNeonGreen does not cross membranes this experiment would serve as a proof of concept that R bodies liberate cargo from endosomal entrapment. We could also try attaching a fluorescent cyclic peptide onto a TAG-RebA/B construct. Cyclic peptides are emerging therapeutic options for a variety of diseases but are not membrane-permeant. They could easily incorporate chemical modifications like a hydrazone junction or fluorophore group, which would enable us to evaluate the benefit our system brings (in temrs of endosomal escape) to real therapeutic options.

---

## Conclusion

Our team has almost brought a novel intracellular delivery system to experimental completion. We developed a novel purification method that can be used with any R body construct, leveraging the innate stability of the R body platform.
We engineered [9 new basic parts and 8 new composite parts](https://2024.igem.wiki/sydney-australia/parts) which are modular and orthogonal and can be easily interchanged to customise our delivery platform. We characterised each composite part by validating conjugation on every R body construct, bar one. We have demonstrated that our product is intrinsically non-cytotoxic and effectively endocytosed at dose-dependent rates. Only one step remains: proving our system enables membrane-impermeant molecules to cross into the cytoplasm. Our platform offers a promising solution to the challenge of endosomal entrapment that has plagued the therapeutics field since its inception.

## Engineering Philosophy

Below, we have documented the philosophical principles that guided our engineering success. We also intend for this to be a resource for future iGEM teams, as an example of how they can apply engineering philosophy to their own work! Learn more on our [Contribution](https://2024.igem.wiki/sydney-australia/contribution) page.

Unfortunately this browser doesn't have a PDF plugin. [See our Engineering Philosophy here instead!](https://static.igem.wiki/teams/5121/philosophy-final.pdf)

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Zhong, Y., Moghaddas Sani, H., Paudel, B. P., Low, J. K. K., Silva, A. P. G., Mueller, S., Deshpande, C., Panjikar, S., Reid, X. J., Bedward, M. J., Van Oijen, A. M., & Mackay, J. P. (2022). The role of auxiliary domains in modulating CHD4 activity suggests mechanistic commonality between enzyme families. *Nature Communications,* 13(1), 7524. https://doi.org/10.1038/s41467-022-35002-0

{% endblock %}


{% extends "base.html" %}
{% block title %}Results{% endblock %}
{% block lead %}On this page you will find the results of every experiment we conducted during our iGEM journey.{% endblock %}
{% block page\_content %}

## Menu

[Summary](#summary)
[Expression & Purification](#Purification)
[Standard expressions](#standard-expressions)
[R body purification](#r-body-purification)
[R body behaviour](#r-body-behaviour)
[Positive controls for amber codon suppression](#positive-controls)
[Genetic Assembly](#genetic-assembly)
[Overlap extension PCR](#overlap)
[Assembly challenges](#assembly-challenges)
[Extension testing](#extension-testing)
[Bioconjugation](#Bioconjugation)
[Thiol-maleimide conjugation](#thiol)
[Copper-catalysed azide-alkyne cycloaddition (CuAAC) conjugation](#copper)
[2-pyridinecarboxyaldehyde (2-PCA) conjugation](#2pca)
[Sortase A conjugation](#sortase)
[*In Vitro* Application](#in-vitro)

## Summary

## R Body Chemotherapeutic Delivery Chassis

Our novel intracellular delivery system is almost entirely experimentally validated. The key results include:

- A **novel purification method** compatible with any R body construct
- **12 new parts** and **8 new composite** parts which are modular and can be easily interchanged to customise the delivery platform
- Characterisation of the new parts and composite parts (see [Parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D#parts-page))
- 4 orthogonal conjugation strategies all validated on their respective R body body constructs
- Demonstration that our product is **intrinsically non-cytotoxic** and **effectively endocytosed** at dose-dependent rates

On this page you will find the results of the experiments we have conducted this year and our interpretation of the data. This page will overlap with the Engineering Success page, the latter presenting a refined summary of our design-build-test-learn cycles and key positive results. The Engineering Success page provides the (iterative) rationale behind our experimental design. Meanwhile this Results page provides additional information about several preliminary optimisation experiments, further detail on purification and cloning, and other miscellaneous data.

---

## Expression & Purification

### Standard expressions

Our team was provided stocks of DH5α containing Reb1 and Reb206 plasmids created by Polka et al. (2016). We amplified the plasmids, transformed into BL21 E. coli and used mNeonGreen fluorescence per cell as a proxy measure of R body expression (Figure 1), with Reb1 serving as a control. It should be noted however that without data on relative rates of fusion versus wildtype RebB monomer expression, rates of monomer incorporation into assembled R bodies, or rates of successful R body assembly given monomer expression, drawing correlation between monomer expression (fluorescence data) and R body yield is only approximate.

![](https://static.igem.wiki/teams/5121/results/results-fig1-final.jpg)

**Figure 1. Fluorescence and growth measurements during R body expression.** A) mNeonGreen fluorescence per cell was used as a proxy for R body expression. Four constructs were expressed in either LB or TPM medium, following which an aliquot was taken and mNeonGreen fluorescence as a function of cell density was measured. Non-fluorescent wildtype Reb1 was used as a negative control. Statistical analysis by one-way ANOVA with Tukey multiple comparisons test, error bars are ±SD, \*\*\*\* p<0.00005, \*\*p<0.005, no significance. B) OD600 measurements of bacterial cultures across different media and constructs. The dashed line indicates the cutoff value of OD600 = 0.4 where cultures were induced with IPTG. C-F) Representative bacterial pellets harvested from each construct. The intense red colour observed in Reb206 and YHReb206 is caused by mCherry gene (refer to Engineering Success for plasmid maps). The Reb1 pellet is a tan colour typical of bacterial pellets.

We observed that codon optimisation did not improve R body yield although it appeared to accelerate the rate of bacterial growth. Enriched media more than doubled R body yield. Expression in TPM and codon-optimisation of the 8 composite parts created in this iGEM project is therefore an important consideration for future entrepreneurship work, particularly with respect to scaling-up operations.

---

### R body purification

Our next set of experiments aimed to replicate the R body purification protocol described in Polka et al. (2016) for a fluorescent R body construct where the RebB monomer is fused to mNeonGreen, named Reb206. The protocol is unconventional as it aims to solubilise everything but the R bodies, the inverse of typical FPLC procedure. It is a harsh protocol that exploits the insolubility and density of R bodies to separate them from cellular components by lysis, enzymatic cleanup, centrifugation and repeated washing with detergent. We could not successfully replicate said protocol for Reb206. We found that all reported protocols use variations of the same key steps, and therefore began optimising these steps until sufficient purity was achieved (Lalucat, 1988; Schrallhammer et al., 2012). We experimented for six months with combinations of longer incubation lengths, multiple enzyme incubations, different wash detergents and concentrations, centrifugation speeds and other parameters. Although we ultimately achieved high purity, we could not observe Reb206 extension in acid. We therefore gained access to a more advanced microscope and repeated purification on wildtype Reb1, which was imaged with DIC and phase contrast microscopy. This finally yielded positive results.

![](https://static.igem.wiki/teams/5121/results/results-fig2.png)

**Figure 2. Effects of different treatments on R body sample purity.** A) Pellet produced by exactly replicating the purification protocol from Polka et al. (2016). The pellet is stringy, hairy, compact and impossible to resuspend. Inset shows the desired consistency and colour of a pure R body pellet. B-C) Introduction of a second lysozyme and DNase incubation step. The sample is clearly contaminated as individual R bodies cannot be distinguished from the refractive mass enveloping them. However the pellet (not shown) can at least be resuspended and is far less stringy or hairy compared to the pellet in panel A. Image produced by 63x phase contrast microscopy on a Zeiss Axio Vert.A1 Fl LED. D-E) The effect of a second lysozyme and DNase incubation step when combined with 1 %(v/v) Triton-X100 washes produces a clear improvement in purity. Panel D demonstrates an R body pellet after one round of overnight lysozyme and DNase incubation followed by MQW and 1% Triton-X100 washes. The pellet is off-white and does not settle well even after centrifugation. Panel E demonstrates a clean, white, settled pellet. F-G) Extending R bodies in pH 3 acetic acid demonstrating a moderate level of contamination. This sample was washed with 1 %(v/v) SDS instead of Triton-X100 and displays areas of high refractive index that are suggestive of contamination. Nevertheless individual R bodies can be resolve and the typical clumping behaviour is observed. H) A clean R body sample prepared via our optimised purification protocol. Key features of this protocol are two rounds of enzymatic cleanup, long overnight incubations and Triton-X100 washes.

These results primarily demonstrate the effect of an iterative experimental process, where successful strategies were preserved and unsuccessful ones discarded. The impact of these strategies were synergistic: for instance, we believe Triton-X100 solubilised more contaminants than SDS and therefore a second enzymatic incubation step proved doubly effective when the optimal detergent was used. It might be that Triton-X100, as a non-ionic detergent with greater micelle diameter, interacts favourably with amphipathic or hydrophobic contaminants than anionic SDS (Chae et al., 2013). Our team is also surprised we were unable to replicate the Polka et al. (2016) protocol. One contributing factor may be the different cell lines used for protein expression: Polka et al. used C43 whereas our team used BL21 cells, which might have altered the identity or quantity of contaminants and necessitated harsher purification.

---

### Purified R body behaviour

Apart from the expected pH-dependent contraction-extension behaviour (see Parts or Figure 2F and 2G), we also observed the time-dependent clumping of R bodies in purified samples. This finding reinforces that vortexing samples may permit smoother sample transfer, and reinforces good microscopy technique. Otherwise we present it as a characteristic behaviour of pure R body samples that we have not yet seen described in the literature.

![](https://static.igem.wiki/teams/5121/results/results-fig3.png)

**Figure 3. Time-dependent clumping of R body samples.** A-C) A purified Reb1 sample clumping over the course of 5 minutes. The refractive white patches in panel C represent end-stage clumps. mage produced by 63x phase contrast microscopy on a Zeiss Axio Vert.A1 Fl LED. D-F) A replicate observation identical to panels A-C.

---

### Positive controls for amber codon suppression

Later in the project, our team expressed and purified most new Reb constructs (Cys-N RebB, RebA-LPETG, etc.) in an identical manner to Reb1 and Reb206 (see above). The TAG-RebA and TAG-RebB constructs for amber codon suppression were the only exceptions. Given our limited experience with amber codon suppression we conducted a positive control using E. coli cotransformed with pEVOL\_AzF and pCDF RFP amber plasmid. The former encodes the p-azido-L-phenylalanylyl-tRNA synthetase and tRNACUA for the amber codon. The latter encodes a modified RFP where the first amino acid is p-azido-L-phenylalanine (AzF). Inspired by Ma et al. (2014) we qualitatively tested whether RFP expression was enhanced by the addition of organic solvents, which helps overcome resistance to AzF uptake through the cell membrane (Figure 4).

![](https://static.igem.wiki/teams/5121/results/results-fig4.png)

**Figure 4. Pellets of RFP-expressing E. coli in amber codon suppression optimisation experiment.** Induction conditions were always 0.2 mM AzF, 0.2 mM IPTG, 0.2% arabinose and an organic solvent. A) E. coli induced with 5% DMSO. B) E. coli induced 5% glycerol. C) E. coli induced with 10% DMSO. D) E. coli induced with 10% glycerol. E) E. coli induced without organic solvent, that is, AzF only. The photo has poor colour representation due to its low light levels, but the pellet is equally pink to panels A-C. F) Control E. coli that were induced in the absence of AzF. The pellet lacks pink colour.

We observed little difference with or without the presence of organic solvent. This may be due to the qualitative nature of our analysis and due to the small culture volumes (2 mL), as Ma et al. (2014) report significant effects on protein yield. Optimising amber codon suppression using organic solvents could be of interest in scaling-up operations, but presently we were hesitant to introduce an untested compound into R body expression medium. We therefore chose to use AzF alone.

The very purification of sizeable pellets from TAG-RebB and TAG-RebA expression cultures suggests that amber codon suppression was achieved, given that the amber codon was positioned close to the N-terminus of RebA or RebB. This alone cannot definitely claim success amino acid misincorporation may produce false positives. However the success of CuAAC conjugation implies that AzF had been incorporated into RebA or RebB monomers. Amber codon suppression can be a challenging technique to implement. Although powerful and easy to execute, its implementation in the lab is commonly hampered by unpredictable variations in incorporation efficiencies at different amber codon positions. Flanking sequences, cell lines, ribosomal fidelity and even permeability of the non-canonical amino acid (ncAA) all influence rates of misincorporation or translation termination. Bartoschek et al. (2021) have developed a predictive tool to identify permissive amber codon sites, which we could use in the future to identify which contextual factors enabled our success with ncAA incorporation.

---

## Genetic Assembly

### Overlap extension PCR

As mentioned in our Engineering Success section, overlap extension PCR was used to create fragments for Gibson assembly. As shown in Figure 4, an insertion sequence can be encoded at any site using two pairs of primers, in each of which there is a primer with an overhang. Standard Gibson assembly can then ligate the short and long fragments (sometimes known as insert and backbone fragments). The small size of the Reb plasmids (~8000 base pairs) was crucial for this bipartite cloning strategy.

![](https://static.igem.wiki/teams/5121/results/results-fig5.png)

**Figure 5. Sample overlap extension PCR strategy.** This example demonstrates how primers with overhangs can be used to insert a sequence at the C-terminus of RebA. A simplified Reb1 template plasmid is shown, with RebA, RebB and kanR genes. Two pairs of primers (green and orange) are designed. In each pair, one primer pairs with a region along the vector backbone (we chose a non-coding region outside kanR) and the other contains an overhang (light green) encoding the insertion sequence (light green) such that the junction between the overhang and primer body is exactly aligned to the C-terminus of RebA. Two separate PCR reactions are run to generate the short fragment and the long fragment, each now terminating with identical backbone sequence (red) or insertion sequence. These two fragments can be Gibson assembled and sequenced to confirm correct assembly.

All our primer designs are tabulated in the [Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D#protocols-page) page and all successfully produced PCR products, which were subsequently Gibson assembled. The Gibson products were transformed, amplified and then sequenced to confirm correct assembly (example in Figure 5). All our Gibson sequencing data is publicly available at this [link](https://benchling.com/s/seq-18pLNNnAGMRQL5zBG6hF?m=slm-gLpIXxDnpw7hcx07ozNT).

![](https://static.igem.wiki/teams/5121/results/results-fig6.png)

**Figure 6. Sample alignment in Benchling of Gibson assembled RebA-LPETG plasmid isolated from three plate colonies.** Alignment was performed against the wildtype Reb1 plasmid. The red highlight demarcates the insert sequence.

In the following drop-down menu we provide details of our entire PCR process for all Cys constructs (Cys-N RebA, Cys-C RebB, etc.), all LPETG constructs (RebA-LPETG, RebB-LPETG) and GGG-mNeonGreen. The PCR gels for TAG-RebA and TAG-RebB are not shown.

## PCR Gel Results

![](https://static.igem.wiki/teams/5121/results/results-fig7-round2.png)

**Figure 7. Our first gel run of PCR amplified fragments for Sortase and Cysteine constructs.** The samples were: (1) Short RebA-LPETG fragment, (2) Long RebA-LPETG fragment (for Sortase A conjugation), (3) Short RebB-LPETG fragment, (4) Long RebB-LPETG fragment, (5) Short Cys-N RebA fragment (for cysteine-maleimide conjugation), (6) and a Long Cys-N RebA fragment. We amplified the three short fragments in one thermocycler, and the long fragments in the other thermocycler.

We found that the shorter fragments had amplified poorly in the old thermocycler, and the longer fragments, amplified in a newer thermocycler, had amplified sufficiently well. The fragments all appeared to be close to the expected size by looking at the 1kb ladder, indicating that at least our fragments had been produced successfully. Suspecting that the PCR conditions for the short fragments was less-than-ideal, we tested a range of Tm temperatures and DMSO conditions in the next PCR round, as well as amplifying and producing the remaining cysteine-ready longer reb constructs.

![](https://static.igem.wiki/teams/5121/results/results-fig8-round-2.png)

**Figure 8. A large testing array for optimising PCR conditions for amplifying short r\RebA and B fragments with CGGGGS and LPETG linkers.**The fragments amplified and run in the gel included: The samples, with corresponding codes in the table, were Cys-N RebA short (A1-6), Cys-C RebA short (B1-6) and long (BL), RebA-LPETG short (C1-6), Cys-N RebB short (D1-6) and long (DL), RebB-LPETG short (E1-6), and Cys-C RebB short (F1-6) and long (FL). Cys-N RebA long was not amplified again because the previous gel showed that it amplified sufficiently. Each short fragment was amplified. Each short fragment was amplified in six different conditions — numbers 1-3 indicate samples amplified at Tms of 53°C, 58°C, and 63°C at 5% DMSO, and 4-6 indicate samples amplified with the same Tms range, at 10% DMSO.

We switched thermocyclers, and found that the short fragments mostly amplified well in the newer thermocycler, with most conditions being quite viable. The long samples, amplified in the older thermocycler but in the same conditions as previous (Tm=58°C, no DMSO) came out poorly — thus, the thermocycler was most likely the culprit in the faulty PCR. We then amplified the remaining long fragments in the new thermocycler in the same conditions.

![](https://static.igem.wiki/teams/5121/results/results-fig9-round2.png)

**Figure 9. PCR of the long fragments that failed to amplify in the previous round.**The lanes are labeled with the same labeling scheme as in the previous figure — BL is Cys-C RebA long, DL is Cys-N RebB long, and FL is Cys-C RebB long. Samples with a were amplified in the newer thermocycler, and samples labeled with b were amplified in the older thermocycler. Sample DLa was improperly pipetted into the fourth well, so it was added in the furthest well instead.

Viewing the amplification results above, both thermocyclers worked similarly well, with all the samples appearing quite clearly on the gel. Thus, it remains unclear what caused the previous amplification failures in the previous gel runs — one possibility is that the older thermocycler was more complicated to configure, and it was then incorrectly programmed for amplification the first two times. In any case, at this point, we had amplified samples of all the long and short fragments for both Sortase A conjugation and cysteine-maleimide conjugation. We pooled the amplified replicates of each sample and ran them on a gel for extraction.

![](https://static.igem.wiki/teams/5121/results/results-fig10-round2.png)

**Figure 10. Amplified samples of all cysteine-maleimide and sortase-ready DNA constructs for gel extraction.**Lane codes corresponds to the previous coding: Cys-N RebA short (A), Cys-C RebA short (B) and long (BL), RebA-LPETG short (C), Cys-N RebB short (D) and long (DL), RebB-LPETG short (E), and Cys-C RebB short (F) and long (FL).

All the samples ran well on the gel. We cut the bands out of the gel, and used a gel extraction kit to purify the DNA for downstream purposes.

We also modified mNeonGreen in a pCDFDuet-1\_His\_mNeon\_TmTP backbone. To make mNeonGreen compatible with sortase A-catalysed conjugation, an N-terminal triglycine motif was added, preceded by a TEV protease recognition sequence to cleave off the N-terminal methionine residue.

![](https://static.igem.wiki/teams/5121/results/results-fig11-round2.png)

**Figure 11. Generation of modified mNeonGreen for compatibility with sortase A conjugations.****A)** PCR of mNeonGreen coding region, with tri-glycine and TEV cleavage site introduced on primer tails. PCR was performed with six replicates, loaded in each lane of the agarose gel. **B)** PCR of mNeonGreen backbone with complementary regions to the insert amplicons. The designed product should be 3.6kb, corresponding to the topmost band. The presence of smaller bands indicates off-target amplification, necessitating gel-purification of the desired PCR product for downstream applications.

---

### Assembly challenges

Below we also report the difficulty of cloning and transforming certain constructs:

1. RebA-LPETG, RebB-LPETG and GGG-mNeonGreen for sortase A bioconjugation were assembled and transformed into DH5α and BL21 on the first attempt.
![](https://static.igem.wiki/teams/5121/results/results-fig7.png)

**Figure 12. Successful transformations into DH5α and BL21.** A) GGG-mNeonGreen Gibson assembly product transformed into DH5α E. coli. B) RebA-LPETG Gibson assembly product transformed into DH5α E. coli. Red circles highlight colonies. C) eSrtA plasmid (Addgene #75144) transformed into DH5α (right) and BL21 (left) E. coli.

2. Cys-C RebB and Cys-N RebB required troubleshooting with different short fragment to long fragment ratios and preparing fresh Gibson mastermix for assembly to work. This helped us obtain Cys-C RebB and Cys-N RebB transformants.
![](https://static.igem.wiki/teams/5121/results/results-fig8.png)

**Figure 13. Successful transformation of Cys-C RebB into DH5α.** The colonies are highlighted by red circles, but appear small and faint.

3. TAG-RebA and TAG-RebB constructs assembled correctly, which we confirmed by running products on a gel, but obstinately refused to transform into DH5α. Troubleshooting by varying the amount of DNA delivered to cells was not fruitful. Transformation was only successful after commercial One Shot™ TOP10 Chemically Competent E. coli from ThermoFisher were used.
![](https://static.igem.wiki/teams/5121/results/results-fig9.png)

**Figure 14. Attempts at transformation of TAG-RebB into DH5α and TOP10 E. coli for plasmid amplification.** A) Failed transformation of TAG-RebB into DH5α using 200 ng of plasmid DNA. B) Successful transformation of TAG-RebB into TOP10 commercial cells using 200 ng of plasmid DNA.

4. Cys-N RebA and Cys-C RebA could not transform into DH5α. We were limited in the supply of commercial competent cells, and given our success with Cys-N RebB and Cys-C RebB chose to save this genetic construct for future efforts.
5. Cotransformation of pEVOL\_AzF with any other plasmid (pCDF RFP amber, TAG-RebA or TAG-RebB) into BL21 proved impossible, regardless of variations in quantities and ratios of DNA. Commercial BL21(DE3) Competent Cells from ThermoFisher however were able to compensate for the inefficiency of the transformation process.

We therefore observe that some of our DNA constructs exhibit poor transformation efficiency or poor assembly, likely for reasons peculiar to each construct.

---

### Extension testing

A key question our team needed to answer is whether our N-terminus or C-terminus modifications influence the extension ability of R bodies. At present, we have confirmed that RebB-LPETG, RebA-LPETG and Cys-C RebB extend readily at pH 5 (Figure 8). Cys-N RebB has not been observed to extend, although an unusual pellet appearance (not shown) and low concentration of R bodies (Figure 8B) leads us to believe a purification error has occurred. We would replicate the protocol to be certain. Cai (2023) proposes a physical model whereby a coil-to-helix transition of the C-termini of R body monomers induces a tension vector change in the lattice structure and unrolls it. We believe our success with preserving extension ability in our C-terminus may therefore lie in the use of linkers that are averse to forming secondary structures (oligoglycine tract in GGGGSC for Cys-C RebB, or Pro in LPETG). This should be an important consideration for further expansion of the R body conjugation toolbox.

![](https://static.igem.wiki/teams/5121/results/results-fig10.png)

**Figure 15. Modified R bodies resuspended in pH 5 HCl-KCl buffer.** All pictures taken under 63x phase contraast with a Zeiss Axio Vert.A1 Fl LED microscope. A) Cys-C RebB samples extend prolifically at pH 5. B) Cys-N RebB has not been observed to extend at pH 5 HCl-KCl. C) RebB-LPETG R bodies extended in pH 5 HCl-KCl buffer. The clumpiness hides individual extended R bodies, but white arrows point out clear examples of individual extended R bodies. D) RebA-LPETG R bodies extended in pH 5 HCl-KCl buffer.

---

## Bioconjugation

### Thiol-maleimide conjugation

Thiol-maleimide conjugation was performed on Cys-C RebB and Cys-N RebB. We observed that Cys-C RebB repeatedly did not conjugate with sulfo-Cy5 maleimide, whereas Cys-N RebB did so readily. This result cannot be a product of chemical accessibility since C-terminus conjugation to mNeonGreen was successful, nor is disulfide bridge formation a likely culprit given that Cys-N RebB can be conjugated. Further investigation is necessary to understand the cause underlying failed conjugation. Other than sulfo-Cy5 maleimide, we successfully conjugated aldoxorubicin to Cys-N RebB for tests in cell culture.

Our team is not aware of any biochemical characterisation technique that can quantify R body concentration and therefore conjugation efficiency. This limits our conclusions to a qualitative answer; whether conjugation is possible or not. For practical applications, conjugation efficiency will need to be defined.

![](https://static.igem.wiki/teams/5121/results/thiol-malemide-conjugation-pellet.png)

**Figure 16. Thiol-maleimide conjugation of sulfo-Cy5 maleimide and aldoxorubicin.** A) Cys-C RebB (left) appears white after three MQW washes whereas Cys-N RebB (right) appears as a bright blue pellet. B) Cys-N RebB (left) compared to a Reb1 control (right) after conjugation. Both samples have been washed thrice with MQW. C) Aldoxorubicin conjugates to Cys-N RebB forming a bright red pellet.

---

### Copper-catalysed azide-alkyne cycloaddition (CuAAC) conjugation

Our first CuAAC reaction onto TAG-RebA and TAG-RebB was unsuccessful. As we were quite confident in ncAA incorporation, we troubleshooted the CuAAC reaction by adding a water-soluble ligand, THPTA, which coordinates Cu(I) and accelerates the reaction by preventing Cu(I) oxidation into Cu(II). We tried this approach as the original CuAAC paper (Rostovtsev et al. 2002) did not utilise a Cu-coordinating agent and reported good yields, and we believed that degassing solutions would sufficiently prevent Cu(I) oxidation. The addition of THPTA however drastically improved the efficiency of the reaction and enabled conjugation.

![](https://static.igem.wiki/teams/5121/results/cuaac-conjugation-pellet.png)

**Figure 17. CuAAC conjugation of sulfo-Cy5 alkyne to TAG-RebB (left) and TAG-RebA (right).** Both samples have been washed thrice with MQW. A control pellet where the CuAAC reaction was conducted without copper catalyst is inset for comparison.

---

### 2-pyridinecarboxyaldehyde (2-PCA) conjugation

Our first conjugation attempt was unsuccessful. After reviewing optimisations reported by Bridge et al. (2021) we concluded that the reaction was temperature sensitive and retried at higher temperatures (whereas the first attempt was conducted at 4°C). This time both our negative control and experimental pellets were blue (Figure 12A). We hypothesized that the sulfo-Cy5 azide dye might associate strongly and non-specifically with R bodies (e.g. due to π-π stacking) and washed thoroughly with 20% ethanol to better solubilise the dye. We discarded very blue supernatants and obtained the expected blue experimental and white control pellets. Given that we discovered, incidentally, that R bodies are stable in 100% ethanol we would recommend a 20% ethanol wash for all conjugation protocols and will undertake this in future cycles.

![](https://static.igem.wiki/teams/5121/results/results-fig18.png)

**Figure 18. Attempts at 2-PCA conjugation with sulfo-Cy5 azide dye.** A) Control 2-PCA conjugation attempt where no Cu catalyst was added (left) compared to experimental 2-PCA conjugation attempt (right). Both pellets have been washed thrice with water but are identically blue, although sulfo-Cy5 azide should not be conjugated to the control pellet. B) Control pellet (left) compared to experimental pellet (right) after three 20% ethanol washes. The control pellet is white whereas the experimental pellet is blue.

---

### Sortase A conjugation

Our modified GGG-mNeonGreen was expressed in BL21 cells. Refer to Protocols for GGG-mNeonGreen expression and GGG-mNeonGreen purification. Both were highly successful (Figure 20) as evidenced by the fluorescent yellow pellets and cell lysates.

![](https://static.igem.wiki/teams/5121/results/results-fig19.png)

**Figure 19. His-Tag Purification of GGG-mNeonGreen.** **A)** Pellets of cells expressing mNeonGreen (left) and wild-type R-bodies (right). **B)** Cell lysates bound to the Ni-NTA matrix during His-Tag purification. **C)** mNeonGreen samples eluted from the Ni-NTA column. The tube on the right contains mNeonGreen that was cleaved from the beads via overnight incubation with the TEV protease. This sample went on to further purification with size exclusion chromatography. The tube on the left was eluted with a high-imidazole concentration buffer. In this sample, mNeonGreen still has an N-terminal his-tag and TEV cleavage site.

![](https://static.igem.wiki/teams/5121/results/results-fig20.png)

**Figure 20. Size Exclusion Chromatography of GGG-mNeonGreen.** **A)** His-Tag-purified mNeonGreen entering the size-exclusion column. **B)** SDS-PAGE of SEC input and the fractions selected for mNeonGreen. Some contaminants are visible in the input column, which have been removed during the purification process. **C)** The fractions were compiled and concentrated into a GGG-mNeonGreen stock solution. This was used in subsequent sortase-A reactions.

Several variants of the sortase enzyme are commercially available. We selected a sortase pentamutant (Addgene #75144), which was engineered via directed evolution to have 140-fold enhancements in ligation activity (Chen et al., 2011). This sortase mutant is translationally fused to an N-terminal polyhistidine tag. This tag was used to perform Ni-NTA affinity chromatography on lysates of cells overexpressing the enzyme. Lysates were incubated with the resin for 3 hours in a binding buffer (50mM Tris pH 7.5, 300mM NaCl, 10mM imidazole). The unbound lysate was collected as a flow-through sample. The resin was washed 3 times with a washing buffer (50mM Tris pH 7.5, 300mM NaCl, 20mM imidazole). Finally, bound proteins were eluted multiple times using an elution buffer (50mM Tris pH 7.5, 300mM NaCl, 500mM imidazole). Aliquots were spared at each stage and run on an SDS-PAGE gel (Figure 8A). The eluted samples were combined and dialysed overnight with the buffer for size exclusion chromatography (25 mM tris pH 7.5, 150mM NaCl). Before size exclusion chromatography (SEC), the sample was concentrated to a volume less than 5mL, suitable for input into the SEC column. The absorbance at 280 nm for each 1mL SEC fraction was measured. Fractions with peaks at the expected size of sortase were collected and run in an SDS-PAGE gel to confirm the presence of sortase A (Figure 8B). SEC visibly removed contaminants enriched during the Ni-NTA chromatography. The pure sortase A samples were then combined and concentrated, forming our stock sortase solution for subsequent conjugation reactions.

![](https://static.igem.wiki/teams/5121/results/results-fig21.png)

**Figure 21. Purification of Sortase A pentamutant.** **A)** Samples collected from various stages of Ni-NTA affinity purification of the his-tagged sortase enzyme. The lysate soluble fraction was loaded into the columns. The sortase enzyme was clearly enriched from the cell lysate, along with other histidine-containing proteins. **B)** Sortase size-exclusion chromatography (SEC) samples. The eluted samples from Ni-NTA chromatography were concentrated and subsequently purified with SEC.

Coincidentally, as we were conducting our conjugation experiments, we discovered that a member of the Structural Biology Group (the group that supplied one of the lab spaces that we used to conduct our experiments) had also been working on protein conjugation using sortase A. As such, we were granted the opportunity to learn some more specific details about sortase A conjugation. In particular, we discovered that sortase A conjugation is a highly reversible reaction and that the most efficient method of pushing the equilibrium to completion is to incorporate an additional GHHHHHH motif following the LPETG sequence (Reed et al., 2020). This would result in cleavage of a 6xHis tag which could then be sequestered by the addition of free Ni2+ ions in solution. Removing one of the products in this manner would greatly inhibit the reverse sortase reaction, resulting in significantly improved conjugation efficiency.

Unfortunately, at this point, we had already designed and produced the LPETG-modified Reb1 constructs and did not have enough time to re-make our plasmids. Nevertheless, Sebastian provided us with a positive control substrate (a truncation of the protein CHD4 - spanning residues 1380-1810 - which possessed a C-terminal LPETG motif) for us to test our sortase enzyme sample with.

We performed a conjugation test using our purified eSrtA sample, GGG-mNeonGreen and Sebastian’s positive control substrate. The reaction was carried out with 10 µM sortase enzyme, 100 µM GGG-mNeonGreen and 100 µM LPETG-substrate, and allowed to incubate at room temperature for 1 hour before being moved to the cold room (4°C) overnight to prevent substrate degradation. The products were analysed by SDS-PAGE (Figure 23), which revealed successful conjugation - evidenced by the appearance of a higher molecular weight product at ~66 kDa, corresponding to the sum of the weights of mNeonGreen (~27 kDa) and the LPETG-substrate (38 kDa) - albeit with very low efficiency (likely < 5%). Despite the low efficiency, this still confirmed that both our sortase and mNeonGreen samples appeared functional for conjugation, giving us confidence to proceed with R body conjugation trials - although with the knowledge that we would have to add a large excess of substrate in an attempt to push the equilibrium forward.

![](https://static.igem.wiki/teams/5121/results/results-fig22.png)

**Figure 22. Sortase conjugation trial to test for functionality of our Sortase A and GGG-mNeonGreen samples.** Sortase A (10µM) was mixed with GGG-mNeonGreen (100 µM) and CHD4(1380-1810)-LPETG (100 µM) in 1x sortase conjugation buffer. The mixture was allowed to incubate at room temperature for 1 hour, before being transferred to 4°C overnight. The reagents and products were analysed by SDS-PAGE.
Sortase A (10µM) was mixed with GGG-mNeonGreen (100 µM) and CHD4(1380-1810)-LPETG (100 µM) in 1x sortase conjugation buffer. The mixture was allowed to incubate at room temperature for 1 hour, before being transferred to 4°C overnight. The reagents and products were analysed by SDS-PAGE.

The method for sortase A ligation onto LPETG-modified R bodies can be found on the Protocols page. In short, we calculated the number of LPETG binding sites using an approximate R body pellet mass and the molar mass of a monomer, and added more than 10-fold molar excess of GGG-mNeonGreen to drive the reaction to completion. This immediately yielded bright yellow pellets and fluorescent R bodies clearly distinguishable under a microscope (Figure 23).

![](https://static.igem.wiki/teams/5121/results/fig-23.png)

**Figure 23. Sortase A ligation of GGG-mNeonGreen with RebA-LPETG and RebB-LPETG.**
A) RebB-LPETG pellet (left) conjugated with GGG-mNeonGreen compared to control RebB-LPETG control pellet (right). No eSrtA was added to the conjugation reaction for the control pellet. Both pellets have been washed thrice with MQW. B) RebA-LPETG conjugated (left) compared to control RebA-LPETG (right). No eSrtA was added during conjugation of the control pellet. C) Fluorescent RebA-LPETG R bodies in MQW. The resolution is insufficient to distinguish individual R bodies but the distinctive clumping pattern of pure R bodies is evident.

Our success with sortase A conjugation is a novel result. It demonstrates successful aqueous-phase bioconjugation to a resuspended insoluble protein, which is typically hindered by limited solvent access to reactive sites and aggregation. R bodies are also one of the largest (if not the largest) natural protein assemblies on which sortase conjugation has been attempted to the extent of our knowledge. For example, Kim et al. (2015) describe “large” 40 kDa protein conjugation, whereas for comparison Cai’s (2023) physical model describes over 800,000 10 kDa monomers being packed into a single R body ribbon. We believe that demonstrating efficient conjugation at this scale demonstrates the feasibility of bioconjugation in a wider range of contexts than previously assumed.

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## *In Vitro* Application

In order to assess endocytic efficiency we collated a large number of images that demonstrate RebA-LPETGGG-mNeonGreen endocytosis into EXPI293 at different dilutions and incubation times. We pelleted 2 mL of resuspended R bodies (see protocol) and diluted it 1:8 and 1:16 then incubated for 24h or 48h in an EXPI293 cell suspension. The cells were visualised under a Zeiss Axio Imager Z1 in trypan blue, which quenches extracellular fluorescence (Figure 24).

![](https://static.igem.wiki/teams/5121/results/fig-24.png)

**Figure 24. Cells incubated with RebA-LPETGGG-mNeonGreen for 48 h.**
A) No trypan blue dye has been added which allows us to visualise extracellular mNeonGreen fluorescence. It is clear that certain cells (white arrows) preferentially endocytose R bodies and accumulate them at concentrations far higher than the surrounding environment. There are certain cells (red arrows) that do not endocytose R bodies. The distribution of R bodies among cells is not a normal distribution. Accumulating high quantities of R bodies apears to have no cytotoxic effect on cells. B) Trypan blue dye has been added which quenches extracellular fluorescence. R bodies are evidently distributed in a vesicular pattern and internalisation ranges from 0 to >10 R bodies per EXPI293 cell.

![](https://static.igem.wiki/teams/5121/results/fig-25.png)

**Figure 25. Endocytic efficiency of RebA-LPETGGG-mNeonGreen constructs.**
A 2 mL pellet of RebA-LPETGGG-mNeonGreen was resuspended and plated at 1:8 and 1:16 dilution into suspension EXPI293 cell culture. The cultures were incubated for 24 h or 48 h. Aliquots of cell culture were collected, mixed with trypan blue at 1:1 ratio and five images were taken of random areas using a Zeiss Axio Imager Z1. The number of R bodies per cell was counted and averaged across all images.

The mean **number of R bodies per cell scaled with dilution factor** averaged **4 R bodies per cell** (when a 2 mL pellet was diluted 1:8 times). However, the mean number of R bodies yields a misleading impression: as seen in Figure 14 the number of R bodies per cell follows a **bimodal** distribution. At high concentration (1:8 dilution), a majority of cells internalised 5-10 R bodies and the remaining proportion internalised 0-1 R bodies (see Figure 15D). The cause and therapeutic implications of this heterogeneity merit further investigation. Figure 24A demonstrates the **intrinsically non-cytotoxic** nature of our platform, matching reports by Pond et al. (1989). We conclude that the cytotoxic activity of R bodies should depend on what cargo it carries, mimicking their role as natural toxin delivery systems in *Paramecia spp.*. Further, the average intracellular accumulation is ostensibly dose-dependent, which is highly promising for therapeutic application. We also believe the potential of R bodies to facilitate internalisation of membrane-impermeant molecules is highly compatible with the development of large macromolecules such as cyclic peptides into therapeutic options.

We also tested an aldoxorubicin-Cys-N RebB conjugate *in vitro*. Our original intention was to use a membrane-impermeant cargo molecule attached to R bodies by an acid-labile or protease-cleavable linker. This would allow us to assess whether R bodies enable endosomal escape *in vitro* (as the endosome acidifies or matures into an endolysosome the cargo should be released after which the R body ruptures the endosomal membrane). However, as deadlines approach there were no candidate molecules that could ship to Australia in time. Aldoxorubicin was available, had a maleimide group and was fluorescent. Effectively, the aldoxorubicin experiment was a repetition of the RebA-LPETGGG-mNeonGreen experiment (Figure 26), the key difference being the presence of an acid-labile linker which would release aldoxorubicin from R bodies in the endosome.

![](https://static.igem.wiki/teams/5121/results/fig-26.png)

**Figure 26. Endocytosis of aldoxorubicin-Cys-N RebB conjugates in EXPI293 cells.**
A-C) DIC microscopy, fluorescence at 567 nm, and both modalities overlaid, respectively, of EXPI293 cells incubated with aldoxorubicin-Cys-N RebB. The R bodies were sterilised with 100% ethanol post-conjugation and then added to EXPI293 cell suspension culture. The cells were incubated at 37°C for 24h. Trypan blue was added in 1:1 ratio to an aliquot of the cell culture to quench extracellular fluorescence. A distinctly vesicular pattern (yellow square in B) overlaid with extensive cytoplasmic diffusion in most live cells is observed. The white arrow indicates a clump of contracted aldoxorubicin-N’ RebB bodies. D) An aliquot of aldoxoubicin-Cys-N RebB

We wanted to know whether the diffuse intracellular fluorescence observed in EXPI293 cells could be attributed to cleavage of aldoxorubicin at its acid-labile hydrazone linker. Therefore we imaged a resuspended sample of aldoxorubicin-Cys-N RebB R bodies that had been stored at room temperature for 24 h in MQW. We observed diffuse fluorescence throughout the solvent which suggested that premature cleavage was releasing aldoxorubicin into solution. This instability is a notorious feature of hydrazone groups (Yousefpour et al., 2019). Aldoxorubicin is highly membrane permeant. Therefore we could not conclude that endosomal release (and subsequent escape) of aldoxorubicin was responsible for the intracellular concentrations observed in EXPI293 cells. Ultimately, we could only conclude that R bodies could deliver aldoxorubicin into cell vesicles.

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Reed, S. A., Brzovic, D. A., Takasaki, S. S., Boyko, K. V., & Antos, J. M. 2020. Efficient sortase-mediated ligation using a common C-terminal fusion tag. Bioconjugate Chemistry. 31(5). https://doi.org/10.1021/acs.bioconjchem.0c00156.

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