Team:Marburg/Cell-Free/In Vivo

In Vivo - In Vitro comparison

Our cell-free chloroplast extract allows the comparison of the effect of genetic parts on protein expression levels without the need for prior transformation. This opens up the possibility of efficient and cheap high-throughput testing of different constructs and even the potential for multiplexing. However, results of this in vitro part analysis are only of value, if the produced results are comparable with the effect of the respective part in vivo. This comparison was demonstrated previously for bacteria and plants. Moore et al. showed comparable behaviour of most parts in E. coli, especially promoter strengths, which demonstrated a clear link in expression data [1]. Strong promoters and terminators in vivo were generally strong in vitro as well. Exceptions were the ribosome binding sites, which quickly became saturated in vitro when used with a strong promoter. For terminators, comparability could only be demonstrated for strong candidates. Schaumberg et al. demonstrated similar results in the comparison of cell-free nuclear-transformed A. thaliana extracts, where reliable predictions for functional synthetic plant parts could be shown [2].

Figure 1: Tobacco plants for the cell-free extracts growing in our greenhouse.

Based on these findings, we aimed for similar levels of comparability of our extract and transformed plants. To confirm this hypothesis, and thus compare the results we gained during testing with our extract in vitro, we set out to transform Tobacco chloroplasts. Four different constructs were transformed into plants, which are equivalent to constructs used in our cell-free extract expression analysis. The transformed plants will be tested in the same way as our extracts, so their expression in vivo can be compared. While the experimental differences between in vitro and in vivo measurements prohibit quantitative comparison, weak and strong signals may be distinguished and a qualitative comparison can be performed. Thus, an effect of the analysed part on protein production may be estimated and candidates affecting expression in the desired way can be chosen. As such, the data generated using the chloroplast extract can be used to greatly reduce the workload of comparing different constructs, as only the top construct candidates need to be cloned into the plant and tested in vivo.

Figure 2: A Tobacco plant growing sterile in a jar for transformation.

Construct Design

As the integration site for the vector used to transform our chloroplasts, trnI/trnA was selected. This site was chosen as it is right behind the end of the 16S rRNA, which is highly transcribed in the chloroplast genome. At the end of the 16S rRNA sequence, no termination occurs. As a consequence, the selected site has the highest readthrough in the chloroplast genome [4, 5]. The choice of this site harbours the highest chance of receiving viable transformants on the first try. It also includes an annotated chromosomal origin of replication (oriC) of the chloroplast genome. The main drawback of this design is that promoter characterisation is difficult due to its limited effect on gene transcription since basal transcription levels are so high.

Other integration sites we considered were trnG/trnfM and trnV/rps12, but these were not favoured since the trnI/trnA site offered highest chances of successful selection. For future experiments, these other sites offer benefits like genetic isolation needed for exact expression measurement.

The utilized aadA selection cassette confers resistance to Spectinomycin and Streptomycin [6]. Fused to the cassette is the gene encoding green fluorescent protein (GFP). Measurement of protein expression is performed with the NanoLuc luciferase bioluminescence platform, just as with the cell-free extract measurements. For normalization, we utilized a cassette encoding the Firefly luciferase (FLuc). This cassette is identical in every measurement and thus the signal strength may be compared, to a degree, between different measurements. The NLuc system was chosen for its enhanced stability, smaller size, and >150-fold increase in luminescence, compared to FLuc and Renilla luciferase (RLuc). The limitations of this system include non-ideal emission for in vivo applications and its unique substrate furimazine [7], however as this system was primarily designed for in vitro testing this is a bearable drawback.

Most of the parts we used to build our constructs were already described to be functional in vivo, although some had to be individually tested by us in order to assess their viability. The parts tested in literature for the normalisation cassette are regulatory elements of the rbcL gene, which is reported to work in Tobacco plastids [8], the 5’ UTR of E. coli phage T7 gene 10, which has been successfully integrated in Tobacco in the past [9], and the 3’ UTR of rrnB, which was tested for E. coli [10] and chloroplasts of Tobacco and Chlamydomonas reinhardtii [11]. For the measurement cassette, the tested 3’ UTR of the rbcl gene was used [12]. Four different 5’ UTRs, which had all been tested in tobacco chloroplasts, were integrated into the construct yielding four different constructs, which we transformed. They were named rps2, psbC, clpP, and psbB ( [13], [14], [15]).

Figure 3: The construct used for our biolistic transformations.
Left to right: The aadA resistance cassette is fused to GFP to enable both antibiotic selection, via Spectinomycin and Streptomycin, and confirmation of transplastomic plants via fluorescence microscopy. Two terminators from the Marburg collection [3] are used as connectors, their function is the reduction of readthrough, which is needed because of the high expression caused by the 16S rRNA, which sits at the 5’-end of the cassette. As a normalisation cassette, Fluc is utilized, while Nluc is used for the measurement.

Transforming the Plants

Generating a chloroplast-transformed plant is anything but a trivial task. A typical leaf mesophyll cell contains 100 chloroplasts and over 1000 plastid genome copies [19]. They need to be altered in a gradual process, which inevitably results in chimeric callus tissue, requiring careful selection over a long time period [6]. This is the reason chloroplast transformation efficiency is among the lowest reported in literature. Typical transformation efficiencies are about one transformant per 50 biolistic bombardments in Tobacco [15], 100 biolistic bombardments in tomato [15] and Arabidopsis [16], or even zero transformants in 3860 biolistic bombardments for Wheat [17].

Figure 4: Depiction of the Tobacco chloroplast transformation process. Assembly of the DNA macrocarrier for leaf bombardment. On the right side, the utilized gene gun is visible.

Chloroplast DNA was first discovered in 1963 [18]. 25 years later, the first transformation was reported. The logical choice in species was the unicellular green algae Chlamydomonas reinhardtii, as the organism only contains a single chloroplast and circa 80 plastid genome copies [19]. The first Tobacco chloroplast transformation was reported five years later in 1993 [20], but after this progress in the field slowed down. While Arabidopsis was successfully transformed another five years later, all regenerated plants were sterile [21] and it took until 2019 for a protocol yielding fertile transplastomic plants to be published [22]. In total, generation of fertile transplastomic plants was achieved for 11 other species and is still anything but trivial [19], [23]).

After surveying relevant literature, we chose to transform our plants, not because it is easy, but because it is hard. Numerous challenges had to be overcome to allow us to receive transformants in the limited timeframe of our iGEM project, as multiple steps had to work on the first attempt. This started with the procedure to generate sterile plant tissue culture. Seeds and media had to be sterilized with chloride to remove contamination, which lowered germination rate considerably. The medium needed to be prepared very carefully regarding the pH, as plant growth would be inhibited by non-solidified media caused by too low pH, but the agar concentration had to be low to keep the medium soft enough for rooting to easily occur. Plant transformation required a specific stage of plant growth to ensure optimal efficiency. For transformation, we used the most popular protocol for Tobacco, biolistic bombardment. This method utilises a gene gun loaded with gold particles bound to DNA, which are then shot into Tobacco leaves. The gold particles enter chloroplasts and thus the DNA is delivered. Vectors utilizing two targeting sequences flanking the inserted genes are used, so the transformation happens via homologous recombination [5]. In total, we bombarded 40 plates with one leaf each.

Figure 5: Tobacco plants growing in sterile jars for biolistic bombardment.
Figure 6: Workflow for the chloroplast transformation. Wild-type plants are grown in a sterile environment. Leaves are cut off and transformed via biolistic bombardment. The gene cassette containing homologous flanks is integrated into the chloroplast genome via homologous recombination. The integration of the cassette results in heteroplasmy, as every mesophyll cell contains a large number of chloroplasts. The potentially transplastomic leaf is cut into pieces and plated on selection medium. In a gradual process under selection pressure, wild-type chloroplasts are replaced by transformed ones. Calli are transferred to fresh plates to keep selection pressure high. Once calli are large enough, imaging and genotyping via PCR and sequencing is possible. Over time, continued selection results in homoplasmy and shoot formation is induced. From this shoot, a whole homoplasmic plant is regenerated.
Figure 7: Green transplastomic callus. The first green callus we received from our transformation was sufficiently large enough to be split for selection and further propagation.

The leaves containing potential transplastomic cells were cut into six pieces each, placed on a regeneration medium containing spectinomycin and left to form calli, which appear over the next 4-12 weeks. Transplastomic lines are identified by the ability to form green calli on the bleached wild-type leaf sections [6]. The combination of the two antibiotics spectinomycin and streptomycin, which are needed to rule out spontaneous antibiotic resistance to spectinomycin through point mutation, inhibits plant growth. Due to this, calli had to be cut into two pieces and separated onto plates containing either only one or both antibiotics, for selection and further growth. From our 40 plates, one callus exhibiting green colour on selection media could be regenerated for now. Another challenge we faced at this point was continuous sterility of plant tissue culture. Fungi readily grow on the utilized RMOP medium and prevention requires careful sterilisation of work equipment, sterile media pouring and working in a sterile environment (laminar flow hood). With these precautions, contaminations could be limited to about one in ten cultures.

Imaging of Transformed Tissue

To elucidate whether transformed fluorescent chloroplasts expressing GFP were present in the callus, imaging was performed. For this, a piece of the callus was taken during separation for selection and further propagation. Cross-sections were prepared and imaged with a fluorescence microscope. We could demonstrate the presence of fluorescent chloroplasts, confirming the first successful chloroplast transformation of an iGEM team ever! As expected, heteroplasmy was observed at this stage. Through further selection pressure over the next months, the fraction of transformed chloroplasts will rise until homoplasmy is achieved.

Figure 8: Imaging of transplastomic callus tissue. Top left: Brightfield, top right: GFP, bottom left: chloroplasts autofluorescence, bottom right: Overlay. Co-localisation is identifiable, suggesting that the fluorescence is indeed produced by chloroplasts. This is supported by the size of bright dots, which is expectedly about 1-2 µm.

Successful NanoLuc Expression - Proof of Concept

After the successful imaging of callus tissue, we performed a proof of concept measurement via the cell-free extract protocol using a piece of the callus instead of a punched piece of leaf tissue. Even with the heteroplasmic callus, it was possible to measure a strong signal. This confirms that the measurement protocol works in transformed plants and that our plants are able to produce a signal, thus we were able to successfully validate our cell-free measurement method! Once a homoplasmic plant will be regenerated, we will repeat this measurement. Another green callus is currently growing on selection medium, once it is large enough we will repeat this measurement to see whether the effect of different UTRs is comparable between cell-free and in vivo measurements. We also plan to do another round of chloroplast transformations in the future to generate additional mutants and thus confirm the comparability of multiple different parts. To learn how much time and effort it took to get a transplastomic Tobacco plant, read our in vivo diary here.

Figure 9: Proof of concept measurement of the successfully transformed chloroplast tissue. Left: Measurement of transformed calli (triplicates), right: wild-type control utilizing an untransformed leaf piece. Mean value utilizing a transformed callus was circa 580 times higher than control, yielding evidence of successful transformation and successful protein expression measurement.

To our Tobacco Transformation Gallery:

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