Team:Marburg/Proof Of Concept

Proof of Concept

Introduction

Discovery, development, and approval of novel crop traits currently takes about 10 to 15 years, about 4 years of which are spent developing proofs of concepts and optimizing genetic constructs[1]. Huge factors contributing to these extensive development times are the inherent slow growth of plants, the immense time needed to regenerate after introduction of genetic constructs or transgenes and the inability to properly prototype genetic constructs in a high-throughput manner. In conjunction with the limited variety of genetic plant parts, more elaborate engineering projects are pretty much impossible at the current time.

When thinking about challenges like the implementation of whole metabolic pathways into plants like the nif (nitrogen fixation pathway) cluster from Klebsiella oxytoca[2], a total of 20 proteins need to be simultaneously expressed in a fine tuned manner[3, 4]. It is in this context that the need for precisely characterized regulatory sequences is becoming althemore clear.

Figure 1: nif cluster of Klebsiella oxytoca
The cluster includes 20 enzymes that need to be present in a very fine tuned balance in order for the cluster to show its full nitrogen fixation capabilities

As already mentioned in our Proposed Implementation, our goal for this year's project is the creation of functioning cell-free extracts from chloroplasts of various plant species and utilizing these to prototype much needed genetic parts for the field of chloroplast engineering. The experiments that were needed in order to present a valid proof of concept included the creation of functioning cell-free extracts from different species and the ability to prototype regulatory sequences in the aforementioned extracts.

Functioning CFE

During the course of our project we were able to create functioning extracts of Tobacco, Spinach and Wheat(see Figure 1). We were able to create extracts of Tobacco and Spinach extracts very early in our project, allowing us to optimize our methods and the reaction mixture used to enable translation in our extracts in order to reach higher levels of expression. In order to evaluate the viability of our extracts we iterated the design of our T7 Universal Test Construct over seven times in order to reach even higher expression levels. The process of how we achieved this can be found on the page Engineering Success.

Figure 2: Successful expression in cell-free extracts of Tobacco, Spinach and Wheat
Luminescence values are given as arbitrary units and the data is presented on a logarithmic scale. The reaction was set up with a total volume of 10µl. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively in order to verify the expression. The measurements were performed using our T7 Universal Test Construct 5.0

By utilizing our highly optimized cell-free extracts we were able to show that regulatory genetic elements can indeed be characterized using our approach. In order to benchmark our cell-free systems we cloned a subset of 11 different 5’UTR parts and 10 different 3’UTR elements including two dummy sequences as a negative control in the positions respectively into our lvl2 dropout vectors. The design of these vectors can be found in the Toolbox part of our Design Page.

The lvl2 measurement vectors include two cassettes: one of which is consistent in order to ratiometrically normalize the expression of the cassette with changing regulatory sequences. Here our consistent transcriptional unit is the Firefly luciferase by which we divide the expression strength of the NanoLuc luciferase with. This was done in order to achieve more accurate comparison of expression between different batches of chloroplast cell-free extracts. If you want to learn more about the normalization of our data, please visit our Measurement Page.

During this experiment we simultaneously wanted to find out if these parts can be used across species. We had this idea when first examining different chloroplast genomes as the regulatory sequences most of the time are very similar between species. This similarity is even more visible when comparing highly conserved regions like the regulatory sequences of the rbcL gene (large subunit of RuBisCo) or the psbA gene (Photosystem II protein D1 precursor). In accordance with our research the parts showed a similar expression pattern across the species border when comparing cell-free expression of Tobacco and Spinach.

Figure 3: Normalized ratio Nluc/Fluc comparison of 11 different 5’UTR sequences in cell-free extracts of Tobacco and Spinach
Luminescence values are given as arbitrary units and the data is presented on a logarithmic scale. The reaction was set up with a total volume of 10µl. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively in order to verify the expression. The measurements were performed using our lvl2 measurement vectors with the displayed parts in the 5’UTR position of the NanoLuc cassette
Figure 4: Normalized ratio Nluc/Fluc comparison of 10 different 3’UTR sequences in cell-free extracts of Tobacco and Spinach
Luminescence values are given as arbitrary units and the data is presented on a logarithmic scale. The reaction was set up with a total volume of 10µl. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively in order to verify the expression. The measurements were performed using our lvl2 measurement vectors with the displayed parts in the 3’UTR position of the NanoLuc cassette

From the part characterization experiment from Tobacco and Spinach we picked 3 of the best performing parts together with 4 endogenous sequences from Wheat and tested their expression in a cell-free system of Triticum aestivum(Figure 3). While some endogenous regulatory elements we built using purified Wheat DNA (psbA, atpB and psaC) did not show any activity at all. The rbcL version of the 5’UTR showed some activity. The best activities were detectable from the 3 parts we identified in the experiment prior.

Figure 5: NanoLuc expression of 7 constructs in cell-free extract of Wheat
Luminescence values are given as arbitrary units and the data is presented on a logarithmic scale. The reaction was set up with a total volume of 10µl. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively in order to verify the expression. The measurements were performed using our lvl2 measurement vectors with the displayed parts in the corresponding positions.

Endogenous Transcription

In all the experiments prior we used the T7 promoter in order to achieve more reliable and stable data. After our optimization of the extracts themselves, we decided to take a closer look at the endogenous transcription machinery, which is purified during our extraction method. For this we evaluated the viability of 5 different promoters in Spinach cell-free extract(Figure 4).

Figure 6: NanoLuc expression of 5 different endogenous promoters in cell-free extract of Spinach
Luminescence values are given as arbitrary units and the data is presented on a logarithmic scale. The reaction was set up with a total volume of 10µl. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively in order to verify the expression. The measurements were performed using our lvl1 measurement vectors differing only in the promoter sequence

We found out that interestingly the 16S promoter core region of the plastid encoded polymerase-promoter seems to function exceptionally well in the Spinach extract. The rbcL promoter region of Tobacco, followed by the long and short version of the 16S promoter also showed promising expression. The psbA promoter exhibited the lowest expression in the Spinach extract. These experiments were only possible due to multiple iteration rounds of optimizing our extraction methods and finding the optimal buffer composition.

Conclusion

During our project we were able to create cell-free systems from a diverse group of plant species including crops. We demonstrated here that our approach of prototyping chloroplast specific regulatory sequences using cell-free systems of chloroplast from diverse plant species is indeed working as intended. On top of this we were able to characterize a vast amount of genetic parts in cell-free extracts of Tobacco, Spinach and Wheat. During further experiments we were even able to break away from the T7 based transcription system and could show that our system can be effectively used to characterize endogenous promoters. This proof of concept is supposed to show the capabilities of our system in a way it can be reliably used in a way to optimize genetic construct designs before implementing them in vivo. We hope that our approach and our highly characterized parts can make a contribution to highly complex engineering projects, such as the implementation of nitrogen fixation into plant chloroplasts.

Sources

  1. Getting a biotech crop to market Phillips McDougall - Croplife International | Croplife ... (n.d.). Retrieved October 17, 2021, from https://croplife.org/wp-content/uploads/2014/04/Getting-a-Biotech-Crop-to-Market-Phillips-McDougall-Study.pdf.
  2. Li, Q., & Chen, S. (2020). Transfer of Nitrogen Fixation (nif) Genes to Non‐diazotrophic Hosts. In ChemBioChem (Vol. 21, Issue 12, pp. 1717–1722). Wiley. https://doi.org/10.1002/cbic.201900784
  3. Temme, K., Zhao, D., & Voigt, C. A. (2012). Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. In Proceedings of the National Academy of Sciences (Vol. 109, Issue 18, pp. 7085–7090). Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.1120788109
  4. Smanski, M. J., Bhatia, S., Zhao, D., Park, Y., B A Woodruff, L., Giannoukos, G., Ciulla, D., Busby, M., Calderon, J., Nicol, R., Gordon, D. B., Densmore, D., & Voigt, C. A. (2014). Functional optimization of gene clusters by combinatorial design and assembly. In Nature Biotechnology (Vol. 32, Issue 12, pp. 1241–1249). Springer Science and Business Media LLC. https://doi.org/10.1038/nbt.3063