What to improve?

In order to achieve the eventual goal of a simple and usable glowing plant certain problems must be addressed. Some of the current barriers to achieving this goal are as follows:

Introduction of the entire LUX operon construct into the plant
The low levels of light produced by the bacterial luciferase
Effects of light production on the growth of the induced plant
Cost and ease of transfection and modifications
Regulation and efficiency of gene expression in plants

Whilst we as a team could not address all of these issues we hope to build a framework in which, by testing various parts of gene constructs and bioluminescent systems, we can quantify and show the best parts available for someone to make a functional glowing plant. In order to achieve this we decided to take advantage of the allowance of type IIS parts and build a golden gate system for transfection of the bacterial luciferase into plant models. Multiple type IIS parts were designed with the below final goals:

Transfection and determination of an luciferase component of the LUX operon (LUXAB) showing improved light output.
Comparative testing and quantifying a new constitutive promoter, the Sugarcane bacilliform virus (ScBv), which shows the advantage of high activity in both monocots and dicots.
Testing and quantifying a new constitutive promoter, the Sugarcane bacilliform virus (ScBv), which shows the advantage of high activity in both monocots and dicots.
Comparative testing of the fluorescent protein Ypet vs GFP
Quantifying the LOV linked riboswitch's location in the 5'UTR effects expression efficiency of GFP.
Testing the effectiveness of a duel construct including riboswitch & the photoreceptive Light-Oxygen-Voltage (LOV) protein, acting as a light sensitive on-off switch for transcription.

Further ideas were to mutate the iLUX protein further using error-prone PCR in order to achieve a product with a more optimal pH and to insert the bacterial Flavin Mononucleotide and Aldehyde genes (LUXCDE) as a single unit using P2A protein separation.

Part Design and approach

Before we could begin with lab work the genes needed to be designed and the method of transfection selected. After much discussion the use of the Ti-plasmid and the plasmid backbones produced by Weber et al. [1] was decided on.

As the IIS system was to be employed any parts to be used required both the addition of BpiI cut sites, which upon digestion and ligation into the level 1 backbone would become BsaI cut sites, but also the removal of any BpiI, BsaI and SapI within the sequence of the part. For the coding parts such as GFP, Ypet, the LOV protein and the riboswitch parts this was not a problem as base replacement within the same codon was possible. However, for other parts such as the promoter care must be taken in order not to disrupt the important functional regions. For the promoter CamV, these changes and the associated testing has already been completed by Engler et al. [2] and as such this sequence was used. The sequence for promoter ScBV was adapted from the work by San‑Ji et al. [3], including shortening to only what they described as the promoting regions. Removing the restriction site was then aided by Peremarti et al.[4] which describes the regions of most importance within promoters. The 5’UTRs and terminators selected were free of the mentioned restrictions sites. The AT1G58420 5’UTR was selected from the paper by Kim et al. [5] as it showed the highest translation efficiency when tested in Nicotiana benthamiana. Four 5’UTRs were designed using this template 3 including the LOV associated riboswitch at different positions (upstream, midstream and downstream) and one base 5’UTR. Three luxAB coding regions were produced with the intention of testing each for efficiency against each other. The two primary luciferase CDS components were the eLUXAB construct produced by Cui et al. [6] and the iLUX construct of Gregor et al. [7]. The eLUXAB construct uses a flexible glycine-serine GS linker to link the two main luciferase subunits together whereas the iLUX was shown to have a sevenfold increase in brightness when expressed in E. coli. . These two modified versions of the bacterial luciferase were then combined changing the specific AA that were thought to have produced this increased fluorescence in iLUX within the sequence of eLUX, producing ieLUX. Furthermore by using the article by Szymczak-Workman et al. [8] a single iLUXAB construct which could be expressed in eukaryotes was produced. All coding regions were also codon optimised for use in Nicotiana benthamiana. The sequence for the riboswitch and LOV protein was kindly provided by Prof. Dr. Günter Mayer laboratory group and was only adapted to include the required restriction sites. Throughout this process the friendly and generous people at Doulix provided support and finally synthesised the parts.

References

[1] - Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. 10.1371/journal.pone.0016765 PubMed 21364738 [2] - Engler et al. - Engler C, Youles M, Gruetzner R, Ehnert TM, Werner S, Jones JD, Patron NJ, Marillonnet S. “A golden gate modular cloning toolbox for plants” ACS Synth Biol. 2014 Nov 21;3(11):839-43. doi: 10.1021/sb4001504. Epub 2014 Feb 20. 10.1021/sb4001504 PubMed 24933124 [3] - Gao, San-Ji, et al. "A novel Sugarcane bacilliform virus promoter confers gene expression preferentially in the vascular bundle and storage parenchyma of the sugarcane culm." Biotechnology for biofuels 10.1 (2017): 172. [4] - Peremarti, A., Twyman, R.M., Gómez-Galera, S. et al. Plant Mol Biol (2010) 73: 363. https://doi.org/10.1007/s11103-010-9628-1 [5] - Kim et al. - Kim, Younghyun, et al. "The immediate upstream region of the 5′-UTR from the AUG start codon has a pronounced effect on the translational efficiency in Arabidopsis thaliana." Nucleic acids research 42.1 (2013): 485-498. [6] - Cui, Boyu, et al. "Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts." PloS one 9.10 (2014): e107885. [7] - Gregor, Carola, et al. "Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging." Proceedings of the National Academy of Sciences 115.5 (2018): 962-967. [8] - Szymczak-Workman et al. Szymczak-Workman, Andrea L., Kate M. Vignali, and Dario AA Vignali. "Design and construction of 2A peptide-linked multicistronic vectors." Cold Spring Harbor Protocols 2012.2 (2012): pdb-ip067876.