Individual Final Project

Shining Proof: Light-Activated Electrophysiological Verification of Plant Transfection'

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Current plant transfection tools

There are well-developed protocols for plant genome editing (CRISPR/Cas9) and post-transcriptional gene silencing, as well as for animal and bacterial systems. However, my interest is in finding the way to deliver genetic engineering components into the mature plant cells to be able to alter their genome or gene expression. Coincidentally, it is challenging in general. The delivery method in this case should also cause minimal tissue damage, not opening the doors to fungal infection, and, simultaneously, overcome ordinary and lignified cell walls.

The most extensively used plant transfection methods are the following:

  1. Agrobacterium tumefaciens vector, which is host specific, induces tissue regeneration response and creates biosafety concerns;
  2. Virus vector, which is host specific and potentially vertically inherited that might be undesirable for some purposes and also creates biosafety concerns;
  3. Biolistics aka gene gun aka particle bombardment — requires expensive equipment, induces tissue regeneration response;
  4. Electroporation, which is possible with access to immature tissues and protoplast.

Nanoparticles look prominent at this background [Liu et al., 2025]. The size also matters here (1—100 nm) and it could be achived using different materials, combining smallness with other properties, e.g. magnetic. This particular project includes peptide-based nanoparticles as a plasmid DNA carier.

Also, transfection requires some confirmation system. Working with more or less developed plant and trying to prolong its life, I lean toward dual-confirmation system, where one or both methods are possible in vivo.

The project aims

  1. Develop peptide-based nanoparticle systems for efficient delivery of genetic constructs into mature plant tissues

  2. Establish a modular dual-validation system co-expressing a GFP reporter and Channelrhodopsin-2 (both are excited by 470 nm light)

  3. Enabling confirmation of transfection via both optical (fluorescence) and functional (light-induced electrophysiological) readouts

Peptide-based nanoparticles (PBNP) composition and advantages

The main components of PBNP are:

  1. Cell-penetrating peptides (CPP) – naturally occured peptides (up to 30 AA) capable of tranferring membrane-impermeable cargo into the cytosol or even organelles [Patel et al., 2025];
  2. Cationic DNA-condensing domain, which mediates PBNP-DNA complex reversible self-assembling and improves the complex stability and cellular uptake;
  3. Endosomal escape domain, which enhances anti-enzymatic protection;
  4. (Optional) Flexible glycine linker
  5. (Optional) Opganelle targeting peptide, e.g. mitochondrion

PBNPs are biocompatible, elegant, and self-assebling. Their modular structure also corresponds the logic of plant organization, so that’s the match.

My PBNP design includes:

  1. BP100 as CPP
  2. Polyarginine domain
  3. Histidine tail
  4. Glycine linkers

The whole sequence:

RRRRRRRRR-GG-KKLFKKILKYL-GG-HHHH

Plasmid DNA

I chose pCAMBIA1304 vector as it already has strong plant CaMV 35S promoter and GFP-GUS reporter system. Although the plasmid is relatively big, it is compatible with nanoparticle technologies. Besides, A. tumefaciens-associated regions could be deleted*.

For my verification systems, I substituted GUS sequence with Channelrhodopsin 2 (ChR2). First, I found ChR2 protein sequence

MDHPVARSLIGSSYTNLNNGSIVIPSDACFCMKWLKSKGSPVALKMANALQWAAFALSVIILIYYAYATWRTTCGWEEVYVCCVELTKVVIEFFHEFDEPGMLYLANGNRVLWLRYGEWLLTCPVILIHLSNLTGLKDDYNKRTMRLLVSDVGTIVWGATAAMSTGYIKVIFFLLGCMYGANTFFHAAKVYIESYHTVPKGLCRQLVRAMAWLFFVSWGMFPVLFLLGPEGFGHLSVYGSTIGHTIIDLLSKNCWGLLGHFLRLKIHEHILLYGDIRKVQKIRVAGEELEVETLMTEEAPDTVKKSTAQYANRESFLTMRDKLKEKGFEVRASLDNSGIDAVINHNNNYNNALANAAAAVGKPGMELSKLDHVAANAAGMGGIADHVATTSGAISPGRVILAVPDISMVDYFREQFAQLPVQYEVVPALGADNAVQLVVQAAGLGGCDFVLLHPEFLRDKSSTSLPARLRSIGQRVAAFGWSPVGPVRDLIESAGLDGWLEGPSFGLGISLPNLASLVLRMQHARKMAAMLGGMGGMLGSNLMSGSGGVGLMGAGSPGGGGGAMGVGMTGMGMVGTNAMGRGAVGNSVANASMGGGSAGMGMGMMGMVGAGVGGQQQMGANGMGPTSFQLGSNPLYNTAPSPLSSQPGGDASAAAAAAAAAAATGAASNSMNAMQAGGSVRNSGILAGGLGSMMGPPGAPAAPTAAATAAPAVTMGAPGGGGAAASEAEMLQQLMAEINRLKSELGE

then, used reversed translation tool

ATGGATCATCCGGTGGCGCGCAGCCTGATTGGCAGCAGCTATACCAACCTGAACAACGGCAGCATTGTGATTCCGAGCGATGCGTGCTTTTGCATGAAATGGCTGAAAAGCAAAGGCAGCCCGGTGGCGCTGAAAATGGCGAACGCGCTGCAGTGGGCGGCGTTTGCGCTGAGCGTGATTATTCTGATTTATTATGCGTATGCGACCTGGCGCACCACCTGCGGCTGGGAAGAAGTGTATGTGTGCTGCGTGGAACTGACCAAAGTGGTGATTGAATTTTTTCATGAATTTGATGAACCGGGCATGCTGTATCTGGCGAACGGCAACCGCGTGCTGTGGCTGCGCTATGGCGAATGGCTGCTGACCTGCCCGGTGATTCTGATTCATCTGAGCAACCTGACCGGCCTGAAAGATGATTATAACAAACGCACCATGCGCCTGCTGGTGAGCGATGTGGGCACCATTGTGTGGGGCGCGACCGCGGCGATGAGCACCGGCTATATTAAAGTGATTTTTTTTCTGCTGGGCTGCATGTATGGCGCGAACACCTTTTTTCATGCGGCGAAAGTGTATATTGAAAGCTATCATACCGTGCCGAAAGGCCTGTGCCGCCAGCTGGTGCGCGCGATGGCGTGGCTGTTTTTTGTGAGCTGGGGCATGTTTCCGGTGCTGTTTCTGCTGGGCCCGGAAGGCTTTGGCCATCTGAGCGTGTATGGCAGCACCATTGGCCATACCATTATTGATCTGCTGAGCAAAAACTGCTGGGGCCTGCTGGGCCATTTTCTGCGCCTGAAAATTCATGAACATATTCTGCTGTATGGCGATATTCGCAAAGTGCAGAAAATTCGCGTGGCGGGCGAAGAACTGGAAGTGGAAACCCTGATGACCGAAGAAGCGCCGGATACCGTGAAAAAAAGCACCGCGCAGTATGCGAACCGCGAAAGCTTTCTGACCATGCGCGATAAACTGAAAGAAAAAGGCTTTGAAGTGCGCGCGAGCCTGGATAACAGCGGCATTGATGCGGTGATTAACCATAACAACAACTATAACAACGCGCTGGCGAACGCGGCGGCGGCGGTGGGCAAACCGGGCATGGAACTGAGCAAACTGGATCATGTGGCGGCGAACGCGGCGGGCATGGGCGGCATTGCGGATCATGTGGCGACCACCAGCGGCGCGATTAGCCCGGGCCGCGTGATTCTGGCGGTGCCGGATATTAGCATGGTGGATTATTTTCGCGAACAGTTTGCGCAGCTGCCGGTGCAGTATGAAGTGGTGCCGGCGCTGGGCGCGGATAACGCGGTGCAGCTGGTGGTGCAGGCGGCGGGCCTGGGCGGCTGCGATTTTGTGCTGCTGCATCCGGAATTTCTGCGCGATAAAAGCAGCACCAGCCTGCCGGCGCGCCTGCGCAGCATTGGCCAGCGCGTGGCGGCGTTTGGCTGGAGCCCGGTGGGCCCGGTGCGCGATCTGATTGAAAGCGCGGGCCTGGATGGCTGGCTGGAAGGCCCGAGCTTTGGCCTGGGCATTAGCCTGCCGAACCTGGCGAGCCTGGTGCTGCGCATGCAGCATGCGCGCAAAATGGCGGCGATGCTGGGCGGCATGGGCGGCATGCTGGGCAGCAACCTGATGAGCGGCAGCGGCGGCGTGGGCCTGATGGGCGCGGGCAGCCCGGGCGGCGGCGGCGGCGCGATGGGCGTGGGCATGACCGGCATGGGCATGGTGGGCACCAACGCGATGGGCCGCGGCGCGGTGGGCAACAGCGTGGCGAACGCGAGCATGGGCGGCGGCAGCGCGGGCATGGGCATGGGCATGATGGGCATGGTGGGCGCGGGCGTGGGCGGCCAGCAGCAGATGGGCGCGAACGGCATGGGCCCGACCAGCTTTCAGCTGGGCAGCAACCCGCTGTATAACACCGCGCCGAGCCCGCTGAGCAGCCAGCCGGGCGGCGATGCGAGCGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGACCGGCGCGGCGAGCAACAGCATGAACGCGATGCAGGCGGGCGGCAGCGTGCGCAACAGCGGCATTCTGGCGGGCGGCCTGGGCAGCATGATGGGCCCGCCGGGCGCGCCGGCGGCGCCGACCGCGGCGGCGACCGCGGCGCCGGCGGTGACCATGGGCGCGCCGGGCGGCGGCGGCGCGGCGGCGAGCGAAGCGGAAATGCTGCAGCAGCTGATGGCGGAAATTAACCGCCTGAAAAGCGAACTGGGCGAA

and optimized codons for A. thaliana avoiding BamHI and EcoRI restriction sites

ATGGATCATCCAGTTGCTAGGTCATTGATCGGTTCTTCTTATACAAATCTTAATAACGGTAGTATCGTTATCCCTTCAGATGCTTGTTTTTGTATGAAGTGGTTGAAGTCTAAGGGATCTCCTGTTGCTCTTAAGATGGCTAATGCTCTTCAATGGGCTGCTTTTGCTCTTTCTGTTATTATCCTTATTTACTACGCTTACGCTACTTGGAGAACTACTTGCGGATGGGAGGAAGTTTACGTGTGTTGTGTTGAGCTCACTAAGGTTGTGATCGAGTTCTTCCATGAGTTCGATGAGCCAGGTATGTTATACCTTGCTAATGGTAATAGAGTACTTTGGTTAAGATATGGTGAGTGGCTTCTTACATGCCCTGTTATTCTTATTCATCTTTCTAATCTCACTGGTCTTAAGGATGATTATAATAAAAGAACTATGAGATTGCTTGTTTCTGATGTTGGTACTATTGTTTGGGGAGCTACTGCTGCTATGTCTACTGGATATATTAAAGTTATCTTTTTTCTTCTTGGATGTATGTATGGAGCAAACACTTTTTTCCATGCTGCTAAAGTTTATATTGAGTCATACCATACAGTCCCTAAGGGACTTTGTAGACAATTGGTCAGGGCAATGGCTTGGCTTTTCTTTGTTTCTTGGGGTATGTTCCCAGTTTTGTTCCTACTCGGACCAGAGGGTTTTGGACATCTTTCAGTGTATGGTTCTACTATCGGACATACAATTATTGATCTCTTATCTAAAAACTGCTGGGGATTGCTCGGTCATTTTCTTAGATTGAAGATTCATGAACATATTTTACTTTACGGAGACATCAGAAAGGTTCAGAAAATTAGAGTTGCTGGAGAGGAATTGGAGGTTGAAACTTTAATGACAGAAGAGGCTCCTGATACTGTGAAAAAGTCTACCGCTCAATATGCTAACAGAGAATCATTTCTTACTATGCGAGATAAACTCAAGGAAAAAGGATTTGAAGTTAGAGCTTCCCTTGATAACTCCGGAATCGATGCTGTTATCAATCATAATAATAACTATAATAATGCTCTTGCTAACGCTGCTGCTGCTGTTGGCAAACCTGGAATGGAGCTTTCTAAATTGGATCATGTTGCTGCTAACGCTGCTGGAATGGGAGGAATAGCTGATCATGTTGCTACAACATCCGGTGCTATTAGTCCTGGTAGAGTTATTTTGGCTGTTCCAGATATTAGTATGGTTGATTACTTTAGAGAACAATTCGCTCAACTTCCTGTTCAATATGAAGTTGTGCCTGCTTTGGGAGCAGATAATGCTGTGCAATTGGTTGTTCAAGCTGCTGGACTTGGTGGATGTGATTTCGTTTTACTTCATCCTGAATTTCTTAGAGATAAGTCTTCTACATCTTTGCCTGCCAGACTGAGATCTATTGGACAACGTGTTGCTGCTTTTGGATGGTCTCCTGTTGGACCTGTTAGAGATCTTATAGAATCTGCTGGATTGGATGGATGGCTCGAAGGACCATCTTTCGGCTTGGGAATTTCACTCCCTAATTTGGCTTCTCTTGTCCTTAGAATGCAACATGCACGTAAAATGGCTGCTATGCTCGGTGGAATGGGTGGTATGTTGGGTTCTAATTTGATGTCTGGATCTGGGGGGGTTGGCCTTATGGGTGCTGGAAGCCCAGGCGGTGGTGGAGGTGCTATGGGAGTTGGAATGACAGGAATGGGAATGGTGGGAACAAATGCGATGGGTAGAGGAGCTGTGGGAAATTCTGTTGCTAATGCTTCTATGGGAGGTGGATCTGCGGGTATGGGTATGGGTATGATGGGAATGGTGGGAGCGGGTGTGGGAGGACAACAGCAGATGGGAGCTAATGGTATGGGTCCTACATCTTTCCAACTTGGATCTAATCCACTTTATAACACTGCTCCTAGTCCTCTCTCTTCGCAACCAGGAGGTGACGCTTCGGCAGCTGCTGCTGCTGCTGCGGCTGCTGCTGCTACTGGGGCTGCTAGTAATTCTATGAATGCTATGCAAGCCGGAGGTTCTGTTAGAAACTCAGGTATACTCGCTGGAGGACTTGGATCTATGATGGGTCCTCCTGGTGCACCAGCTGCGCCTACTGCTGCTGCTACAGCTGCTCCTGCTGTTACAATGGGTGCTCCAGGTGGTGGAGGAGCTGCTGCTAGTGAGGCTGAAATGCTTCAACAACTTATGGCTGAGATTAATAGATTAAAGTCTGAGCTTGGCGAA

Then, I uploaded both pCAMBIA1304 and optimized ChR2 sequences to Benchling, where I replaced GUS with ChR2. At this stage, I didn’t make any other changes to sequence**. This is a map of modified plasmid backbone where gene of interest could be inserted: modified-pCAMBIA1304-sequence modified-pCAMBIA1304-sequence

  • Alternative prominent approach is to design custom minicircle DNA, which lacks antibiotic resistance genes and some other bacterial sequences [Almeida et al., 2020]. ** Other possible changes: add P2A (2A peptides) between GFP and ChR2 sequences to induce ribosomal skipping during translation; include some third reporter that would be destroyed by successful insertion of gene of interest, wich would be easy to detect in vivo, though I didn’t come up with any idea of it.

The experiment plan

  1. Order peptide and DNA constructs

  2. Prepare plasmid DNA solution

  3. Prepare cationic peptide solution

  4. Mix at optimized N/P ratio (nitrogen/phosphate ratio)

  5. Incubate for self-assembly

  6. Direct transfer to mature plant (syringe w/o a needle OR water supply)

  7. Check transfection result using imaging and electrophysiological assay