Project Description | Thailand-RIS

Project Discussion with P’Gift

Our Goals

Transfer the gene for PETase from a bacteria (i.e. Ideonella sakaiensis, among others) to a eukaryotic multicellular organism (i.e. Nicotiana benthamiana); previous work has only been done with microalgae, which are unicellular
Enable the plant chassis to produce PETase and excrete it outside the individual cell

Signal Peptide

Once the gene for PETase has been successfully transferred to N. benthamiana via Agrobacterium mediated transformation and is being expressed by the plant, the PETase enzyme will be produced within the plant cells
This necessitates a signal peptide to direct the enzymes outside the cell and to the desired places
While the gene for PETase also has a signal peptide, as it comes from a prokaryote, we don’t know how it will function inside of a plant, which is much different from a bacteria
So, the sequence for the signal peptide will be replaced with the signal peptide for alpha-amylase, which is from another plant (Oryza sativa Japonica Group)
Once the PETase is produced by the cell and has been directed to the correct place, the signal peptide will be cleaved off, and therefore does not affect the actual enzyme
*bioinformatics: predicting protein structure (modeling) based on the gene (not necessary, but can be used to prove that the cleavage happens)

Codon Usage

Reverse translation is the process of taking an amino acid sequence and translating it back to a nucleotide sequenc
When we do so, we have to be mindful of the codon usage of the organism we want to transfer the gene to; multiple codons correspond to the same amino acid, however different organisms prefer a certain codon for an amino acid, and this is especially true for more distantly related organisms like bacteria and plants
The codons are optimized for N. benthamiana, so that our chassis can correctly express the gene and in sufficient quantities

Ordering the Gene

Either the nucleotide sequence or the amino acid sequence is sent to a company that synthesizes genes, and the gene is given as a vector to ensure the stability of the nucleotides (so that they don’t break down or mutate? during transport)
The vector we ordered is pCAMBIA, which has a right boundary (RB) and left boundary (LB) that marks the part of the gene that will be integrated into the plant genome via agrobacterium mediated transformation (choosing the right type of vector is important, as different vectors are more suitable for different organisms/purposes)

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