Difference between revisions of "Part:BBa K5293014"

Latest revision as of 00:29, 2 October 2024

pHREAC_eGFP_ER

This part is a component of a larger collection designed by the 2024 iGEM uOttawa team. These plasmids were developed to facilitate the screening of subcellular localizations in Nicotiana benthamiana and maximise transient expression of recombinant proteins, leveraging the plant's eukaryotic system to perform post translational modifications. All parts within this collection can safely be used in a BSL 1 laboratory to the best of our knowledge. By incorporating signal peptides, we aimed to direct proteins to specific organelles such as the chloroplast, vacuole, cytoplasm, apoplasm and endoplasmic reticulum, optimising recombinant protein accumulation and stability for efficient biomanufacturing.

The plasmids we designed provide a comprehensive, user-friendly toolkit for the plant synthetic biology community, enabling rapid and versatile screening of protein localization while improving the scalability of plant-based peptide production. Each plasmid contains carefully designed elements to maximise target protein concentrations and streamline purification processes, making them suitable for both beginners and experienced users. They were designed for and used to perform agrobacterium mediated infiltration of Nicotiana benthamiana.

Information on each of the 5 plasmids can be found here:

Endoplasmic Reticulum - BBa_K5293014

Vacuole - BBa_K5293015

Chloroplasts - BBa_K5293016

Apoplasm - BBa_K5293017

Cytoplasm - BBa_K5293018

For detailed information on the design of each plasmid, including the rationale behind specific components, please refer to the Design tab.

These plasmids were extensively tested with various inserts, primarily focusing on the expression of fusion proteins such as His-Linker-mScarlet-Linker-TEV-Semaglutide (BBa_K5293006) and His-Linker-TEV-Semaglutide (BBa_K5293005) for the biomanufacturing of GLP-1 Agonists. Notably, the plasmid targeting the chloroplasts (BBa_K5293016) yielded the highest protein expression levels, which is why our results page contains the most information on this plasmid. Our experiences with this plasmid, as well as the others, are thoroughly documented in the Experience tab.

We designed these plasmids using Benchling for easy DNA synthesis, enabling any iGEM team to quickly begin experimentation. To use these plasmids, simply order your CDS of interest as a gene block and assemble it using Gibson Assembly. We recommend ordering the entire plasmid with mCherry (BBa_K5293011) inside the SapI restsriction sites. The Lac promoter was used as its leaky nature allows unsuccessful colonies to express mCherry and successful transformations can be easily delected by the absence of mCherry expression.

Sequence and Features

RB / LB T-DNA - Right and left borders of the sequence that will be inserted into the plant’s genome by Agrobacterium

CaMV 35S - Cauliflower Mosaic Virus 35S promoter, allowing constitutive expression in plants (Benfey & Chua, 1990)

SapI Sites - Recognition sequence for the SapI restriction enzyme to allow digestion and insertion of the Gene of Interest

NOS Terminator - The Nopaline Synthase terminator is commonly used for transgene expression in plants (de Felippes & Waterhouse, 2023)

NOS Promoter - Bacterial Nopaline Synthase promoter commonly used for constitutive transgene expression in plants (Kummari et al., 2020)

eGFP - Constitutively expressed by the transgenic plants, this green fluorescent protein will allow transformation screening (Cormack et al., 1996)

KanR - Kanamycin resistance gene permits bacterial selection for those who have successfully incorporated the plasmid

ER localization tag - The KDEL and PRR1b sequences encode for a signal peptide that directs the proteins to the endoplasmic reticulum, where it is retained.

• iGEM Part Registry Notes *

The entire annotated plasmid backbone is registered as a Basic Part. Although it could be subdivided into smaller functional units (promoters, CDS, RBS, terminators, etc.), it remains consistent across the collection for biofarming purposes and serves as the "backbone" functional unit. The localization tags are registered individually as Basic Parts, and their combination creates the composite parts. Each component of the plasmid backbone is documented and registered for further reference and use.