Part:BBa_K5293016
pHREAC_eGFP_pLac_mCherry_CHL
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.
Information on each of the 5 plasmids can be found here:
Endoplasmic Reticulum - BBa_K5293014
Vacuole - BBa_K5293015
Chloroplasts - BBa_K5293016
Cytoplasm - BBa_K5293017
Apoplasm - 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-mScarlet-TEV-Semaglutide (BBa_K5293006) and His-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.
- 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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2326
Illegal EcoRI site found at 4024
Illegal XbaI site found at 922
Illegal SpeI site found at 5870
Illegal PstI site found at 8527
Illegal PstI site found at 10399 - 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2326
Illegal EcoRI site found at 4024
Illegal NheI site found at 3235
Illegal SpeI site found at 5870
Illegal PstI site found at 8527
Illegal PstI site found at 10399
Illegal NotI site found at 5724 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2326
Illegal EcoRI site found at 4024
Illegal BglII site found at 1289
Illegal BglII site found at 2552
Illegal BglII site found at 2572
Illegal BglII site found at 9342
Illegal BamHI site found at 928 - 23INCOMPATIBLE WITH RFC[23]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2326
Illegal EcoRI site found at 4024
Illegal XbaI site found at 922
Illegal SpeI site found at 5870
Illegal PstI site found at 8527
Illegal PstI site found at 10399 - 25INCOMPATIBLE WITH RFC[25]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2326
Illegal EcoRI site found at 4024
Illegal XbaI site found at 922
Illegal SpeI site found at 5870
Illegal PstI site found at 8527
Illegal PstI site found at 10399
Illegal NgoMIV site found at 3036
Illegal NgoMIV site found at 4194
Illegal NgoMIV site found at 4720
Illegal NgoMIV site found at 5596
Illegal NgoMIV site found at 5720
Illegal NgoMIV site found at 6747
Illegal NgoMIV site found at 7338 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
blah blah blah
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