Plasmid_Backbone

Part:BBa_K5293017:Design

Designed by: Arnaud Boudigou   Group: iGEM24_uOttawa   (2024-09-23)


pHREAC_eGFP_APO


Assembly Compatibility:
  • 10
    INCOMPATIBLE 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
  • 12
    INCOMPATIBLE 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 NotI site found at 5724
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE 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
  • 25
    INCOMPATIBLE 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 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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.


Plasmid Map

apoplast-plasmidmap.png

Figure 1: Plasmid Map of BBa_K5293017 containing the pLac-mCherry insert (BBa_k5293011)


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)

• PR1b: Localization tags allow recombinant protein subcellular targeting

Targeting proteins to the apoplast only requires the tobacco PR1b signal peptide sequence which directs them to the secretory pathway (Dixon et al.,1991). These proteins will migrate from the ER, ‎through the Golgi Apparatus and then be packed in secretory vesicles whose content get released in the apoplast by exocytosis.

• SapI Site: Recognition sequence for the SapI restriction enzyme to allow digestion and insertion of the Gene of Interest

• mCherry: Red constitutive fluorescent protein present in the insertion site to be excised during cloning for post-cloning red/white screening (Shaner et al., 2004)

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

• NSs Protein: Viral protein suppressor used to limit RNA silencing from the plant’s immune system (Takeda et al., 2002)

• 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

Colony Screening

The Lac promoter and mCherry are not apart of these parts. They are contained within the SapI sites and are excised out when your gene of interest is cloned in. However due to the leaky nature of the Lac promoter, you can plate the colonies without IPTG, saving costs, and still see red-white colony screening.

pinkspots.png

Figure 2: Transformed colonies of the plasmid showing successful colonies (white) and unsuccessful transformations (red) on LB-Kanamycin media with no IPTG.

References

Benfey, P. N., & Chua, N.-H. (1990). The Cauliflower Mosaic Virus 35S Promoter: Combinatorial Regulation of Transcription in Plants. Science, 250(4983), 959–966. https://doi.org/10.1126/science.250.4983.959

Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173(1), 33–38. https://doi.org/10.1016/0378-1119(95)00685-0

Dixon, D. C., Cutt, J. R., & Klessig, D. F. (1991). Differential targeting of the tobacco PR‐1 pathogenesis‐related proteins to the extracellular space and vacuoles of crystal idioblasts. The EMBO Journal, 10(6), 1317–1324. https://doi.org/10.1002/j.1460-2075.1991.tb07650.x

de Felippes, F. F., & Waterhouse, P. M. (2023). Plant terminators: The unsung heroes of gene expression. Journal of Experimental Botany, 74(7), 2239–2250. https://doi.org/10.1093/jxb/erac467

Kummari, D., Palakolanu, S. R., Kishor, P. B. K., Bhatnagar-Mathur, P., Singam, P., Vadez, V., & Sharma, K. K. (2020). An update and perspectives on the use of promoters in plant genetic engineering. Journal of Biosciences, 45(1), 119. https://doi.org/10.1007/s12038-020-00087-6

Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N. G., Palmer, A. E., & Tsien, R. Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. Red fluorescent protein. Nature Biotechnology, 22(12), 1567–1572. https://doi.org/10.1038/nbt1037

Takeda, A., Sugiyama, K., Nagano, H., Mori, M., Kaido, M., Mise, K., Tsuda, S., & Okuno, T. (2002). Identification of a novel RNA silencing suppressor, NSs protein of Tomato spotted wilt virus. FEBS Letters, 532(1–2), 75–79. https://doi.org/10.1016/s0014-5793(02)03632-3