Plasmid_Backbone

Part:BBa_K5293014:Design

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


pHREAC_eGFP_ER


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2334
    Illegal EcoRI site found at 4032
    Illegal XbaI site found at 930
    Illegal SpeI site found at 5878
    Illegal PstI site found at 8535
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2334
    Illegal EcoRI site found at 4032
    Illegal NheI site found at 3243
    Illegal SpeI site found at 5878
    Illegal PstI site found at 8535
    Illegal NotI site found at 5732
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2334
    Illegal EcoRI site found at 4032
    Illegal BglII site found at 1297
    Illegal BglII site found at 2560
    Illegal BglII site found at 2580
    Illegal BglII site found at 9350
    Illegal BamHI site found at 936
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2334
    Illegal EcoRI site found at 4032
    Illegal XbaI site found at 930
    Illegal SpeI site found at 5878
    Illegal PstI site found at 8535
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2334
    Illegal EcoRI site found at 4032
    Illegal XbaI site found at 930
    Illegal SpeI site found at 5878
    Illegal PstI site found at 8535
    Illegal NgoMIV site found at 3044
    Illegal NgoMIV site found at 4202
    Illegal NgoMIV site found at 4728
    Illegal NgoMIV site found at 5604
    Illegal NgoMIV site found at 5728
    Illegal NgoMIV site found at 6755
    Illegal NgoMIV site found at 7346
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.


Plasmid Map

endoplasmic-plasmidmap.png

Figure 1: Plasmid Map of BBa_K5293014 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 + KDEL: Localization tags allow recombinant protein subcellular targeting

Targeting proteins to the endoplasmic reticulum requires the tobacco PR1b signal peptide sequence, which directs them to the secretory pathway (Dixon et al., 1991). The KDEL sequence then specifies the retention of the proteins within the ER (Dean & Pelham, 1990).

• 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

Dean, N., & Pelham, H. R. (1990). Recycling of proteins from the Golgi compartment to the ER in yeast. The Journal of Cell Biology, 111(2), 369–377. https://doi.org/10.1083/jcb.111.2.369

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