Plasmid_Backbone

Part:BBa_K5299002

Designed by: Maria Nefeli Stoupa   Group: iGEM24_Thessaly   (2024-09-22)


pSEVA23g19[g1]

Introduction

pSEVA23g19[g1] is a modular plasmid from the SEVA (Standard European Vector Architecture) collection, specifically designed for genetic engineering in Gram-negative bacteria. The "23" indicates that it carries a kanamycin resistance gene for selection, while "3g" refers to the pBBR1 origin of replication, which supports stable replication across a broad range of bacterial species. The "[g1]" tag specifies that the plasmid's cargo is 1AI2, associated with level 1 Golden Standard reactions [1].

Figure 1: pSEVA23g19[g1] map. Features and Restriction Sites.

=Usage and Biology

Golden Standard

The Golden Standard assembly system is designed with a highly structured approach to plasmid construction, following a set of rules similar to those found in the SEVA format. It organizes plasmid vectors into three fundamental components: the origin of replication, the selection marker, and the cargo. Each of these components is minimized to retain only the essential DNA sequences required for functionality, ensuring that plasmid constructs are as efficient as possible [1].

Figure 2: Standard European Vector Architecture.

Our team successfully cloned all of our basic parts into the pUPD2 vector by using the enzyme BsmbI. pUPD2 is a vector designed for the GoldenBraid (GB) DNA assembly system and serves as a versatile Level 0 vector, enabling the initial cloning of genetic parts, such as promoters, coding sequences, and terminators. It is designed for seamless and modular cloning using Type IIS restriction enzymes like BsaI and BsmBI, allowing for the precise assembly of transcriptional units in subsequent steps. pUPD2, originally designed for the GoldenBraid (GB) system, is also compatible with the Golden Standard assembly method due to the presence of BsaI restriction sites [2].The compatibility of pUPD2 with both the GoldenBraid and Golden Standard assembly systems enabled us to efficiently create our transcriptional units. So, after assembling the genetic parts in pUPD2, we successfully transferred the transcriptional units into the pSEVA23g19[g1] plasmid. For the cloning procedure, we utilized E. coli DH5α cells, and subsequently tested the expression of the transcriptional units in P. putida. The pBBR1 origin of replication, present in pSEVA23g19[g1], is compatible with both E. coli and P. putida, ensuring stable plasmid replication and expression across both hosts [3].

Figure 3: Assembly of Genetic Parts in pUPD2 and Successful Transfer of Transcriptional Units into pSEVA23g19[g1] Plasmid

References

[1] Blázquez, B., León, D. S., Torres-Bacete, J., Gómez-Luengo, Á., Kniewel, R., Martínez, I., Sordon, S., Wilczak, A., Salgado, S., Huszcza, E., Popłoński, J., Prieto, A., & Nogales, J. (2023). Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria. Nucleic acids research, 51(19), e98. https://doi.org/10.1093/nar/gkad758

[2] Dusek, J., Plchova, H., Cerovska, N., Poborilova, Z., Navratil, O., Kratochvilova, K., Gunter, C., Jacobs, R., Hitzeroth, I. I., Rybicki, E. P., & Moravec, T. (2020). Extended Set of GoldenBraid Compatible Vectors for Fast Assembly of Multigenic Constructs and Their Use to Create Geminiviral Expression Vectors. In Frontiers in Plant Science (Vol. 11). Frontiers Media SA. https://doi.org/10.3389/fpls.2020.522059

[3] Jahn, M., Vorpahl, C., Hübschmann, T., Harms, H., & Müller, S. (2016). Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR. Microbial cell factories, 15(1), 211. https://doi.org/10.1186/s12934-016-0610-8


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 56
    Illegal EcoRI site found at 333
    Illegal XbaI site found at 306
    Illegal XbaI site found at 709
    Illegal SpeI site found at 771
    Illegal PstI site found at 294
    Illegal PstI site found at 721
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 56
    Illegal EcoRI site found at 333
    Illegal SpeI site found at 771
    Illegal PstI site found at 294
    Illegal PstI site found at 721
    Illegal NotI site found at 46
    Illegal NotI site found at 739
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 56
    Illegal EcoRI site found at 333
    Illegal BamHI site found at 312
    Illegal BamHI site found at 703
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 56
    Illegal EcoRI site found at 333
    Illegal XbaI site found at 306
    Illegal XbaI site found at 709
    Illegal SpeI site found at 771
    Illegal PstI site found at 294
    Illegal PstI site found at 721
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 56
    Illegal EcoRI site found at 333
    Illegal XbaI site found at 306
    Illegal XbaI site found at 709
    Illegal SpeI site found at 771
    Illegal PstI site found at 294
    Illegal PstI site found at 721
    Illegal NgoMIV site found at 2101
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 678
    Illegal BsaI.rc site found at 94


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Categories
Parameters
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