Plasmid

Part:BBa_K5044044:Design

Designed by: Chuming Chen   Group: iGEM24_HUBU-China   (2024-10-01)


Plastid transformation vector for kiwifruit by HUBU-China


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 129
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 129
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 118
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 129
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 129
    Illegal NgoMIV site found at 1846
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2091
    Illegal BsaI.rc site found at 818


Design Notes

Design Considerations for pQQC7 During the detailed design of the pQQC7 plasmid, several key considerations were taken into account to ensure its functionality, stability, and efficiency in plastid transformation. Here are the specific design considerations for pQQC7:

Promoter Strength and Specificity: psbA Promoter (aadA): The psbA promoter from Chlamydomonas reinhardtii was chosen for its strong and constitutive expression in chloroplasts. This ensures that the aadA gene is robustly expressed, providing reliable selection of transformed plants. Prrn Promoter (GFP): The tobacco 16S rRNA promoter (Prrn) was selected for its high transcriptional activity in chloroplasts, ensuring strong and consistent expression of the GFP reporter gene. Translation Efficiency: 5' UTR (T7g10): The 5' untranslated region from gene10 of bacteriophage T7 (T7g10) was included to enhance translation efficiency. This sequence has been shown to stabilize mRNA and facilitate ribosome binding, leading to higher protein yields. Homologous Recombination Sequences: LHRR and RHRR: The left and right homologous recombination regions (LHRR and RHRR) were carefully designed to match the target sequences in the kiwifruit chloroplast genome. These regions ensure precise integration between the trnfM and trnG genes, reducing the likelihood of random insertions and increasing the chances of achieving homoplasmy (uniformity of the plastid genome). Selectable Marker: aadA Gene: The aminoglycoside 3′-adenylyltransferase (aadA) gene was chosen as the selectable marker because it confers resistance to spectinomycin. This allows for the efficient selection of transformed plants on media containing spectinomycin. Reporter Gene: GFP Gene: The Green Fluorescent Protein (GFP) was included as a visual marker to confirm successful transformation and to monitor gene expression. The GFP fluorescence can be easily detected using standard fluorescence microscopy, facilitating the screening of transformed tissues. Plasmid Backbone: Cloning Flexibility: The pQQC7 vector was designed with multiple restriction enzyme sites (SacI, KpnI, SalI, SpeI, BlnI, ApaI, SphI) to provide flexibility for cloning and modification. This allows researchers to easily insert or replace genes of interest. Stability and Compatibility: The backbone was derived from pBluescript II KS(+) and pYY11, which are well-characterized and stable vectors. This ensures that the pQQC7 plasmid is compatible with standard molecular biology techniques and can be propagated and maintained efficiently in bacterial hosts. Transformation Efficiency: Biolistic Transformation: The vector was designed to be compatible with biolistic (particle bombardment) methods, which are commonly used for plastid transformation. The size and structure of the plasmid were optimized to ensure efficient delivery and integration into the chloroplast genome. Regulatory Compliance: Safety and Regulations: The design of pQQC7 complies with regulatory standards for genetically modified organisms (GMOs). The use of well-characterized and safe components, such as the aadA selectable marker and the GFP reporter, ensures that the vector is suitable for both research and potential commercial applications. Codon Optimization: Chloroplast Codon Usage: The sequences of the aadA and GFP genes, as well as the regulatory elements, were codon-optimized for the chloroplast to ensure efficient translation and high protein expression levels. Homogeneity and Stability: Avoidance of Gene Silencing: The chloroplast genome is known for its capacity to maintain multiple transgenes without gene silencing. The design of pQQC7 takes advantage of this property, allowing for the stable and long-term expression of introduced genes.


Source

The pQQC7 plasmid is a synthetic construct designed for plastid transformation in kiwifruit (Actinidia chinensis). The various components of the pQQC7 vector are derived from different sources:

Selectable Marker (aadA): Gene: aadA (aminoglycoside 3′-adenylyltransferase) Promoter: psbA promoter from Chlamydomonas reinhardtii Source: The aadA gene and its associated regulatory elements are commonly used in plant transformation vectors. The psbA promoter is derived from the chloroplast genome of Chlamydomonas reinhardtii, which is known for its strong and constitutive expression in the chloroplast. Accession Number: [Insert NCBI accession number for the Chlamydomonas reinhardtii chloroplast genome, if available] Reporter Gene (GFP): Gene: Green Fluorescent Protein (GFP) Promoter: Tobacco plastid 16S rRNA promoter (Prrn) 5' UTR: 5′ untranslated region from gene10 of bacteriophage T7 (T7g10) Source: The GFP gene is a widely used reporter gene. The Prrn promoter is derived from the tobacco (Nicotiana tabacum) plastid genome, and the T7g10 5' UTR is derived from the bacteriophage T7. Accession Numbers: Tobacco 16S rRNA Promoter (Prrn): [Insert NCBI accession number for the tobacco plastid genome, if available] Bacteriophage T7 (T7g10 5' UTR): [Insert NCBI accession number for bacteriophage T7, if available] Homologous Recombination Sequences: Left Homologous Recombination Region (LHRR): 1,092 bp Right Homologous Recombination Region (RHRR): 1,185 bp Source: These sequences are amplified from the kiwifruit (Actinidia chinensis) chloroplast genome to ensure precise integration between the trnfM and trnG genes.

References