pABDP2
(Plasmid #249501)

• Purpose: Bidirectional chloroplast promoter (BDP2) enabling co-expression for transformation of Chlamydomonas reinhardtii photosynthetically deficient strain CC4388
• Depositing Lab: Isabel Desgagné-Penix
• Publication: Engineering bidirectional chloroplast promoters for tunable co-expression of multiple genes in microalgae (unpublished)

Backbone

• Vector backbone: pASAPI
• Backbone manufacturer: Chlamydomonas Resource Center; http://www.chlamycollection.org
• Backbone size w/o insert (bp): 6378
• Total vector size (bp): 9158
• Modifications to backbone: The 3′ untranslated region (3′ UTR) of the psbA gene was added
• Vector type: Bacterial Expression, Synthetic Biology ; Chlamydomonas reinhardtii chloroplast genome
• Selectable markers: Photosynthesis-based selection by restore of the psbH gene

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: NEB Stable E. coli strain can also be used
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: mVenus (yellow/green fluorescence)
• Alt name: mVenus
• Species: Aequorea victoria
• Insert Size (bp): 719
• Mutation: Change of F46L, F64L, M153T, V163A, S175G
• GenBank ID: GenBank: AAZ65844 GenBank: AAZ65844
• Promoter: Bidirectional chloroplast promoter enabling co-expression of the endogenous chIL and petB genes

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: complete plasmid sequencing
• 3′ sequencing primer: complete plasmid sequencing

Gene/Insert 2

• Gene/Insert name: tdTomato (red fluorescence)
• Alt name: tdTomato
• Species: Discosoma sp.
• Insert Size (bp): 1431
• Mutation: tdTomato results from the fusion of two copies of the dTomato gene, an optimized version of the red fluorescent protein DsRed derived from the coral Discosoma sp.
• GenBank ID: GenBank: AAV52169 GenBank: AAV52169
• Promoter: Bidirectional chloroplast promoter enabling co-expression of the endogenous chIL and petB genes

Cloning Information for Gene/Insert 2

• Cloning method: Gibson Cloning
• 5′ sequencing primer: complete plasmid sequencing
• 3′ sequencing primer: complete plasmid sequencing

Resource Information

• Supplemental Documents:
  • pABDP2.gb

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

The plasmid pABDP2 was constructed to enable the simultaneous expression of two transgenes in the Chlamydomonas reinhardtii chloroplast, driven by the endogenous bidirectional promoter BDP2. These vector is designed for transformation of the photosynthetically deficient strain CC4388, in which the psbH gene, encoding an auxiliary core subunit of Photosystem II, is disrupted. Following homologous recombination at the psbN–trnE2 locus, the vector restore psbH expression and consequently recover photoautotrophic growth. Selection is therefore achieved through restoration of photosynthetic function, eliminating the need for antibiotic resistance markers.

The depositor confirms that discrepancies between the depositor's sequence and Addgene's sequences have no functional consequences. The BDP2 sequence was amplified directly from the chloroplast DNA of Chlamydomonas reinhardtii strain CC4388, and BLASTs to OZ318231.1.