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View additional product information for GeneArt® Synechococcus Protein Expression Kit - FAQs (A24243)
12 product FAQs found
The promoter for pSyn_1 is the Psc. Psc is a weak constitutive promoter that drives basal expression of your gene of interest. The promoter for pSyn_6, on the other hand, is the psbA promoter (PpsbA). PpsbA is a strong constitutive promoter of the psbA gene (encoding photosystem II protein D1) from Synechococcus elongatus, driving the high level expression of your gene of interest.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please view the following articles that have some discussion on the advantages of chloroplast transformation versus nuclear transformation:
- Fischer et al. (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7:152-158.
- Kindle et al. (1991) Engineering the chloroplast genome: Techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 88:1721-1725.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The promoter for pSyn_1 is the Psc. Psc is a weak constitutive promoter that drives basal expression of your gene of interest. The promoter was taken from the solanesyl diphosphate synthase gene from Synechocystis sp. Strain PCC 6803. Note: Use of this weak constitutive promoter is a good fit for applications that are hindered by strong expression, such as pathway engineering or complementation of mutant genes normally expressed at low levels (Simkovsky et al., 2012).
The promoter for pSyn_6 is the psbA promoter (PpsbA). PpsbA is a strong constitutive promoter of the psbA gene (encoding photosystem II protein D1) from Synechococcus elongatus, driving high-level expression of your gene of interest. The pSyn-6 vector also includes a ribosome-binding site (GAAGGAG) for efficient initiation of translation, as well as a stop codon. A 6xHis TEV and a V5-His epitope tag is also included in the vector backbone for detection/purification of the gene of interest. NS1 (neutral site 1) homologous recombination sites are present in both vectors for the integration of the vector into the Synechococcus elongatus genome.
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After 6 months of storage, the Synechococcus did not lose their competency. We have not yet tested longer storage points.
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A double homologous recombination event occurs between the Neutral Site 1 in the genome and between NSa and NSb in the plasmid. This results in the gene of interest and spectinomycin resistance integrating into the genome within the Neutral Site 1.
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We no longer offer any strains. Our reagents and protocols were developed using Synechococcus elongatus strain PCC 7942, a model cyanobacterium.
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We recommend our Synechococcus kit (A24230), where the integration is directed to the Neutral Site 1 of the algae genome.
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The cell growth can be measured by OD750 and the linear range will be between 0.2 and 1.2 (in 1 cm lightpass). If too high, please dilute them and measure again. It usually takes 5-6 days for full growth. An accurate measure can be taken using the Countess Cell Counter or Tali Cytometer from Thermo Fisher Scientific. The formula to calculate cell number is as follows:
Cell concentration (cells/mL) = (OD750 - 0.088)/9 × 10e8 = (OD750 - 0.088)/(9 × 10-8)
Please note, our COA states that “240 µl of frozen cells are thawed out and cultured in 6 mL of Gibco TAP media at 28 degrees C and 50 µE light. Optical density is measured at 750 nm following 6 days of growth. Optical density must reach 0.6. Viability testing is performed on a minimum of 6 vials per lot.”
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GeneArt gene synthesis was used to create the plasmid backbones synthetically. Also, GeneArt is the umbrella brand for synthetic biology, which is a large area in which these kits will be used.
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Yes, unfortunately, silencing is a big problem in algae. The extent of silencing depends on the sequence of the gene and the toxicity of the protein that expresses from that gene to the cell.
The engineering kit is designed for random insertion of your gene of interest, which can lead to your gene being lost quickly. The protein expression kit, which contains the pChlamy_4 vector, is designed so that proteins are expressed as transcriptional fusions with the bleomycin/zeocin resistance gene sh-ble (Rasala et al., 2012). The self-cleaving sequence for the 2A peptide from the foot-and-mouth disease virus (FMDV) is placed between the antibiotic resistance gene and the gene of interest. It encodes a short ~20 amino acid sequence that mediates proper cleavage to yield two discrete proteins. With this system we have seen positive transformants maintain high expression levels for much longer than with other systems, even after many passages with or without selection pressure.
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It is a stable transfection.
The average expected time constant is 15-20 milliseconds. We have a Neon electroporation protocol in the manual.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.