Difference between revisions of "Part:BBa K2924000"

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[[File:Pcpc560_fluorescence_time.png|400px|thumb|left|<i>Fig. 2: A: Fluorescence of the cultures. B: Fluorescence of the cultures normalized by optical density. Fluorescence was measured at 𝝺<sub>ex/em</sub> 527 nm/512 nm. Fluorescence was measured compared to the empty vector control to control for autofluorescence of the cells. Sterile BG11 was used as a blank to measure autofluorescence of the medium. Measurements were carried out in technical triplicates, standard deviations are shown.</i>]]
 
<i>Fig. 2: A: Fluorescence of the cultures. B: Fluorescence of the cultures normalized by optical density. Fluorescence was measured at 𝝺<sub>ex/em</sub> 527 nm/512 nm. Fluorescence was measured compared to the empty vector control to control for autofluorescence of the cells. Sterile BG11 was used as a blank to measure autofluorescence of the medium. Measurements were carried out in technical triplicates, standard deviations are shown.</i>
 
<i>Fig. 2: A: Fluorescence of the cultures. B: Fluorescence of the cultures normalized by optical density. Fluorescence was measured at 𝝺<sub>ex/em</sub> 527 nm/512 nm. Fluorescence was measured compared to the empty vector control to control for autofluorescence of the cells. Sterile BG11 was used as a blank to measure autofluorescence of the medium. Measurements were carried out in technical triplicates, standard deviations are shown.</i>
  

Revision as of 18:32, 15 October 2019


Pcpc560 Promoter

A strong promoter for protein expression in cyanobacteria. It is made up of two predicted promoters and 14 transcription factor binding sites, as well as an RBS. The sequence stems from the cpcB gene from Synechocystis sp. PCC 6803

Usage and Biology

The Promoter Pcpc560 was discovered in 2013 by Jie Zhou et al.1 at the Chinese academy of sciences of Beijing. The name derives from the cyanobacterial gene that it originally expresses, phycocyanin B-subunit (cpcB). Zhou et al used a region of 560 bases upstream of this gene, which contains two predicted promoters and 14 predicted transcription factor binding sites (TFBSs). Negative transcription TFBSs are usually located between 500-1000bp upstream of the gene, so this region was left out of the Pcpc560 construct1. The promoter carries an RBS, so it can be cloned directly in front of the gene of interest. They tested the promoter for the expression of two genes with enzymatic activity, the native enzyme crotonyl-CoA-specific trans-enoyl-CoA reductase (Ter) and D-lactate dehydrogenase from E. coli K-12. In both cases, protein yields of 15% of soluble protein were reported. If the promoter found by Zhou et al. makes these high yields possible for any expressed protein, cyanobacteria might become a viable chassis for protein production, especially since they do not need any carbon source and do not produce CO2 emissions. The promoter is already present in the registry as BBa_K1968001 and was submitted by the team of Edinburgh in 2016, which was not used in Synechocystis. However, BBa_K1968001 differs slightly from this promoter in its sequence and since the function of a promoter is strictly sequence-dependant, this Pcpc560 promoter is submitted as a new part.

References

1: Zhou, Jie, et al. "Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria." Scientific reports 4 (2014): 4500.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

The characterization data shown on this page is taken from the experimental data of the composite part BBa_K2924034, in which the Pcpc560 promoter expresses mVenus as a reporter gene in the pSDHY plasmid.

Fig. 1: Optical density of the cultures at 750 nm, the usual wavelength for cell density measurements of cyanobacteria. Measurements were carried out in triplicates, standard deviations are shown.


Fig. 2: A: Fluorescence of the cultures. B: Fluorescence of the cultures normalized by optical density. Fluorescence was measured at 𝝺ex/em 527 nm/512 nm. Fluorescence was measured compared to the empty vector control to control for autofluorescence of the cells. Sterile BG11 was used as a blank to measure autofluorescence of the medium. Measurements were carried out in technical triplicates, standard deviations are shown.

Fig. 2: A: Fluorescence of the cultures. B: Fluorescence of the cultures normalized by optical density. Fluorescence was measured at 𝝺ex/em 527 nm/512 nm. Fluorescence was measured compared to the empty vector control to control for autofluorescence of the cells. Sterile BG11 was used as a blank to measure autofluorescence of the medium. Measurements were carried out in technical triplicates, standard deviations are shown.

The empty vector control (EVC) grew faster than the cultures with the heterologous protein, suggesting strong expression leading to a metabolic burden. The cells expressing mVenus had a higher metabolic burden because the gene was codon-optimized for Synechocystis , while the a-s1-casein gene was codon-optimized for E. coli. The fluorescence per OD750 decreased over time, likely due to limitations in light and nutrients, which force the cells to put more energy into photosynthetic pigments. The expression was shown to be strong and constitutive, creating a metabolic burden for the cells, but resulting in very high fluorescence. To gain information about the localization of the expressed protein, some cells were imaged in a fluorescence microscope.

Fig. 3: Fluorescence microscopy image of the mVenus expressing Synechocystis cells. Autofluorescence of the phycocyanin in the phycobilisomes is shown in magenta, fluorescence of mVenus is shown in green.

The expression of mVenus is strictly intra-cellular and is not localized to any specific location in the cell. After protein extraction, the soluble protein of the cells was analyzed by SDS-PAGE

Fig. 4: SDS-PAGE of soluble fraction of cyanobacterial protein, extracted from the cultures after 2 days. The SDS-PAGE was run at 45mA for 90 minutes and then stained with Coomassie blue.

Between 25 and 35 kDa in the soluble fraction, a band is visible for mVenus, which is not visible in the EVC. mVenus has a molecular weight of 26.9 kDa, so the band is in the correct area. The very intense bands just above 15 kDa is likely cpcB, a protein from the phycobiliprotein complex, which is used to collect light energy. The protein band for the heterologous protein is approximately the second most intense band in the protein solution, suggesting a strong expression and potentially high yields.


The promoter was also successfully used to express a bovine milk protein in Synechocystis sp. PCC 6803 in the composite part BBa_K2924036.