Part:BBa_K2924034

Pcpc560 + α-s1-casein + T1/T7 Terminator

Strong constitutive cyanobacterial promoter P_cpc560 expressing a-s1-casein with the T1/T7 double terminator.

===Usage and Biology=== The promoter was cloned into the pSHDY plasmid. The pSHDY plasmid is an RSF1010-based, low-copy self-replicating vector derived from pVZ321 and has a broad host range, which can ensure the conjugation from E. coli to cyanobacteria and other microorganisms¹. After testing the strength of the promoter with a fluorescent reporter mVenus, it was used to express a protein from cow’s milk, a-s1-casein in Synechocystis sp. PCC 6803.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1204
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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Characterization

The Synechocystis cells were grown in 30 ml BG-11 medium at 150 rpm, 1% CO₂ and 80 µmol photons per second and square meter (80 µE).

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.

Based on the growth of the different Synechocystis strains expressing a empty vector control, mVenus or ɑ-s1-casein it is possible to say, that heterologous protein are strongly expressed leading to a metabolic burden, which is shown in decreased growth (Fig. 1). The cells expressing mVenus likely 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.

Fig. 2: SDS-PAGE of cyanobacterial protein, separated into soluble and insoluble protein fractions. The gel was run at 220V, 400A for 45 minutes and then stained with a Coomassie blue dye.

After 2 days’ growth, samples of 20 OD units were taken and used for protein measurements and SDS PAGE. The Synechocystis cells were disrupted using glass beads to shred the cells in a Precellys® 24 homogeniser. The cell extract was centrifuged to obtain a pellet of insoluble protein and a supernatant of soluble protein, which were separated (Cell disruption protocol).

The samples were used for an SDS-PAGE. A-s1-casein is a protein of approx. 25.4 kDa size.

In Synechocystis strains expressing ɑ-s1-casein a specific protein band was visible slightly under 25 kDa, which was not detected in the empty vector control (Fig. 2). This band is very likely the ɑ-s1-casein protein, which ran slightly lower than expected. It is visible to a smaller degree in the insoluble protein fraction as well.

The example result of ɑ-s1-casein indicates, that bovine milk proteins can be successfully produced in Synechocystis. They are mostly located in the soluble fraction of the cells. However in the future they need to be produced and purified in large scales in order to examine if the organism is suitable for industrial production.

References

1: Behle, A., Saake P., Axmann, I. M. "Comparative analysis of inducible promoters in cyanobacteria." bioRxiv (2019): 757948.