Difference between revisions of "Part:BBa K2924034"

Revision as of 21:12, 19 October 2019

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

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

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.

Usage and Biology

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.

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.

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.

A band was visible slightly under 25 kDa, which was not visible in the empty vector control. This band is very likely the a-s1-casein protein, which ran slightly lower than expected. It is visible to a smaller degree in the insoluble protein fraction as well. 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]

</html>

References

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

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 ===Usage and Biology===
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-The <i>Synechocystis</i> cells were grown in 30 ml <a href="dx.doi.org/10.17504/protocols.io.7kmhku6">BG-11 medium</a> at 150 rpm, 1% CO<sub>2</sub> and 80 µmol photons per second and square meter (80 µE). </html>
+The <i>Synechocystis</i> cells were grown in 30 ml <a href="https://www.protocols.io/view/recipe-for-standard-bg-11-media-7kmhku6">BG-11 medium</a> at 150 rpm, 1% CO<sub>2</sub> and 80 µmol photons per second and square meter (80 µE). </html>
 [[File:Pcpc560_growth_effects.png|400px|thumb|right|<i><b>Fig. 1:</b> 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.</i>]]
 <html><p align="justify"> </html>
@@ Line 19: / Line 19: @@
 <br><br><br><br><br><br><br><br><br><br><br><br>
 <html><p align="justify"> </html>
-After 2 days’ growth, samples of 20 OD units were taken and used for protein measurements and SDS PAGE. The <i>Synechocystis</i> 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 <html><a href="dx.doi.org/10.17504/protocols.io.ps6dnhe">(Cell disruption protocol)</a></html>.
+After 2 days’ growth, samples of 20 OD units were taken and used for protein measurements and SDS PAGE. The <i>Synechocystis</i> 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 <html><a href="https://www.protocols.io/view/cell-lysis-and-extraction-of-total-protein-from-sy-ps6dnhe">(Cell disruption protocol)</a></html>.
 <html><p align="justify">
-The samples were used for an <a href="dx.doi.org/10.17504/protocols.io.8evhte6">SDS-PAGE</a>. A-s1-casein is a protein of approx. 25.4 kDa size.
+The samples were used for an <a href="https://www.protocols.io/view/sds-page-8evhte6">SDS-PAGE</a>. A-s1-casein is a protein of approx. 25.4 kDa size.
 <p align="justify">
 A band was visible slightly under 25 kDa, which was not visible in the empty vector control. This band is very likely the a-s1-casein protein, which ran slightly lower than expected. It is visible to a smaller degree in the insoluble protein fraction as well.