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This year the iGEM team Düsseldorf has designed many new BioBrick parts. One of these parts is BBa_K2924036. It contains the cyanobacterial promoter P_cpc560 (BBa_K2924000) which is used for the expression of the gene of the fluorescent protein mVenus (BBa_K2924035) with the T1/T7 double terminator (BBa_B0015).

The strong and constitutive 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 Escherichia coli to cyanobacteria and other microorganisms¹. To test the strength of the P_cpc560 promoter (BBa_K2924000), it was cloned in front of mVenus (BBa_K2924035), a fluorescent protein originally isolated from Aequorea victoria with improved brightness. The sequence was provided codon-optimized for Synechocystis sp. PCC 6803. All experiments were carried out in Synechocystis sp. PCC 6803, into which the plasmid was conjugated by triparental mating with a transformed E. coli strain.

To test the strength of the P_cpc560 promoter (BBa_K2924000), it was cloned in front of mVenus (BBa_K2924035), a fluorescent protein originally isolated from Aequorea victoria with improved brightness. The sequence was provided codon-optimized for Synechocystis sp. PCC 6803. All experiments were carried out in Synechocystis sp. PCC 6803, into which the plasmid was conjugated by triparental mating with a transformed E. coli strain.

Fig. 1: Optical density of the cultures at 750 nm, the usual wavelength for cell density measurements of cyanobacteria. The empty vector control (EVC) grew faster than the cultures with the heterologous protein, suggesting strong expression leading to a metabolic burden. Measurements were carried out in triplicates.

Based on the growth of the different Synechocystis strains expressing a empty vector control (EVC), 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).

Fig. 2: A: Fluorescence of the Synechocystis cultures. B: Fluorescence of the Synechocystis cultures normalized by optical density. Fluorescence was measured at 527 nm while shining light of 512 nm onto the cells. Fluorescence was measured compared to the empty vector control to control for autofluorescence of the cells. Clean BG11 was used as a blank to measure autofluorescence of the medium. Measurements were carried out in technical triplicates.

The fluorescence per OD₇₅₀ decreased over time (Fig. 2, B), likely due to limitations in light and nutrients, which force the cells to put more energy into photosynthetic pigments, but still remains high in comparison to the non fluorescent control. .

After 2 days’ growth, 10 ml samples 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. The protein content of the fractions was quantified by a Bradford protein assay.

Fig. 3: Protein produced per cell after 2 days of growth, determined by Bradford assay.

The total amount of protein produced by the cells with P_cpc560 expressing mVenus did not differ significantly from the empty vector control (Fig. 3). The fraction of insoluble protein was larger in those cultures however, suggesting that not all mVenus is present as soluble protein, as the protein folding apparatus might be overloaded by the strong expression. Since mVenus was expected in the soluble fraction SDS-PAGE and protein detection by western blot were executed with this fraction (Fig. 4 and 5).

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.

The gel showed no difference between the control and the mVenus cultures in the insoluble protein. Between 25 and 35 kDa in the soluble fraction, a band is visible for mVenus, which is not visible in the control (Fig. 4). mVenus has a molecular weight of 26.9 kDa, so the band is in the correct area.

To confirm the identity of the protein, a western blot was performed with the soluble protein fraction (Fig. 5). Three clones containing pSDHY P_cpc560 + mVenus were used for expression and protein extraction. The Western blot was carried out with an anti-gfp antibody.

Fig. 5: Western Blot of soluble protein. The image was created by merging the visible light image of the ladder with the chemifluorescence image of the bands.

The western blot shows no band for the control, while showing three distinct bands in all mVenus colonies (Fig. 5). The middle band corresponds to the expected size of mVenus. The other bands might be misfolded or partly-degraded protein respectively.

To determine where mVenus is located in the cells confocal microscopy images were taken. The mVenus protein was located evenly in the cytosol of the cells (Fig. 6)

Fig 6: 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.

CRIPSRi-dCas9 knock-down experiments

The mVenus gene was used to test the effectiveness of a CRISPRi-dCas9 knock-down, which was kindly provided to us by Yao et al.(2015) ². This dCas9 has a mutated cutting site, so it only binds to the target gene determined by the sgRNA without cutting it. In this way, a gene can be inducibly down-regulated. Two clones were used to test the down-regulation system (Fig. 7 and 8).

Fig. 7: mVenus fluorescence in Synechocystis over time, normalised to the optical density, after induction with 500 µM aTc.

As seen in Fig. 7, the optimal activity of the sgRNA/dCas9 complex, which can be seen in the decrease of the fluorescence, is after 24 h after induction with 500 µM anhydrotetracycline (aTc).
For further comparison, the decrease of fluorescence of several biological and technical replicates of induced and uninduced control and knockdown (KD) Synechocystis sp. PCC 6803 strains was tested and compared (Fig. 8).

Fig. 8: Total fluorescence compared with different controls after 24h after induction with 500µM aTc. Empty vector control (EVC) and positive control (mVenus) are pictured alongside the knock-down (KD) strain.

In Fig. 8 a decrease of fluorescence can be seen in the induced mVenus KD strain compared to the uninduced mVenus KD strain. In contrast, the induced mVenus KD strain still shows fluorescence compared to the EVC strain. This proves the function of the CRSPRi/dCas9-system ².

References

1. Behle, A., Saake P., Axmann, I. M. "Comparative analysis of inducible promoters in cyanobacteria." bioRxiv (2019): 757948.
2. Yao, Lun, et al. "Multiple gene repression in cyanobacteria using CRISPRi." ACS synthetic biology 5.3 (2015): 207-212.