Device

Part:BBa_K4831013:Design

Designed by: Aleksi Branders   Group: iGEM23_ABOA-Turku   (2023-10-03)


IPTG inducible merA and merB (P. aeruginosa) expression device for Synechocystis sp. PCC6803


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1498
    Illegal EcoRI site found at 4178
    Illegal SpeI site found at 1511
    Illegal PstI site found at 3732
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1498
    Illegal EcoRI site found at 4178
    Illegal SpeI site found at 1511
    Illegal PstI site found at 3732
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1498
    Illegal EcoRI site found at 4178
    Illegal BglII site found at 1612
    Illegal XhoI site found at 1481
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1498
    Illegal EcoRI site found at 4178
    Illegal SpeI site found at 1511
    Illegal PstI site found at 3732
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1498
    Illegal EcoRI site found at 4178
    Illegal SpeI site found at 1511
    Illegal PstI site found at 3732
    Illegal NgoMIV site found at 3042
    Illegal NgoMIV site found at 3090
    Illegal NgoMIV site found at 4001
    Illegal AgeI site found at 3328
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This device and all its parts are designed specifically for Synechocystis sp. PCC6803. That means that the codon presence is similar to Synechocystis native genes. This device is asembled with a golden gate inspired assembly system, leaving spesific 4 bps scars between every part.

Source

The merA gene was native to Synechocystis. This gene is the main reason we chose this specific cyanobacterium for our project. The homologs from P.aeruginosa were mainly chosen by compatibility with Synechocystis in mind. The merB gene was native to E. coli and we theorized it could work well with the merA gene. The homologs from P.aeruginosa were mainly chosen by compatibility with Synechocystis in mind.

The genes were codon-optimized with CodonWizard to ensure proper translation in the host cell [5].

References

[1] Guerrero, F., Carbonell, V., Cossu, M., Correddu, D. and Jones. P. R. (2012) Ethylene synthesis and regulated expression of recombinant protein in Synechocystis sp. PCC 6803. PLoS ONE 7(11):e50470.http://dx.doi.org/10.1371/journal.pone.0050470

[2] Thiel, K., Mulaku, E., Dandapani, H., Nagy, C., Aro, E-M., Kallio, P. (2018) Translation efficiency of heterologous proteins is significantly affected by the genetic context of RBS sequences in engineered cyanobacterium Synechocystis sp. PCC 6803. Microbial Cell Factories 17(1):34. https://doi.org/10.1186/s12934-018-0882-2

[3] Thiel K, Patrikainen P, Nagy C, Fitzpatrick D, Pope N, Aro EM, Kallio P. (2019) Redirecting photosynthetic electron flux in the cyanobacterium Synechocystis sp. PCC 6803 by the deletion of flavodiiron protein Flv3. Microbial Cell Factories, 18: 189. https://doi.org/10.1186/s12934-019-1238-2

[4] Nagy, C., Thiel, K., Mulaku, E., Mustila, H., Tamagnini, P., Aro, E-M, Pacheco, C. C., Kallio, P. (2021) Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number. Microbial Cell Factories 20, 130. https://doi.org/10.1186/s12934-021-01622-2

[5] Rehbein, P., Berz, J., Kreisel, P., & Schwalbe, H. (2019). "CodonWizard" - An intuitive software tool with graphical user interface for customizable codon optimization in protein expression efforts. Protein expression and purification, 160, 84–93. https://doi.org/10.1016/j.pep.2019.03.018