Device

Part:BBa_K4831013

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


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

Overview

In our project we designed a new improved methylmercury detoxification system in Synechocystis using two mercury degrading enzymes: Mercuric reductase (MerA) and Alkylmercury lyase (MerB). To achieve this, we assembled a 7-fragment construct with the two genes of interest (merA and merB) regulated by the same lac-based inducible promoter with a “leaky transcription”. This expression device is specifically designed for Synechocystis sp. PCC6803.

The construct contains an IPTG inducible lac promoter variant, a ribosomal binding site S3 followed by the merA-gene (from Pseudomonas aeruginosa), a ribosomal binding site S5 followed by the merB-gene (from Pseudomonas aeurignosa), a transcription terminator sequence, and a spectinomycin resistance gene for antibiotic selection. The construct can be used to enhance your systems resistance to methyl mercury and mercuric compounds or for bioremediation as we did in our project.

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 assembled with a Golden Gate-inspired assembly system, leaving specific 4 bps scars between every part.

Usage and Biology

MerB mediates the first steps of degradation in various organomercurial salts. It catalyzes the protonolysis reaction where a proton (H+) attacks the organic mercury compound, breaks the carbon-mercury bond and leads to the formation of ionic mercury (Hg2+) as well as a respective organic compound. In the case of methylmercury the addition of carbon-mercury bond breaking protons (H+) leads to the formation of methane and ionic mercury (Hg2+). MerA subsequently detoxifies ionic mercury (Hg2+) with the help of a NADPH substrate. With many reactions in heterotrophic organisms, NADPH is often a limiting factor, making the autotrophic Synechocystis an optimal candidate to catalyze this reaction.

Source

Multiple places. RBS are native high expression genomic translation units. Go check individual part documents for more specific details.


Sequence and Features


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]

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


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