Device

Part:BBa_K4831016

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


IPTG inducible merB and YFP reporter 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 study only the effectiveness of merB in catalyzing the protonolysis reaction of methylmercury into Hg(II), we assembled a 7-fragment construct with two genes (merB and sYFP2) regulated by the same lac-based inducible promoter with a “leaky transcription.” The Yellow fluorescent protein produced by sYFP2 can be used to indicate the relative translation efficiency. 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 sYFP2-gene, a ribosomal binding site S5 followed by the merB-gene (from Escherichia coli) and a transcription terminator sequence. The construct can be used to enhance your systems resistance to methyl mercury and mercuric compounds.

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 specific 4 bps scars between every part.

NOTE! This composite doesn't translate YFP correctly since we werent able to edit the original parts sequence to delete the starting codon. Now there is a staring codon in the rbs s3 and in the gene itself. If you use this composite, make sure to delete the starting codon of SYFP2. Then the frame shit and rbs will work fine.

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+).

YFP, a fluorecent reporter protein can be used to study relative translation efficiency by measuring relative fluorescence values normalized to OD750 nm.

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 2754
    Illegal XbaI site found at 2735
    Illegal XbaI site found at 3248
    Illegal SpeI site found at 1511
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1498
    Illegal EcoRI site found at 2754
    Illegal SpeI site found at 1511
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1498
    Illegal EcoRI site found at 2754
    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 2754
    Illegal XbaI site found at 2735
    Illegal XbaI site found at 3248
    Illegal SpeI site found at 1511
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1498
    Illegal EcoRI site found at 2754
    Illegal XbaI site found at 2735
    Illegal XbaI site found at 3248
    Illegal SpeI site found at 1511
  • 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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