Part:BBa_K4831015
IPTG inducible merA (Synechocystis) expression device with a EFE as a reporter gene
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 the effectiveness of only merA in reducing mercuric ions(Hg2+) into elemental mercury Hg0, we assembled a 7-fragment construct with two genes (merA and EFE) regulated by the same lac-based inducible promoter with a “leaky transcription”. The ethylene-forming enzyme produced by EFE can be used to indicate the relative translation efficiency. This expression device is specifically designed for Synechocystis sp. PCC6803.
The device contains an IPTG inducible lac promoter variant, a ribosomal binding site S3 followed by the merA-gene (from Synechocystis sp. PCC6803), a ribosomal binding site S5 followed by the EFE-gene and a transcription terminator sequence. The device 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 assembled with a Golden Gate-inspired assembly system, leaving specific 4 bps scars between every part.
Usage and Biology
MerA 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.
Ethylene forming enzyme (EFE) enables the conversion of intracellular 2-oxoglutarate into ethylene, which can be quantitatively measured by gas chromatography (GC). [1,2]
Source
Multiple places. RBS are native high expression genomic translation units. Go check individual part documents for more specific details.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1498
Illegal SpeI site found at 1511 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1498
Illegal SpeI site found at 1511 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1498
Illegal BglII site found at 1612
Illegal XhoI site found at 1481 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1498
Illegal SpeI site found at 1511 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1498
Illegal SpeI site found at 1511
Illegal NgoMIV site found at 3788 - 1000COMPATIBLE 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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