Coding

Part:BBa_K4831012

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


A synthetic codon-optimized EFE-gene encoding ethylene forming enzyme for Synechocystis sp. PCC6803

Overview

A synthetic codon-optimized EFE-gene encoding EFE for Synechocystis sp. PCC6803. The modified EFE gene codes for Ethylene forming enzyme, which enables the conversion of intracellular 2-oxoglutarate into ethylene, which can be quantitatively measured by gas chromatography (GC). The EFE-gene can thus be utilized as a reporter gene.

Design Notes

The gene has been codon-optimized to work in Synechocystis sp. PCC6803. The sequence is modified with overhangs for position 5 to fit a type IIs assembly with 7 fragments using BbsI. BbsI site removed from EFE. The stop codon is included in the gene sequence. One codon has been changed to GAG from GAA to avoid an illegal restriction site (Sap1).

NOTE! The starting codon ATG has been removed to be compatible with our composite part (reading frame shift).

Source

A synthetic codon-optimized EFE-gene encoding EFE for Synechocystis sp. PCC6803. EFE has previously been used as a reporter gene in several studies [1, 2, 4].

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 328
  • 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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