Coding

Part:BBa_K2114010

Designed by: Wladislaw Stroukov   Group: iGEM16_Freiburg   (2016-10-08)


GST_HA_G4S_CotG

N-terminal fusion of glutathione S-transferase to spore crust gene cotG by a flexible GGGGS linker.


Usage and Biology

cotG spore gene for surface display [Ref: ] GST converts CDNB+GSH Usage: 3A assembly this part + appropriate promoter into pBS1C [Ref: B4 Mascher Lab]


    • Contributions Below Completed by Brian Baxter of IGEM Concordia 2020



  • Part Purpose


- This part is primarily served for targeted drug delivery in bacillus subtilis. Other possible uses when considering the site specific enzymatic capabilities of the part include metabolite production and bioremediation.



  • Genetic connection of components


- Glutathione S-transferase (gene) - HA epitope tag - GGGGS linker - CotG (gene)



  • Components


1) cotG


- cotG is a gene of Bacillus subtilis which codes for an exterior protein member of its spore coat. It acts to assemble some of the outer proteins of the spore coat. This protein's functionality is dependent upon phosphorylation by a still unknown enzyme. This is not a conserved protein, however, most spore forming bacteria have a protein of similar functionality recognized by a positively charged region containing randomly coiled tandem repeats.


2) glutathione S-transferase


- Glutathione-S-transferase (GST) catalyzes the nucleophilic conjugation of glutathione (GSH) with many types of electrophilic substrates. This enzyme will catalyze a reaction on the spore coat causing proteins to become ‘sticky’ allowing spores to attach to surfaces, an action which allows the activation of prodrugs on site.


3) HA epitope tag


- Epitope tags allow for easier detection, purification and isolation of proteins. The Human influenza hemagglutinin (HA) is a glycoprotein, its incorporation in recombinant proteins has shown to not affect the activity of proteins. The HA-tag allows binding to an epitope-specific nanobody (single-domain antibody), this is the ‘targeting’ part of the device.


- Sequence: 5' TAC CCA TAC GAT GTT CCA GAT TAC GCT 3' Or 5' TAT CCA TAT GAT GTT CCA GAT TAT GCT 3'


4) GGGGS linker


- Gly-Gly-Gly-Gly-Ser is an amino acid chain used to connect two parts of a device. This linker is referred to as a ‘fold breaking linker’ which allows expression from a fusion breaker protein partner

- The serine offers polar character which improves solubility helping to prevent aggregation. The role of a linker is to provide additional flexibility in the region. The simplicity of the amino acids is to not allow the formation of secondary structures. As listed under



  • Central Reaction and Usage


A) GST converts CDNB+GSH


- Enzyme: Glutathione-S-transferase (GST) - Enzymatic target: Glutathione (GSH) - Substrate: 1-chloro-2,4-dinitrobenzene (CDNB)


B) 3A assembly of the - part + appropriate promoter - into pBS1C


- The team suggested approach for using this part is to use the Biobrick 3A assembly methodology to incorporate the part with an appropriate promoter into pBs1C, a biobrick compatible empty integrative vector designed for Bacillus Subtilis. As mentioned under the part design page added by the part creator, the HA epitope tag and GGGGS linker were added by primer extension and PCR. pGEX-6P-1 vector was selected to amplify the glutathione-S-transferase gene.



Characterization

I) Expression: WB II) Surface localization: III) FACS

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 688
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 85



References:

1. Laborde, E., P. Ruzza, A., GS. Zhao, X., WH. Habig, M., J. Lu, Y., W. Zhao, M., . . . R. Haaften, G. (1970, January 01). Simple method for visual detection of glutathione S -transferase activity and inhibition using cysteamine-capped gold nanoparticles as colorimetric probes. Retrieved September 02, 2020, from https://link.springer.com/article/10.1007/s13404-015-0171-3

2. Radeck, J., Kraft, K., Bartels, J., Cikovic, T., Dürr, F., Emenegger, J., . . . Mascher, T. (2013, December 2). The Bacillus BioBrick Box: Generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. Retrieved September 02, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177231/

3. Popp, P., Dotzler, M., Radeck, J., Bartels, J., & Mascher, T. (2017, November 8). The Bacillus BioBrick Box 2.0: Expanding the genetic toolbox for the standardized work with Bacillus subtilis. Retrieved September 02, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678133/

4. Saggese, A., Isticato, R., Cangiano, G., Ricca, E., & Baccigalupi, L. (2016, May 15). CotG-Like Modular Proteins Are Common among Spore-Forming Bacilli. Retrieved September 02, 2020, from https://jb.asm.org/content/198/10/1513

5. Zablotowicz, R., Hoagland, R., Locke, M., & Hickey, W. (1995, March). Glutathione-s-transferase activity and metabolism of glutathione conjugates by rhizosphere bacteria. Retrieved September 02, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1388388/

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