Team:Tec-Chihuahua/Parts

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Parts

Basic Parts Used

T7 Promoter and RBS [BBa_K525998] This unregulated T7 promoter, which includes an integrated RBS, has high levels of transcription when the T7 RNA polymerase is present; thus, expression of the BioBrick™ for AtPFN1 is under the control of IPTG in E. coli BL21 (DE3) strains.

Double Terminator from E. coli rrnB [BBa_B0015] This double terminator consists of T1 form E. coli rrnB, which consists of a 64 bp stem-loop, and TR from coliphage T7, which is a transcription terminator. It was used for the construction of our genetic circuitry, which needed an efficient and reliable terminator.

Promoter (LacI regulated) [BBa_R0010] This is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites, one for CAP protein and the other for LacI protein. Since Lacl can be inhibited by IPTG, this promoter can be induced by this reagent. It was used for the construction of our circuitry, which needed an efficient and regulated promoter.

Basic Parts Added to the Registry

Erv1p [BBa_K2959002] This DNA sequence codes for Erv1p, a protein from Saccharomyces cerevisiae capable of oxidizing thiol groups in proteins and catalyzing disulfide bond formation.1 For a successful expression in E. coli, it’s indispensable to avoid interactions that result in aggregation of folding intermediates. The usage of Erv1p is a mechanism for the formation of disulfide bonded recombinant proteins in prokaryotic cells.2

PsDef1 [BBa_K2959003] This defensin, also known as Pinus sylvestris Defensin 1, causes morphological changes in fungi mycelium when interacting with the sphingolipid membrane on fungal cells, disrupting the membrane integrity. It also possesses antimicrobial activity in Gram-positive and Gram-negative bacteria.3

WAMP1b [BBa_K2959006] Antimicrobial peptide that comes from Triticum kiharae seeds, consists of 116 amino acids which includes 10 cysteine residues that form consecutive disulfide bonds. This makes it a highly stable molecule. It is capable of inhibiting fungalysins from phytopathogenic fungi acting as a defense mechanism in plants against fungal infections.5,6

AtPFN1 [BBa_K2959009] Also known as Arabidopsis thaliana Profilin 1, this protein is a profilin extracted from the plant Arabidopsis thaliana which means that it is an actin binding protein. It penetrates the cell wall and membrane of fungal cells, generating reactive oxygen species and mitochondrial superoxide triggering cell apoptosis.7

Composite Parts Added to the Registry

Expressible PsDef1 [BBa_K2959004]

- (LacI promoter) + (RBS) + (6x His-Tag) + (PsDef1) + (Double Terminator)
- (BBa_R0010) + (BBa_B0034) + (BBa_K2959003) + (BBa_B0015)

This BioBrick™ contains the necessary genetic circuitry to be induced by IPTG and express Defensin 1 from Pinus sylvestris. It consists of a LacI regulated promoter, RBS, the coding sequence of PsDef1 as a fusion protein with a 6x His-Tag for purification by metal affinity chromatography, and a double terminator. This BioBrick™ causes morphological changes in fungi mycelium disrupting the membrane integrity.4

Expressible PsDef1 with Erv1p [BBa_K2959005]

- (LacI promoter) + (RBS) + (6x His-Tag) + (PsDef1) + (RBS) + (Erv1p) + (Double Terminator)
- (BBa_R0010) + (BBa_B0034) + (BBa_K2959003) + (BBa_K2959002) + (BBa_B0015)

This composite part consists of LacI regulated promoter, RBS, coding sequence of PsDef1 as a fusion protein with a 6x His-Tag, a second ribosome binding site, coding sequence for Erv1p and a double terminator. Expression can be positively regulated by the addition of lactose or IPTG thanks to its promoter. Erv1p helps with proper folding since it catalyzes the formation of disulfide bonds.1

Expressible WAMP1b [BBa_K2959007]

- (LacI promoter) + (RBS) + (6x His-Tag) + (WAMP1b) + (Double Terminator)
- (BBa_R0010) + (BBa_B0034) + (BBa_K2959006) + (BBa_K2959002) + (BBa_B0015)

Composite part consisting of a LacI regulated promoter, ribosome binding site, coding sequence for WAMP1b as a fusion protein with a 6x His-Tag for purification by metal affinity chromatography, and a double terminator. This construct allows the positive expression of the peptide using IPTG or lactose thanks to its LacI promoter. This peptide can successfully bind to chitin and acts as a plant defense mechanism against fungi.6

Expressible WAMP1b with Erv1p [BBa_K2959008]

- (LacI promoter) + (RBS) + (6x His-Tag) + (WAMP1b) + (RBS) + (Erv1p) + (Double Terminator)
- (BBa_R0010) + (BBa_B0034) + (BBa_K2959006) + (BBa_B0015)

This composite part consists of LacI regulated promoter, RBS, coding sequence of WAMP1b as a fusion protein with a 6x His-Tag, a second ribosome binding site, coding sequence for Erv1p, and a double terminator. Expression can be positively regulated by the addition of lactose or IPTG thanks to its promoter. Erv1p helps with proper folding since it catalyzes the formation of disulfide bonds.1

Expressible AtPFN1 [BBa_K2959010]

- (T7 Promoter) + (RBS) + (6x His-Tag) + (WAMP1b) + (Double Terminator)
- (BBa_K525998) + (BBa_K2959006) + (BBa_B0015)

Composite part that consists of a T7 promoter, ribosome binding site, coding sequence for AtPFN1 as a fusion protein with a 6x His-Tag for purification by metal affinity chromatography, and a double terminator. Expression can be positively regulated by the addition of IPTG thanks to its promoter. This construct generates a protein capable of penetrating the cell wall and membrane of fungal cells, triggering cell apoptosis.7

References

  1. Lee, J. E., Hofhaus, G., & Lisowsky, T. (2000). Erv1p from Saccharomyces cerevisiae is a FAD‐linked sulfhydryl oxidase. FEBS letters, 477(1-2), 62-66. doi: 10.1016/s0014-5793(00)01767-12.
  2. Hatahet, F., Nguyen, V. D., Salo, K. E., & Ruddock, L. W. (2010). Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli. Microbial cell factories, 9(1), 67. doi: 10.1186/1475-2859-9-673.
  3. Bulat I. Khairutdinov, Elena A. Ermakova, Yuri M. Yusy- povych, Elena K. Bessolicina, Nadezhda B. Tarasova, Yana Y. Toporkova, Valentina Kovaleva, Yuriy F. Zuev, Irina V. Nesmelova, NMR structure, conformational dynamics, and biological activity of PsDef1 defensin from Pinus sylvestris, BBA- Proteins and Proteomics (2017), doi:10.1016/j.bbapap.2017.05.0124.
  4. Khairutdinov, B. I., Ermakova, E. A., Yusypovych, Y. M., Bessolicina, E. K., Tarasova, N. B., Toporkova, Y. Y., ... & Nesmelova, I. V. (2017). NMR structure, conformational dynamics, and biological activity of PsDef1 defensin from Pinus sylvestris. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1865 (8), 1085-1094. doi:10.1016/j.bbapap.2017.05.0125.
  5. Dubovskii, P. V., Vassilevski, A. A., Slavokhotova, A. A., Odintsova, T. I., Grishin, E. V., Egorov, T. A., & Arseniev, A. S. (2011). Solution structure of a defense peptide from wheat with a 10-cysteine motif. Biochemical and Biophysical Research Communications, 411(1), 14–18. doi: 10.1016/j.bbrc.2011.06.0586.
  6. Odintsova, T. I., Vassilevski, A. A., Slavokhotova, A. A., Musolyamov, A. K., Finkina, E. I., Khadeeva, N. V., … Egorov, T. A. (2009). A novel antifungal hevein-type peptide fromTriticum kiharaeseeds with a unique 10-cysteine motif. FEBS Journal, 276(15), 4266–4275. doi:10.1111/j.1742-4658.2009.07135.x7.
  7. Park, S. C., Kim, I. R., Kim, J. Y., Lee, Y., Kim, E. J., Jung, J. H., ... & Lee, J. R. (2018). Molecular mechanism of Arabidopsis thaliana profilins as antifungal proteins. Biochimica et Biophysica Acta (BBA)-General Subjects, 1862 (12), 2545-2554. doi:10.1016/j.bbagen.2018.07.028

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