Composite

Part:BBa_K5023014

Designed by: Lucas Daniel Ovelar Vargas   Group: iGEM23_UNILA-LatAm   (2023-10-08)
Revision as of 20:25, 10 October 2023 by Lucasovelar (Talk | contribs)


PAR_Ble_SP7_PHL7_Ter

This composite part have: PAR promoter, the bleomycin/zeocin resistance sequence, signal peptide, the PHL7 enzyme and the terminator. Designed to express and secrete PHL7 in Chlamydomonas reinhardtii, utilizing pJP32 backbone. This circuit was originally designed by our advisor João Molino.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 698
    Illegal PstI site found at 1572
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal NheI site found at 1211
    Illegal PstI site found at 698
    Illegal PstI site found at 1572
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 2043
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 698
    Illegal PstI site found at 1572
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 698
    Illegal PstI site found at 1572
    Illegal NgoMIV site found at 1079
    Illegal NgoMIV site found at 1140
    Illegal NgoMIV site found at 1357
    Illegal NgoMIV site found at 1458
    Illegal NgoMIV site found at 2370
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1808


File:Mfold-K3002006-1.png


Introduction to the Mhetygua Project

The Mhetygua project, initiated for the IGEM Global 2023 edition, represents a pioneering endeavor to combat the pressing issue of microplastic pollution. Spearheaded by a dedicated group of students, the project titled "MHETYGUÁ: algae as devices for the degradation of aquatic pollutants" seeks to harness the potential of algae, specifically Chlamydomonas reinhardtii, as a bio-tool for microplastic degradation. Beyond the scientific pursuit, the project embodies a broader vision of fostering environmental awareness and education, aiming to catalyze a shift towards sustainable practices and a reduced plastic footprint in the Latin American region.

Commentary on the Genetic Circuit Construction: Circuit Number 4

The genetic circuit, labeled as Circuit Number 4, is a unique design aimed at achieving the expression and secretion of the enzyme PHL7 in the microalgae Chlamydomonas reinhardtii. This circuit was originally conceptualized by our esteemed advisor, João Molino. The construction of this circuit involves the following key components:

  1. PAR Promoter: The PAR promoter initiates the transcription process. Recognized for its light-inducible properties, it allows for the modulation of gene expression based on light conditions, making it particularly effective in Chlamydomonas reinhardtii.
  2. Bleomycin/Zeocin Resistance Sequence: This sequence provides resistance against the antibiotics bleomycin and zeocin. Its inclusion ensures that only algal cells with the integrated genetic circuit can thrive in the presence of these antibiotics, facilitating the selection of desired traits.
  3. Signal Peptide: Essential for the secretion of the synthesized enzyme, the signal peptide guides the PHL7 protein towards the secretory pathway, ensuring its release into the surrounding environment.
  4. PHL7 Enzyme: PHL7 is the focal enzyme of this circuit. Once expressed and secreted, it is expected to perform its designated biochemical functions, contributing to the overarching goals of the Mhetygua project.
  5. Terminator: The terminator sequence marks the end of the transcription process, ensuring that the genetic expression is both stable and efficient.

The entire circuit is housed within the pJP32 backbone. This vector is chosen for its compatibility with Chlamydomonas reinhardtii, ensuring that the circuit is successfully integrated and expressed within the algal cells.



Mhetyguá Parts

PAR Promoter

We chose the PAR promoter because it is a promoter sequence commonly used in Chlamydomonas reinhardtii for the expression of transgenes. The promoter in question is light-inducible, and its activity is modulated by the availability of light-harvesting complexes, specifically the phycobilisome complex. PAR is relatively strong, and its expression can be adjusted by changing the intensity and duration of light exposure. This promoter is a combination of the HSP70A (upstream) and RBCS2 promoters; no heat shock is required as a transcription activator. Several characteristics of this promoter suggest its usefulness as a tool to enhance transgene expression in this alga. It can, by itself, confer high inducibility to a transgene.

Reference: Schroda, M., Blöcker, D., & Beck, C. F. (2000). The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant Journal, 21(2), 121-131. <a href="https://doi.org/10.1046/j.1365-313x.2000.00652.x">https://doi.org/10.1046/j.1365-313x.2000.00652.x</a>

Ble Resistance Gene

The bleomycin resistance gene confers resistance to the antibiotic bleomycin and zeocin. In the context of Chlamydomonas reinhardtii the bleomycin resistance of cells harboring the Sh ble gene is attributed to the action of the bleomycin-binding protein. This protein binds to bleomycin, sequestering it and preventing it from exerting its antibiotic effect on the cell. This resistance mechanism is particularly useful in genetic engineering experiments. By introducing the Sh ble gene alongside other genes of interest into Chlamydomonas reinhardtii, researchers can easily select for cells that have taken up the foreign DNA by exposing them to bleomycin. Only the cells that have incorporated the Sh ble gene (and by extension, the other genes of interest) will survive, allowing for efficient selection.

Reference: Gatignol, A., Durand, H., & Tiraby, G. (1988). Bleomycin resistance conferred by a drug‐binding protein. FEBS Letters, 230.

SP7 Signal Peptide

For the constructs expressed in Chlamydomonas reinhardtii, we used the signal peptide SP7 which has been recently characterized in a paper by João Molino. SP7 is a novel protein identified in the flagella of Chlamydomonas reinhardtii. The flagella are whip-like appendages that protrude from the cell body and are vital for motility and sensory functions. Which confers an interesting capacity to secrete recombinant proteins.

Reference: Molino JVD, de Carvalho JCM, Mayfield SP (2018) Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii. PLoS ONE 13(2): e0192433. <a href="https://doi.org/10.1371/journal.pone.0192433">https://doi.org/10.1371/journal.pone.0192433</a>

FAST-PETase Enzyme

FAST-PETase is derived from the original sequence of the PETase enzyme, with several modifications introduced through directed evolution. The FAST-PETase sequence is approximately 590 amino acids in length and consists of several domains, including a signal peptide sequence for secretion (twin-arginine translocation (Tat) signal peptide), a catalytic domain, and a carbohydrate-binding module. The catalytic domain is responsible for breaking the ester bonds in PET, while the carbohydrate-binding module helps anchor the enzyme to the plastic surface. Directed mutations have enhanced the enzyme's ability to bind to PET and break it down into its components more quickly. Therefore, we chose FAST-PETase because it is capable of breaking down PET at a faster rate than the original PETase enzyme.

Reference: Lu, H., Diaz, D.J., Czarnecki, N.J. et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 604, 662–667 (2022). <a href="https://doi.org/10.1038/s41586-022-04599-z">https://doi.org/10.1038/s41586-022-04599-z</a>

Linker

Linker, the most basic function of linkers in recombinant proteins is to covalently join functional domains (e.g., flexible linkers or rigid linkers) or release them under desired conditions (clickable linkers). Linkers can also provide many derived functions, such as improving the folding and stability of recombinant proteins, enhancing the expression of fusion proteins, and they can enhance the bioactivity of fusion proteins. The linker we chose binds to the C-terminal region of MHETase to the N-terminal of PETase, with flexible glycine-serine linkers of 12 total residues (Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly).

References:
KNOTT, B. C. et al. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proceedings of the National Academy of Sciences of the United States of America, v. 117, n. 41, p. 25476–25485, 13 out. 2020. <a href="https://doi.org/10.1073/pnas.2006753117">https://doi.org/10.1073/pnas.2006753117</a>
CHEN, X.; ZARO, J. L.; SHEN, W.-C. Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, v. 65, n. 10, p. 1357–1369, out. 2013. <a href="http://dx.doi.org/10.1016/j.addr.2012.09.039">http://dx.doi.org/10.1016/j.addr.2012.09.039</a>

MHETase

MHETase is an enzyme capable of breaking down MHET into its monomers, terephthalic acid (TA) and ethylene glycol (EG), through the cleavage of the ester bond. The enzyme is composed of two domains: a large N-terminal domain and a small C-terminal domain, connected by a linker region. The N-terminal domain contains the enzyme's catalytic site and is responsible for substrate recognition and binding, while the C-terminal domain plays a role in regulating the enzyme's activity.

Reference: Maity, W., Maity, S., Bera, S. et al. Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes. Appl Biochem Biotechnol 193, 2699–2716 (2021). <a href="https://doi.org/10.1007/s12010-021-03562-4">https://doi.org/10.1007/s12010-021-03562-4</a>

PHL7 Enzyme

PHL7 is a thermophilic polyester hydrolase isolated from a plant compost metagenome along with six homologs (PHL1-6)4. It rapidly hydrolyzes amorphous PET at 70°C, producing terephthalic acid (TPA) and ethylene glycol (EG), outperforming all previously reported PET hydrolytic enzymes, including modified variants.

Reference: RICHTER, P. K. et al. Structure and function of the metagenomic plastic-degrading polyester hydrolase PHL7 bound to its product. Nature Communications, v. 14, n. 1, p. 1905, 5 abr. 2023. <a href="https://doi.org/10.1038/s41467-023-37415-x">https://doi.org/10.1038/s41467-023-37415-x</a>

RPL23 Terminator

RPL23 is a termination signal used to terminate the transcription of the RPL23 gene. This sequence signals the RNA polymerase to dissociate from the DNA template and release the newly synthesized RNA molecule. The RPL23 terminator sequence has been found to work well in terminating transcription in a variety of organisms and is often used as a standard terminator sequence in genetic engineering.

Reference: LÓPEZ‐PAZ, C. et al. Identification of Chlamydomonas reinhardtii endogenous genic flanking sequences for improved transgene expression. The Plant Journal, v. 92, n. 6, p. 1232–1244, 18 nov. 2017. <a href="https://doi.org/10.1111/tpj.13731">https://doi.org/10.1111/tpj.13731</a>

mCherry Reporter

mCherry is a popular fluorescent protein derived from Discosoma sp. and is frequently used as a reporter gene in various organisms due to its bright red fluorescence. In the context of Chlamydomonas reinhardtii, a model green alga, mCherry has been employed as a valuable tool for genetic engineering. The expression level of mCherry can be correlated with the activity of promoters or other genetic elements, aiding in the analysis of gene expression patterns in Chlamydomonas.

Reference: ARIAS, C. et al. Semicontinuous system for the production of recombinant mCherry protein in Chlamydomonas reinhardtii. Biotechnology Progress, v. 37, n. 2, 20 nov. 2020.

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