Part:BBa_K5487003:Design
T7 Promoter + lac operator + RBS + Lpp-OmpA + Z1-PETase + MHETase
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1913
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1913
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 533
Illegal BamHI site found at 1796 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1913
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1913
Illegal NgoMIV site found at 1481
Illegal NgoMIV site found at 1595
Illegal NgoMIV site found at 2051
Illegal AgeI site found at 1665 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
Designing a composite sequence like the one housed in the pET30a vector, which includes elements such as the T7 Promoter, lac operator, RBS, Lpp-OmpA, Z1-PETase, and MHETase, involves several critical considerations to ensure successful expression and functionality of the recombinant protein. Here are some key design considerations:
Regulatory Element Compatibility:
Ensuring that the T7 promoter and the lac operator are compatible and function correctly in the host organism, BL21 (DE3), to allow for tight regulation of gene expression. Ribosome Binding Site (RBS) Design:
Designing an RBS that optimally matches the translation machinery of the host, ensuring efficient translation initiation of the downstream genes. Gene Fusion and Expression:
Ensuring that the fusion of Lpp-OmpA with Z1-PETase and MHETase does not adversely affect the folding, stability, or functionality of the individual proteins. Signal Peptide Function:
Confirming that the Lpp signal peptide effectively targets and translocates the fusion protein to the bacterial outer membrane in BL21 (DE3). Protein Solubility and Stability:
Designing the fusion protein to maintain solubility and stability within the bacterial cell, preventing aggregation or degradation. Orientation and Exposure:
Ensuring the correct orientation of the Z1-PETase and MHETase on the bacterial surface to facilitate their function, such as substrate binding and catalysis. Codon Usage:
Optimizing codon usage to match the host's preference for efficient translation and to potentially enhance expression levels. Mutagenesis and Protein Engineering:
Considering the need for site-directed mutagenesis to improve protein characteristics, such as enhancing stability, activity, or surface exposure. Inducer Concentration:
Determining the optimal concentration of IPTG to induce expression without causing toxicity to the cells or reducing the cell's growth rate. Protein Purification and Handling:
Including tags or designing the construct in a way that facilitates downstream purification and handling of the recombinant protein. Sequence Verification:
Verifying the sequence of the construct through DNA sequencing to ensure there are no errors introduced during the cloning process. Bioinformatics Analysis:
Utilizing bioinformatics tools to predict the structural and functional impact of the fusion on the proteins and the overall stability of the construct. Safety Considerations:
Designing the construct to minimize any potential hazards, such as allergenicity or toxicity, especially if the protein will be used in industrial or medical applications. Scalability:
Considering the scalability of the expression system, ensuring that the construct performs well not only in small-scale cultures but also in larger bioreactors if needed. Environmental Impact:
Assessing the environmental impact of the construct, particularly if the bacteria are intended for release or application in the environment.
Source
Plasmid Description: The composite part is a synthetic construct assembled within the pET30a expression vector. This vector is a commonly used plasmid derived from the pET series, which are based on the ColE1 origin of replication and are designed for high-level expression of cloned genes in bacterial systems, particularly in E. coli. The constructed plasmid is intended for research and biotechnological applications, particularly for the expression and surface display of the Z1-PETase and MHETase fusion protein on BL21 (DE3) cells for biodegradation of PET plastics.