Part:BBa_K5487005:Design
T7 Promoter + lac operator + RBS + Lpp-OmpA + sfGFP
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- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 547
Design Notes
Name: Membrane-Targeted Superfolder Green Fluorescent Protein (sfGFP) Expression Cassette
Function: This composite part is designed to test the functionality of the Lpp-OmpA membrane targeting sequence by fusing it with the superfolder green fluorescent protein (sfGFP). The construct allows for the expression of sfGFP targeted to the bacterial inner membrane, facilitating the assessment of membrane protein expression and localization.
Components and Their Roles:
T7 Promoter: A strong, inducible promoter derived from the T7 bacteriophage. It provides a high level of transcription when induced with isopropyl β-D-1-thiogalactopyranoside (IPTG).
lac operator: A DNA sequence that, in conjunction with the lac repressor, enables the controlled expression from the T7 promoter. It ensures that the promoter is off in the absence of IPTG and can be turned on in a controlled manner.
Ribosome Binding Site (RBS): A critical sequence for the initiation of protein translation, it recruits ribosomes to the mRNA, ensuring efficient translation of the sfGFP gene.
Lpp-OmpA: A fusion protein construct composed of the lipoprotein signal peptide Lpp and the outer membrane protein A (OmpA). This sequence is designed to direct the sfGFP to the bacterial membrane, allowing for the assessment of membrane targeting.
sfGFP: A bright, fast-folding variant of the green fluorescent protein (GFP) from Aequorea victoria. It serves as a reporter protein to visualize the expression and localization of the Lpp-OmpA fusion.
Optimal Conditions:
Temperature: Typically grown at 37°C for optimal bacterial growth and protein expression. Media: LB broth or other rich media supplemented with the appropriate antibiotics. Induction: IPTG is added to a final concentration of 0.1-1 mM to induce expression from the T7 promoter. Application:
Protein Localization: To determine if the Lpp-OmpA sequence can successfully direct sfGFP to the bacterial membrane. Fluorescence Assay: To verify the expression of the fusion protein by observing GFP fluorescence under a fluorescence microscope. Membrane Protein Expression: To serve as a model system for the expression of other membrane proteins fused to sfGFP. Usage in Projects:
Transformation: Transform the construct into a suitable bacterial host, such as E. coli BL21 (DE3). Cultivation: Grow the transformed cells in appropriate media with antibiotics. Induction: Induce expression with IPTG, and allow the culture to grow for a period to allow protein expression. 4.Fluorescence Observation: Observe the cells under a fluorescence microscope to check for GFP fluorescence, indicating successful membrane targeting. Safety Considerations:
Handle the bacterial cultures and recombinant DNA with appropriate biosafety precautions. Use personal protective equipment (PPE) and follow good microbiological practices. Storage and Stability:
Store bacterial glycerol stocks at -80°C for long-term preservation. Maintain cell cultures under stable conditions to prevent loss of recombinant protein functionality.
Source
Plasmid Vector: pET30a
Component Origins:
T7 Promoter:
Source: The T7 Promoter is a synthetic element derived from the T7 bacteriophage. It is widely used in molecular biology for driving high levels of gene expression in bacterial systems, particularly in E. coli. lac operator:
Source: The lac operator sequence is originally from the Escherichia coli lac operon, which is a well-studied genetic system. It is used here to provide inducible control of the T7 promoter. Ribosome Binding Site (RBS):
Source: The RBS is a synthetic sequence designed to optimize translation initiation efficiency in bacterial systems. It is tailored to match the preferences of the host organism's ribosomes. Lpp-OmpA:
Source: Lpp: The lipoprotein signal peptide Lpp is derived from various bacterial species and is used for directing proteins to the bacterial membrane. OmpA: The OmpA sequence comes from the outer membrane protein A of E. coli, which serves as a stable anchor for the fusion protein in the outer membrane. sfGFP (superfolder Green Fluorescent Protein):
Source: sfGFP is a variant of the Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria. It has been engineered for improved folding and brightness, making it a popular choice as a fluorescent reporter in living cells. Construction Details:
The composite part is assembled synthetically using molecular cloning techniques. The individual components are combined in a specific order within the pET30a vector to create the final construct. The assembly process involves several steps, including PCR amplification of individual parts, restriction enzyme digestion, ligation, and transformation into E. coli. The final construct is verified through DNA sequencing to ensure the correct order and orientation of the components. Expression Host:
BL21 (DE3): This E. coli strain is commonly used for the expression of recombinant proteins. It contains the T7 RNA polymerase gene under the control of the lacUV5 promoter, allowing for the induction of the T7 promoter with IPTG. Application:
The composite part is designed for testing the functionality of the Lpp-OmpA membrane targeting sequence by fusing it with sfGFP. The construct allows for the expression of sfGFP targeted to the bacterial inner membrane, facilitating the assessment of membrane protein expression and localization. Safety and Handling:
As with all recombinant DNA work, appropriate biosafety procedures should be followed, including the use of personal protective equipment and proper disposal of waste materials. Storage:
The plasmid is typically stored as transformed bacterial glycerol stocks at -80°C for long-term preservation or as isolated plasmid DNA for short-term use.