Part:BBa_K2384015

Repressor gene xylR+Strong PxylA promoter+RBS+Lpp-Omp-MBP(Pb)+His-tag

Xylose-inducible expression system: strong PxylA promoter and repressor gene xylR; after xylose addition, the repressor is released from the PxylA that activates transcription initiation.(BBa_K733002) They have been designed for high expression in B. megaterium.when fused with N-terminal fragment of Outer membrane protein A (OmpA), the lead metal binding peptide(MBP)will be translocated and displayed on the surface of bacteria, which will contribute a lot to increase the mental binding capacity when combined will other collection parts. In the Lpp-Omp display system with potential for displaying macromolecular proteins,it contains signal peptide and the first nine amino acids of Lpp and forty-sixth to 159th amino acids of OmpA.The former contains all the information that carries the protein to the outer membrane and anchors it to the inside of the outer membrane,and the latter can anchor the target fusion protein to the outside of the membrane.The MBP that follows it can be expose. PbrR is a member of the MerR family of metal-sensing regulatory protein, acts as an effective Pb(II) capturer. According to earlier research, the DNA binding domain and metal binding domain can function individually and the constructed peptide can form a stable dimer with its mercury and lead binding affinity remaining. This year our parts were doing codon optimisation.

Usage and Biology

Optimization Report

Gene Name: Lpp-OmpA+MBP(Pb)

Expression System:Bacillus megaterium

Gene Length:939 (bp)

1.Codon Used Adjustment The best value is 1 for sequence optimization.

Figure 1:Before Codon Adjustment

Figure 2:After Codon Adjustment

2.Codon Used Distribution Show the relative codon used distribution

Figure 3:Before Optimization

Figure 4:After Optimization

3.GC Content: The comparison of GC content between original sequence and optimized sequence

Figure 5:Before Optimization

Figure 6:After Optimization

Figure 7:The expression of Lpp-OmpA-MBP detected using SDS-PAGE

Figure 8:Plasmid digested by PvuII

Figure 9:Our genetic circuit

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 847
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]