Part:BBa_K5133001

Ribosome binding site (RBS, from plasmid pJL1)

Group: GEC-China (iGEM 2024, team number: #5133)

Brief introduction

This basic part is derived from plasmid pJL1 (Addgene: #69496)^[1], including a conserved ribosome binding site (RBS) as 5'-aagaaggaga-3'^[2]. The plasmid pJL1 is commonly used for the sfGFP expression for cell-free protein synthesis (CFPS). Hence, this part is used for the construction of three composite parts: BBa_K5133004 (sfGFP generator), BBa_K5133006 (Microcin H47 generator), and BBa_K5133008 (Microcin M generator), for CFPS in our project.

Design and characterization

The plasmid design of this biological part is shown as Figure 1, assembled with iGEM standard backbone pSB1C3. To demonstrate the correctness of DNA sequence, Results of Sanger sequencing for BBa_K5133004 showing the successful assembly among T7 promoter, RBS, and sfGFP (Figure 2).

Figure 1. Design of basic part BBa_K5133000, generated by SnapGene.

Figure 2. Validation the DNA sequence of BBa_K5133000 by Sanger sequencing, generated by SnapGene.

Usages

This part is used for the construction of three composite parts: BBa_K5133004 (sfGFP generator), BBa_K5133006 (Microcin H47 generator), and BBa_K5133008 (Microcin M generator), for CFPS in our project.

DNA sequence (from 5' to 3')

atcccgcgaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaacttt

Red font: conserved T7 promoter

References

[1] https://www.addgene.org/69496/

[2] Conrad, T. et al. Maximizing transcription of nucleic acids with efficient T7 promoters. Communications Biology 3, 439 (2020). doi: 10.1038/s42003-020-01167-x

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]