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 in vitro sfGFP expression of cell-free protein synthesis (CFPS)^[3]. 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 validate the correctness of DNA sequence, result of Sanger sequencing for BBa_K5133004 show the successful assembly among T7 promoter (BBa_K5133000), RBS (this part), and sfGFP (BBa_K5133002) (Figure 2).

Figure 1. Design of basic part BBa_K5133001, generated by SnapGene.

Figure 2. Validation of DNA sequence 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')

aagaaggagatatacat

Red font: RBS

References

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

[2] Hausjell, J. et al. The effects of lactose induction on a plasmid-free E. coli T7 expression system. Bioengineering 7, 8 (2020). doi: 10.3390/bioengineering7010008

[3] Ba, F. et al. Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology. Biotechnology Journal 19, 2300327 (2024)). doi: 10.1002/biot.202300327

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]