Coding

Part:BBa_K3122000

Designed by: Laura Sanchez Lara   Group: iGEM19_MADRID_UCM   (2019-10-16)


AEGIS's lamB display system for Gram- bacteria


E. coli LamB is one of the best characterized and most flexible heterologous peptide carriers. This is due to its extracelular domains, consisting mainly of long loops. One of these loops can tolerate insertions of rather long sequences, between amino acid residues 153 and 154 (Sousa, 1996).

Our group have adapted this functional display system to type IIS enzymes grammar, and called it AEGIS's lamB display system for Gram-negative bacteria. This part was designed to use the outer membrane protein lamB of E. coli K12 strain to express any tag or short sequence to the extracellular medium on its specific permissive loop. The advantages of this approach are notable: not only can it be used for bacteria with cell wall ensuring its right functioning, but also it is really simple and easy to use following MoClo standard.

To see the design of this part go to the Design tab.

Biology

LamB is maltoporin precursor of Gram-negative bacteria. This outer membrane proteins family is involved in transport of maltodextrins across the outer membrane. Besides, LamB is the receptor of several different bacteriophagues as lambda fague. Gene sequence of LamB [Escherichia coli str. K-12 substr. MG1655] was obtained from EcoGene:EG10528.

Characterization

Characterization of this part has been conducted by building a transcriptional unit BBa_K3122005 and performing Lambda fague assays. This transcriptional unit was assembled in pARK1 alpha plasmid including the following parts:

  • BBa_K3122003: Promoter J23107 adapted to type IIS assembly
  • BBa_K2656009: RBS  B0030 adapted to type IIS assembly
  • BBa_K3122000: LamB coding sequence. This part is our AEGIS' lamB display system.
  • BBa_K2656026: Terminator B0015 adapted to type IIS assembly


How to use it

This new display system that we created can be used by future iGEM teams on their MoClo reactions with the desired tag to create level 1 constructs. The only requirement is to adapt the tag sequence to the new scars designed.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 152
    Illegal AgeI site found at 464
    Illegal AgeI site found at 1229
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 467
    Illegal BsaI.rc site found at 461

References

C. Sousa, A. Cebolla and V. de Lorenzo, "Enhanced metalloadsorption of bacterial cells displaying poly-His peptides", Nature Biotechnology, vol. 14, no. 8, pp. 1017-1020, 1996. Available: 10.1038/nbt0896-1017 [Accessed 20 October 2019].

Judging

The sharp design of this part allows a perfect scarless ligation of the desired tag into the permissive loop of LamB, type IIS adapted sequence, in the same one-pot reaction where MoClo is ocurring. It guarantees perfect expression of the tag.

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