Coding

Part:BBa_K4447005

Designed by: Rafael Garcia Gomez   Group: iGEM22_TecMonterrey_GDL   (2022-09-30)


Bacterial Expression System for the Detection of Erythromycin based on FRET

Expression system based on FRET biosensor for the detection of erythromycin. This system is suitable for cloning in pSB1C3 as the plasmid backbome, which propagates the BioBrick part and is widely used by other iGEM teams.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal XhoI site found at 3157
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3812
    Illegal SapI site found at 961


Usage and Biology

The usage of genetic constructs that are designed to produce a protein, either inside or outside a cell, is important in several ways, such as increasing the expression rate. With this BioBrick, we are generating a bacterial expression platform that is capable of generating several copies of our protein of interest, EryK, coupled with the FRET system for the detection of erythromycin.

Our expression platform is composed of the following elements: an inducible promoter, which is regulated in response to specific stimuli; a ribosome-binding site to bind the ribosome for the initiation of translation; and a transcription terminator that mediates the release of the transcript RNA from the translational complex. Finally, we concluded the following parts were the most optimal to use for our composite part: 

•BBa_I0500: Inducible pBAD/araC promoter. Expression by the pBAD promoter can be regulated tightly by induction and 
subsequent inhibition. In the presence of L-arabinose and low glucose concentrations, transcription initiation occurs. 

•BBa_J61101: Ribosome-binding site from Anderson Library. Anderson (2006) states that these RBS are suitable for 
general protein expression in Escherichia coli or other prokaryotes. Results from TU Delft (2010) measured RBS strength, 
concluding that BBa_J61101 is the strongest of a library composed of six different ribosome-binding sites from the Anderon 
Library.

•BBa_B0010: rrnB T1 terminator from Escherichia coli. BBa_B0010 is a transcriptional terminator consisting of a 64-
base-pair stem loop. Orosz et al. (1991) state that this region was shown to be an efficient terminator in isolated form.

References

[1]. Orosz, A., Boros, I., & Venetianer, P. (1991). Analysis of the complex transcription termination region of the Escherichia coli rrnB gene. European journal of biochemistry, 201(3), 653–659. https://doi.org/10.1111/j.1432-1033.1991.tb16326.x
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