Device

Part:BBa_K4593020

Designed by: Jianfei Song   Group: iGEM23_BNDS-China   (2023-10-08)
Revision as of 03:39, 11 October 2023 by Jianfei Song (Talk | contribs) (→‎Overview)

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S. aureus in vivo elimination apparatus for E. coli

Overview

This part contains 3 genetic circuits: an S. aureus quorum sensing (QS) detection system (BBa_I746101), an Endolysin combinative expression system (BBa_K4593019), and a self-lysing release system (BBa_I746104 and BBa_K4593001).

As the final intended product of our project's S. aureus elimination module, this device could constitutively express S. aureus targeting endolysins (a set of enzymes that specifically lyses S. aureus) and detect the presence of S. aureus QS signal. If the QS signal molecule is detected, the self-lysing enzyme composite (Spn1s_LysRZ) will be activated, and the engineered cell will lyse itself to release the endolysin that eliminates pathogenic S. aureus.

Fig.1 An abbreviated demonstration of the single cell gene circuit of S. aureus in vivo elimination device. Pro 1 and Pro 3 are both constitutive promoters. RBS and terminator are omitted for the sake of space.

Endolysin expression circuit

This circuit could constitutively express 3 types of S. aureus targeting endolysin-LysDZ25, ClyC, and Lys GH15-that have maximum efficiency conditions complementary with each other. This ensures that at least one type of endolysin will be functional under various conditions in the human digestive tract. The lytic efficiency of the endolysins with respect to concentration is being characterized separately in our project. For detailed information, see the following basic parts:

LysDZ25: BBa_K4593000

ClyC: BBa_K4593002

LysGH25: BBa_K4593003

QS-detection circuit

This circuit could constitutively express AgrA and AgrC, two proteins that transduce the QS signal. Specifically, AgrC is a membrane kinase that recognizes extracellular AIPs (QS molecule of S. aureus). After AgrC detects the QS signal, AgrA will be subsequently phosphorylated by it, gaining the ability to activate the transcription of genes downstream promoter P2.

Release circuit

The circuit allows the engineered bacteria to express the self-lysing enzyme complex (Spn1s_LysRZ) under the presence of S. aureus QS signal. Specifically, phosphorylated AgrA could activate the transcription activity of the P2 promoter, enabling the Spn1s_LysRZ located downstream to be transcribed.

For detailed characterization of Spn1s_LysRZ, see BBa_K4593001

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 3468
    Illegal NheI site found at 3491
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 418
    Illegal BglII site found at 6040
    Illegal BamHI site found at 1302
    Illegal XhoI site found at 2591
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 5684
    Illegal NgoMIV site found at 6524
    Illegal NgoMIV site found at 7198
    Illegal AgeI site found at 3090
    Illegal AgeI site found at 3228
    Illegal AgeI site found at 5553
    Illegal AgeI site found at 6668
    Illegal AgeI site found at 6896
    Illegal AgeI site found at 7067
    Illegal AgeI site found at 7180
  • 1000
    COMPATIBLE WITH RFC[1000]


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