DNA

Part:BBa_K4195000

Designed by: Honglin Song   Group: iGEM22_XMU-China   (2022-09-26)


OmpA 5'UTR-OmpA 0.83-hfq binding sequence


Encoding the siRNA sequence which can silence the ompA gene in E. coli BL21(DE3). Use BBa_I0500 to silence the ompA gene in E. coli BL21(DE3).

Biology

In E. coli, the omp system is precisely regulated. OmpA, an OM porin containing a PG interaction motif, adds noncovalent stability to the envelop of Gram-negative bacteria(1). The high stability of envelop is obviously disadvantageous for OMV massive secretion. In order to avoid the effects of endogenous ompA, we need a way to silence this gene. RNAi was used to silence it.

RNAi design can use TACE system. In the TACE system, a gene loop for transferring the DNA sequence corresponding to the siRNA, where the GGA Cassette can be replaced by the sequence encoding the siRNA according to the GGA assembly standard. OmpA 5’-UTR can protect siRNA from degradation, and hfq binding sequence can improve the binding efficiency of siRNA and target mRNA.

Using the siRNA design software provided by Team: Bielefeld-CeBiTec in iGEM 2018: siRCon, the siRNA sequence design for endogenous ompA in E. coli BL21(DE3) was used for RNAi. The number 0.83 means the silence probability (up to 1.0).


Usage

We assembled the inducible promoter (BBa_I0500), the part (OmpA 5'UTR-ompA 0.83-hfq binding sequence) and the double terminator (BBa_B0015) on the expression vector pSB1C3 to get the composite part BBa_K4195104 by standard assembly (Fig. 1). Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), and the positive transformants were confirmed by chloramphenicol, colony PCR and sequencing.

T--XMU-China--fig.1 siRNA for OmpA circuit.png

Fig. 1 Gene circuit of ompA RNAi sequence.


Characterization

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1890bp (lane K4195104).

T--XMU-China--fig.2 BBa K4195104 colony PCR BL21(DE3).png

Fig. 2 The result of colony PCR. Plasmid pSB1C3.


Then, the colony with the corrected sequence was cultivated to verify its hypervesiculation ability. We purified and quantitated the OMV of experimental and control group, whose gene circuit only contains the promoter BBa_I0500. The experimental result is shown on Fig. 3. We can draw the conclusion that the composite part BBa_K4195104 can’t achieve hypervesiculation ability compared with control group.

T--XMU-China--fig. 3NC vs OmpA 0.83-OMV secretion.png

Fig. 3 Normalized protein concentration and OD600 of experimental and control group.


Reference

1. C. Schwechheimer, C. J. Sullivan, M. J. Kuehn, Envelope control of outer membrane vesicle production in Gram-negative bacteria. Biochemistry 52, 3031-3040 (2013).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 216
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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