Composite

Part:BBa_K4195107

Designed by: Xiaoping Yu   Group: iGEM22_XMU-China   (2022-09-27)
Revision as of 15:08, 12 October 2022 by Rynnie (Talk | contribs)


I0500-OmpA 5'UTR-Lpp 1.0-hfq binding sequence-B0015


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


Biology

In E. coli, Lpp is an abundant lipoprotein which can covalently cross-links the OM and PG, providing structural envelope integrity and stability(1). The high stability of envelope is obviously disadvantageous for OMV massive secretion. In order to avoid the effects of endogenous lpp, 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 lpp in E. coli BL21(DE3) was used for RNAi. The number 1.0 means the silence probability (up to 1.0).


Usage

We assembled the inducible promoter (BBa_I0500), the part (OmpA 5'UTR-lpp 1.0-hfq binding sequence) and the double terminator (BBa_B0015) on the expression vector pSB1C3 to get the composite part BBa_K4195107 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. 1siRNA for Lpp circuit.png

Fig. 1 Gene circuit of lpp RNAi sequence.


Characterization

1. Agarose gel electrophoresis (AGE)

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1890bp (lane K4195107).

T--XMU-China--fig. 2BBa K4195107(Lpp1.0,colony PCR,BL21(DE3)) .png

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

2. Hypervesiculation ability

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. Observing the difference of OD600 between two groups, we can guess that bacterial membrane structure’s stability decreases in experimental group and draw the conclusion that the composite part BBa_K4195107 has the hypervesiculation ability compared with control group.

T--XMU-China--fig. 3NC vs Lpp 1.0-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
    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 1434
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


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