Difference between revisions of "Part:BBa K4195000"
(→Reference) |
|||
| (One intermediate revision by one other user not shown) | |||
| Line 4: | Line 4: | ||
| − | Encoding the siRNA sequence which can silence the ompA gene in ''E. coli'' BL21(DE3). Use <partinfo>BBa_I0500</partinfo> to silence the ompA gene in ''E. coli'' BL21(DE3). | + | Encoding the siRNA sequence which can silence the ''ompA'' gene in ''E. coli'' BL21(DE3). Use <partinfo>BBa_I0500</partinfo> to silence the ''ompA'' gene in ''E. coli'' BL21(DE3). |
===Biology=== | ===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. | + | 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. | 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). | + | 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). |
| Line 21: | Line 21: | ||
[[File:T--XMU-China--fig.1 siRNA for OmpA circuit.png|300px]] | [[File:T--XMU-China--fig.1 siRNA for OmpA circuit.png|300px]] | ||
| − | '''Fig. 1 Gene circuit of ompA RNAi sequence.''' | + | '''Fig. 1 Gene circuit of ''ompA'' RNAi sequence.''' |
| Line 37: | Line 37: | ||
[[File:T--XMU-China--fig. 3NC vs OmpA 0.83-OMV secretion.png|300px]] | [[File:T--XMU-China--fig. 3NC vs OmpA 0.83-OMV secretion.png|300px]] | ||
| − | '''Fig. 3 Normalized protein concentration and | + | '''Fig. 3 Normalized protein concentration and OD<sub>600</sub> of experimental and control group.''' |
Latest revision as of 09:12, 12 October 2022
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.
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).
.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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 216
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]