Difference between revisions of "Part:BBa K3332098"
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<partinfo>BBa_K3332098 short</partinfo> | <partinfo>BBa_K3332098 short</partinfo> | ||
| − | Encoding the siRNA squence which can silence the phnF and phnJ gene in E.coli BL21(DE3). | + | Encoding the siRNA squence which can silence the phnF and phnJ gene in ''E.coli'' BL21(DE3).Use <partinfo>BBa_K823005</partinfo> to silence the phnF gene in ''E.coli'' BL21 (DE3). |
| − | + | ===Biology=== | |
| − | === | + | |
| + | In ''E. coli'', the phn system is precisely regulated. The protein PhnF gene encoding can directly inhibit the expression of other genes in the phn system. But this is obviously disadvantageous for our experiment. At the same time, C-P lyase is actually composed of expression products of genes other than phnJ, so there is site competition between endogenous phnJ and exogenously transformed phnJ. In order to avoid the effects of endogenous phnF and phnJ, we need a way to silence these two genes. We determined to silence genes using RNAi. | ||
| + | |||
| + | 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 iGEM2018: siRCon, the siRNA sequence design for endogenous phnF and phnJ in ''E. coli'' BL21(DE3) was used for RNAi. The number 0.97 and 0.94 means the silence probability (up to 1.0). | ||
| + | |||
| + | ===Usage=== | ||
| + | |||
| + | |||
| + | The plasmid with this sequence was transformed into ''E. coli'' DH5α & ''E. coli'' BL21(DE3), and the correct recombinant one was confirmed by chloramphenicol, enzyme-cut identification and sequencing. We used this part to characterize the influence of our RNAi sequence toward target sequences. | ||
| + | |||
| + | ===Characterization=== | ||
| + | '''1. Agarose Gel Electrophoresis''' | ||
| + | |||
| + | When we were building this circuit, enzyme-cut identification was used to certify the plasmid was correct. We used the ''EcoR''I and ''Pst''I to cut the plasmid, then we got the target separate fragment-823bp (lane 1). | ||
| + | <table><tr><th>[[File:T--XMU-China2020--BBa K3332098 1.png|thumb|500px|Fig.1 The result of plasmid cut with enzyme ''EcoR''I and ''Pst''I. Plasmid: pSB1C3.]]</th><th></table> | ||
| + | |||
| + | '''2. qPCR''' | ||
| + | |||
| + | QPCR was used to determine the expression intensity of the target gene (phnJ & phnF). 16S gene was used as an internal reference. | ||
| + | <table><tr><th>[[File:T--XMU-China2020--BBa K3332098 2.png|thumb|500px|Fig.2 The expression intensity of phnJ and phnF in the bacteria with RNAi plasmid.]]</th><th></table> | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Latest revision as of 22:30, 27 October 2020
RNAi sequence of phnF(0.97)-RNAi sequence of phnJ(0.94)
Encoding the siRNA squence which can silence the phnF and phnJ gene in E.coli BL21(DE3).Use BBa_K823005 to silence the phnF gene in E.coli BL21 (DE3).
Biology
In E. coli, the phn system is precisely regulated. The protein PhnF gene encoding can directly inhibit the expression of other genes in the phn system. But this is obviously disadvantageous for our experiment. At the same time, C-P lyase is actually composed of expression products of genes other than phnJ, so there is site competition between endogenous phnJ and exogenously transformed phnJ. In order to avoid the effects of endogenous phnF and phnJ, we need a way to silence these two genes. We determined to silence genes using RNAi.
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 iGEM2018: siRCon, the siRNA sequence design for endogenous phnF and phnJ in E. coli BL21(DE3) was used for RNAi. The number 0.97 and 0.94 means the silence probability (up to 1.0).
Usage
The plasmid with this sequence was transformed into E. coli DH5α & E. coli BL21(DE3), and the correct recombinant one was confirmed by chloramphenicol, enzyme-cut identification and sequencing. We used this part to characterize the influence of our RNAi sequence toward target sequences.
Characterization
1. Agarose Gel Electrophoresis
When we were building this circuit, enzyme-cut identification was used to certify the plasmid was correct. We used the EcoRI and PstI to cut the plasmid, then we got the target separate fragment-823bp (lane 1).
2. qPCR
QPCR was used to determine the expression intensity of the target gene (phnJ & phnF). 16S gene was used as an internal reference.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 438
Illegal NheI site found at 461 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 281
Illegal XhoI site found at 712 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]