Difference between revisions of "Part:BBa K3332031"
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===Biology=== | ===Biology=== | ||
| − | In ''E. coli'', the phn system is precisely regulated. PhnF gene encoding protein 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. | + | In ''E. coli'', the phn system is precisely regulated. ''PhnF'' gene encoding protein 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, 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, Hfq binding sequence can improve the binding efficiency of siRNA and target mRNA. | ||
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When we were building this circuit, enzyme-cut identification was used to certify the plasmid was correct. We used the ''Eco''R I and ''Pst'' I to cut the plasmid, then we got the target separate fragment-428bp (lane K3332075). | When we were building this circuit, enzyme-cut identification was used to certify the plasmid was correct. We used the ''Eco''R I and ''Pst'' I to cut the plasmid, then we got the target separate fragment-428bp (lane K3332075). | ||
| − | <table><tr><th>[[File:T--XMU-China2020--BBa K3332029.png|thumb|500px|Fig.1 The result of plasmid cut with enzyme ''Eco''R I and ''Pst''I . Plasmid: pSB1C3.]]</th><th></table> | + | <table><tr><th>[[File:T--XMU-China2020--BBa K3332029.png|thumb|500px|Fig.1 The result of plasmid cut with enzyme ''Eco''R I and ''Pst'' I . Plasmid: pSB1C3.]]</th><th></table> |
===Sequence and Features=== | ===Sequence and Features=== | ||
Revision as of 02:03, 28 October 2020
OmpA 5'UTR-phnF 0.75-Hfq binding sequence
Encoding the siRNA squence which can silence the phnF 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. PhnF gene encoding protein 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, 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 in E. coli BL21(DE3) was used for RNAi. The number 0.75 means the silence probability (up to 1.0).
Usage
we ligased the strong promoter (BBa_J23101), the part (OmpA 5'UTR- phnF 0.75-Hfq binding sequence) and the strong terminator (BBa_J61048) on the expression vector pSB1C3 to get the composite part BBa_K3332075 by standard assembly. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), and the correct recombinant one was confirmed by chloramphenicol, enzyme-cut identification and sequencing.
Characterization
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-428bp (lane K3332075).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 218
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]