Part:BBa_K3332077

J23101-OmpA 5'UTR-phnF 0.69-Hfq binding sequence-J61048

Encoding the siRNA squence which can silence the phnJ gene in E.coli BL21(DE3).Use K823005 to silence the phnJ 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 phnJ in E. coli BL21(DE3) was used for RNAi. The number 0.69 means the silence probability (up to 1.0).

Usage

We ligased the promoter (BBa_J23101), the part (OmpA 5'UTR- phnJ 0.69-Hfq binding sequence) and the strong terminator (BBa_J61048) on the expression vector pSB1C3 to get the composite part BBa_K3332077 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

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-428bp (lane K3332077).

Fig.1 The result of plasmid cut with enzyme EcoRI and PstI . Plasmid: pSB1C3.

2. 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).

Fig.1 The result of plasmid cut with enzyme EcoR I and Pst I. Plasmid: pSB1C3.

3. qPCR

QPCR was used to determine the expression intensity of the target gene (phnJ & phnF). 16S gene was used as an internal reference.

Fig.2 The expression intensity of phnJ and phnF in the bacteria with RNAi plasmid.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 281
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 294
  
• 1000
  COMPATIBLE WITH RFC[1000]