Part:BBa_K4169030

Cre-reverse loxP system:working when you need to express genes in the other direction

In the presence of theophylline, subsequent expression of Cre is induced. In addition, the promoter and RBS between the two reverse loxP's will be flipped so that it expresses the gene in the other direction.

Usage and Biology

When our engineered bacteria sense theophylline, it expresses Cre, which recognizes the reverse repeat sequence at both ends of the loxP site and binds to form a dimer, which then binds to a dimer at another loxP site to form a tetramer. In this way, Cre mediates sequence flips between reverse loxPs.

Agarose Gel Electrophoresis

First, we identified this part by agarose gel electrophoresis.  We transferred it to DH5α and performed PCR verification, as shown in Figure 1, loxP, promoter, RBS target band size is 205 bp, and the actual electrophoresis results met the target band size;  figure 2 below, promoter, theophylline-induced opening riboswicth, Cre target band size is 1323 bp, the actual electrophoresis results met the target band size.

Figure 1. Agarose gel electrophoresis analysis of Cre.

SDS-PAGE

Second, we identified Cre by SDS-PAGE. We identified that Cre can be expressed after we add theophylline induction.

Figure 3. SDS-PAGE analysis of Cre.
Through SDS-PAGE analysis, We found that DH5α with the Cre plasmid transformed had two bands between 35 kDa and 40 kDa, but only one band in DH5α without the transformed plasmid. Cre's molecular mass size is 38 kDa，We identified that this part can produce Cre.

Functional Parameters

To verify the functionality of our part, we had the upstream gene of the promoter replaced with red fluorescence，and then transferred our plasmid into Escherichia coli DH5α and measured the intensity of red fluorescence with a microplate reader. Unfortunately, our bacteria did not produce red fluorescence, and upon analysis, it was conjectured that the transcriptional loxP formed a strong secondary structure and could not translate the red fluorescence protein behind it.

Sequence and Features

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
  Illegal NheI site found at 1292
  Illegal NheI site found at 1315
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 454
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 39
  Illegal AgeI site found at 140
  
• 1000
  COMPATIBLE WITH RFC[1000]