Part:BBa_K3769102

Cre-vvd

VVD, a photosensitive protein, is derived from the fungus Neurospora crassa, and homodimerizes under blue light in 400-490nm and separates in the dark. Cre is a commonly used tyrosine recombinase from the P1 bacteriophage that excises DNA flanked by loxP sites.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 141
  Illegal BglII site found at 1021
  Illegal XhoI site found at 855
  Illegal XhoI site found at 1735
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2176
  Illegal AgeI site found at 2659
  
• 1000
  COMPATIBLE WITH RFC[1000]

Demonstrate the function of Cre-VVD

We constructed pET30a-pT7-lacO-loxP-T-T-loxP-sfGFP and cotransformed it with pBbS5a-nCre-Vvd-Vvd-cCre into BL21(DE3). We set up five experimental groups (Figure 1) and measured the green fluorescence of them using a microplate reader. As we can see in Figure 2, cells that were cotransformed with both plasmids and received blue light showed a strong signal of green fluorescence, as strong as the positive control. In contrast, cells that were cotransformed with both plasmids and kept in dark showed a much lower signal. This indicated that blue light did activate the Cre-VVD system, which excised the terminators between two loxP sites and resulted in more sfGFP expression; however, it also meant that even without blue light, the Cre-VVD system could still form active dimers. Future work includes optimizing the Cre-VVD system and making it harder to form spontaneous dimers. Another curve representing cells that contained pET30a-pT7-lacO-loxP-T-T-loxP-sfGFP plasmid showed some expression of sfGFP, indicating that this plasmid itself had leaky expression. One thing we can do about this leakiness would be swapping the current terminators for even stronger terminators to prevent unwanted transcription from this construct before the Cre-VVD system comes to work. After this experiment, we analyzed the length of the core region of the pET30a-pT7-lacO-loxP-T-T-loxP-sfGFP plasmid of these cells to verify the excision of terminators. We amplified the region from promoter to terminator using the first and second experimental groups as templates. We then ran a gel to check their sizes. As shown in Figure 3, lane 1 is the linear DNA amplified using cells that were cotransformed with both plasmids and received blue light, while lane 2 is the linear DNA amplified using cells that had both plasmids but were kept in dark. The linear DNA in Lane 2 is 1113 bp in size, indicating that the terminators between two loxP sites were still there; on the other hand, the linear DNA in Lane 1 is only 913 bp in size, suggesting that the terminator region was excised from the plasmid. Combining both fluorescence and gel results, we showed that the Cre-VVD system worked as expected.

Figure 1. Experimental setup

Figure 2. A time-course result showing the Cre-VVD system is working.

Figure 3. An agarose gel showed the excision of terminators between two loxP sites. Lane 1: Opto-Cre-Vvd+IPTG+Light，Lane 2: Opto-Cre-Vvd+IPTG-Light.