Part:BBa_K2598025

lacZ under CGG-regulated promoter pCGG

This part contains lacZ under a CGG-regulated promoter pCGG, T7 RNAP sigma fragment CGG(BBa_K2598011) can direct T7 core fragment to pCGG, inducing the expression of downstream genes. A strong terminator is added to avoid recombination.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 3112
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 5
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Figure 1 shows the relationship between the wavelength of light exposed on liquid medium and the intensity of BFP, GFP and RFP E. coli expressed from left figure to right figure respectively. We got the data through flow cytometer and analyzed it to get the figure. The y-axis is the number of cells, and the x-axis is fluorescence intensity. And every color is E. coli that grows for 8 hours under the light of the corresponding wavelength. We can see E. coli has the highest blue fluorescence expression under blue light from the left graph. And We can also see E. coli has the highest green and red fluorescence expression under green light and right light from the middle and right graph respectively. So this figure proves that our system and our parts can work well.

Figure 1:Relationship between the wavelength of light exposed on liquid medium and the intensity of BFP, GFP and RFP E. coli expressed from left figure to right figure respectively

Characterization

Figure 2 shows the relationship between fluorescence intensity and excitation wavelength. The x-axis is wavelength of 10h illumination. The solid medium gradually emerged and the y-axis is RGB figure of fluorescence in illuminated solid medium. This curve illustrates how our system responses to different excitation wavelength, which perfectly meets our expectation. So this figure proves that our system and our parts can work well.

Figure 2:Relationship between fluorescence intensity and excitation wavelength

Characterization

Figure 3 shows colors we got from the solid medium exposed under light, in which E. coli producing fluorescent protein grows. When E .coli producing fluorescent protein are exposed under uniform light of single wavelength, the solid medium gradually emerged corresponding colors. And using color picker, we get many pure colors with predominant continuity.

Figure 3: Colors we got from the solid medium, in which E. coli producing fluorescent protein grows, exposed under light

Characterization

We explored the relationship between fluorescent intensity and illumination intensity, which affects the shade of the color. Figure 4 shows the red fluorescent intensity of ecoli under light of 620-630nm wavelength with different illumination intensity. We found when illuminated under around 400-600lux light, we can get the most red fluorescence. We also explored the relationship between green and blue fluorescent intensity of ecoli under light of 515-530nm wavelength and 460-470nm wavelength respectively and illumination intensity. The results were similar, that is, moderate intensity of light is most favorable for E. coli to express fluorescence.

Figure 4:The relationship between the red fluorescent intensity of ecoli under light of 620-630nm wavelength and illumination intensity

Characterization

Figure 5 shows results of gel electrophoresis of various parts after PCR with primer VF2\VR. The distance between primer binding sites and both ends of the parts are approximately 150 bp, thus rendering the product about 300 bp longer. The picture is edited to show a more compact photo.

Figure 5: Results of gel electrophoresis of various parts after PCR with primer VF2\VR