Part:BBa_K5276025

Register 0

A118B-AA_lac promoter_trc promoter_A118B-GG_PhiC31B-AA_A118P-GG_J23111_Rha promoter_PhiC31P-AA_A118P-AA_RBS_GFP

Description

Register 0 is a unit of expression consisting of four promoters(constant promoter J23111, trc promoter, and inducible promoter lac operon, Rha promoter) with different directions, Three pairs of DNA recognition sites(A118B-GG_A118P-GG, A118B-CC_A118P-CC, PhiC31B-AA_PhiC31P-AA), a Ribosome binding site, and msfGFP.

Figure 1: Diagram of Register 0 Plasmid

Register 0 is a regulation part that can be used to verify the feasibility of our Register system; it can verify whether two or more promoters can work together and whether it is effective to take regulatory proteins like LacI apart from promoters like P_lac.

Results

To verify whether two or more promoters can work together, in our Register parts, we first construct Register 0 plasmid as an example, which consists of some overlapping and orthogonal recombinase recognition sites, four promoters(constant promoter J23111, trc promoter, and inducible promoter lac operon, Rha promoter) with different directions. We used Register 0 to verify whether two promoters in a series can achieve the AND gate function and whether the constant promoter in the opposite direction will interfere with the promoter expressing the forward direction. According to previous experiments, the distance between the promoter and the start codon should be as close as possible, so we placed the necessary elements of the inducible promoter such as LacI, rhaS, rhaR outside the recombinase sites and only placed these promoter in the register region.

Figure 2: Map of Register 0 plasmid

After the plasmid was successfully constructed, we transferred the Register 0 plasmid into BL21 (DE3) competent cells and induced them by adding 1mM IPTG and 0.3% rhamnose at a final concentration after the cells grew to the logarithmic growth phase. Then we recorded the time span and fluorescence intensity as output. The results are as follows.

Figure 3. AND GATE test of Lac operon and rhamnose promoter.

The single Register 0 without recombinase input can be an excellent AND gate. The figure above shows that IPTG and RHA have a relatively ideal induction effect, indicating that our promoters work properly. Since the distance between the Lac operon and the start codon is farther than that of the rhamnose promoter, the Lac operon has a smaller effect on promoting the transcription of mRNA when the rhamnose promoter is working.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 118
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 489
  Illegal NheI site found at 512
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 118
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 118
  Illegal AgeI site found at 1128
  Illegal AgeI site found at 1251
  
• 1000
  COMPATIBLE WITH RFC[1000]