Part:BBa_K4307040

attB-promoter-attP & Ser integrase

Serine integrase, adapted from bacteriophage, is capable of catalyzing site-specific recombination between two attachment sites (attP and attB) and inverting the DNA sequence flanked by these two opposing sites. We use Bxb1 integrase, an integrase found in Mycobacteriophage Bxb1 in our project.

This time, we combined attP and attB, the attachment sites of serine integrase with OR1-OR2, the part that has promoter function in the right operator (OR) of phage genome.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1040
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 980
  Illegal BamHI site found at 1231
  Illegal BamHI site found at 1609
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4
  Illegal NgoMIV site found at 2024
  Illegal AgeI site found at 2384
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 24
  Illegal BsaI site found at 55
  Illegal BsaI.rc site found at 150
  Illegal BsaI.rc site found at 831
  

Characterization

The following figure demonstrates our successful construction.

Figure 1: The construction results of attB-promoter-attP and Ser integrase.

Fluorescence assay was done to characterize the biobrick.

To ensure the inversion effect of Bxb1 integrase, we locate mCherry and EGFP upstream and downstream of OR1-OR2 promoter respectively.

The gene of Bxb1 integrase was integrated with pBAD promoter. The fluorescence signal of mCherry was recorded at 16th hour after induction with 1% (w/v) arabinose in this experiment. Besides the fluorescence, the OD600 was measured in order to normalize the fluorescence signal per cell. All groups were carried out twice to do a statistical analysis. Different experiments were induced in one 96 well plate. The culture temperature was 37℃ in this trial.

Figure 2: Characterization of serine integrase function by fluorescence spectrophotometry.

qPCR assay was done to characterize the biobrick.

We applied quantitative RT-PCR to measure the inversion efficiency of serine integrase. The primers used in qPCR were designed to amplify sequences in between only after inversion caused by integrase. We analyzed the inversion efficiency of serine integrase by measuring the Ct values of qPCR before and after arabinose induction. The influence of bacterial amount on the Ct values of target gene was blanked by measuring the Ct value of a reference gene (resistance marker gene on the plasmid vector).

After intricate analysis, we summarized valid data and performed regression analysis to test our detection. (Table 1) Even though the exact inversion efficiency cannot be determined, relative magnitude demonstrated that our system induced a two-fold increase in the ratio of inversed promoter sequence (Figure 3).

Table 1: qPCR results of serine integrase characterization.

Induction temperature (℃)	Induction time (h)	Target sequence Ct (unit)	Reference sequence Ct (unit)
Group 1	Induced	32.9	32.18
	Not induced	35.07	31.72
Group 2	Induced	14.195	14.655
	Not induced	14.91	13.615
Group 3	Induced	14.905	15.28
	Not induced	14.975	15.025
Group 4	Induced	13.834	14.705
	Not induced	14.785	13.325
Regression analysis	Induced	- 0.8791 * p + q - 0.8961 = 0
	Not induced	- 0.9299 * p + q - 1.5752 = 0

Figure 3: Characterization of serine integrase inversion efficiency by RT-qPCR. An exemplary qP graph (left) and the regression results (right).

Conclusion

We successfully expressed the integrase from bacteriophage in bacteria, and the results of the fluorescence assay showed that integrase could have obvious inversion effect, but the action time was a little bit long.